Leptomonas sp. s93-3 and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a strain of Pseudochlorella limnetica S93‑3 and application thereof. One of the purposes of the application is to provide a strain of Pseudochlorella limnetica Coelastrella tenuitheca ) strain S93‑3 The strain is preserved in China Center for Type Culture Collection on November 28, 2025, and the preservation number is CCTCC NO: P202541. S93‑3 The strain provides important strain resources and application approaches for developing new heavy metal pollution remediation technologies which are environment-friendly and have strong synergies, and has wide prospects in agricultural safety production and ecological environment governance.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a thin-walled monostellar algae. S93-3 And its applications. Background Technology

[0002] Cadmium (Cd) is a non-essential element for the human body, widely present in the natural environment, often existing in compound form and difficult to degrade. It is one of the most biotoxic heavy metals. Soil pollution sources are diverse, including industrial metal smelting, domestic sewage discharge, and fertilizer use. Cadmium has a strong tendency to accumulate in plants and animals; in humans, even low concentrations can cause harm, for example, by binding to sulfhydryl-containing proteins, inhibiting enzyme activity, and thus causing disease. Many grain crops, such as rice, have a strong bioaccumulation capacity for heavy metals, especially cadmium, which is most prominent among cereal crops. Cadmium enters the human body through food, water, and air and accumulates in the body. Therefore, reducing the total cadmium content and decreasing the available cadmium content in arable land is crucial.

[0003] To address this challenge, efficient and eco-friendly restoration strategies need to be developed. 2+ Remediation employs various methods, such as filtration, sedimentation, adsorption, coagulation, ion exchange, and photocatalysis. Among these technologies, biosorption, utilizing microorganisms as biosorbents, has shown significant advantages, including high cost-effectiveness, ease of operation, and environmental safety. Among various biosorbents (such as bacteria, fungi, yeast, and algae), microalgae stand out as a low-cost and environmentally friendly option, effectively removing Cd by utilizing nitrogen and phosphorus in wastewater as nutrients. 2+ As photosynthetic microorganisms, microalgae are easy to cultivate and remain effective even as dead-cell biosorbents. Unlike traditional methods, biosorbents produce minimal sludge, reducing waste management challenges. Furthermore, the biomass used in biosorbents is typically renewable and reusable, further enhancing sustainability. Biosorbents are a highly efficient and sustainable method for removing Cd. 2+ Repair method.

[0004] Currently, various microalgae strains have shown resistance to Cd. 2+ The biosorption effect, for example Neochloris- oleoabundans、 Chlamydomonas, Scenedesmus sp. Microalgae, such as Chlorella, are examples of algae that possess Cd remediation capabilities. While many microalgae resources have been shown to have Cd remediation capabilities, most remain in the theoretical research stage, and their Cd bioremediation mechanisms need further elucidation. Therefore, exploring more Cd-tolerant and Cd-remediation-capable algal strains, verifying their Cd remediation functions, and elucidating their potential mechanisms are of great significance.

[0005] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the inventors studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0006] This invention belongs to the field of microbial technology, specifically relating to a thin-walled monostellar algae. S93-3 And its applications.

[0007] One of the objectives of this invention is to provide a thin-walled monostellar algae ( Coelastrella tenuitheca ) strain S93-3 This strain was deposited at the China Center for Type Culture Collection on November 28, 2025, with accession number CCTCC NO: P202541.

[0008] One of the objectives of this invention is to provide the aforementioned thin-walled monostellar algae. S93-3 The ITS, whose sequence is shown in SEQ ID NO.1.

[0009] One of the objectives of this invention is to provide the aforementioned thin-walled monostellar algae. S93-3 Applications in the cultivation of cadmium-tolerant plants.

[0010] One of the objectives of this invention is to provide the aforementioned thin-walled monostellar algae. S93-3 Applications in reducing cadmium concentrations in the environment.

[0011] According to a preferred embodiment, thin-walled monostellatium... S93-3 It can be mixed with other excipients permitted by the product to prepare a formulation for application.

[0012] One objective of this invention is to provide a method for mitigating cadmium toxicity in plants under cadmium stress, comprising the following steps: utilizing the aforementioned thin-walled monostellatium. S93-3 Plants were treated with bacterial suspensions.

[0013] One of the objectives of this invention is to provide the aforementioned thin-walled monostellar algae. S93-3 Applications include reducing the inhibitory effect of cadmium on plant growth, or preparing products that can reduce the inhibitory effect of cadmium on plant growth.

[0014] One of the objectives of this invention is to provide the aforementioned thin-walled monostellar algae. S93-3 Applications in alleviating cadmium toxicity in plants under cadmium stress, or applications in preparing products for alleviating cadmium toxicity in plants under cadmium stress.

[0015] One of the objectives of this invention is to provide the aforementioned thin-walled monostellar algae.S93-3 Applications in promoting plant biomass increase under cadmium stress, or in the preparation of products that promote plant biomass increase under cadmium stress.

[0016] One of the objectives of this invention is to provide the aforementioned thin-walled monostellar algae. S93-3 Applications in the preparation of cadmium-resistant microbial pesticides or wastewater treatment formulations suitable for plants.

[0017] One objective of this invention is to provide a method for mitigating cadmium toxicity in plants under cadmium stress, which utilizes the aforementioned thin-walled monostellate algae. S93-3 Plants were treated with bacterial suspensions.

[0018] According to a preferred embodiment, the treated plant is a watered plant. Preferably, the above-mentioned thin-walled monostellatium is used. S93- 3 OD produced by strain 600 The seed culture was prepared at a concentration of 0.5 μL, and then inoculated into cadmium-contaminated samples and cultured at 25–37°C for 1–7 days. The final cell concentration (OD) of the strain in the cadmium-contaminated samples was determined. 600 The value is 0.5. Preferably, the culture is carried out at 28°C for 7 days.

[0019] According to a preferred embodiment, the plant is rice or lettuce.

[0020] One objective of this invention is to provide a formulation for reducing cadmium concentration in the environment, comprising the aforementioned thin-walled monostellate algae. S93-3 The formulation also contains excipients.

[0021] According to a preferred embodiment, the concentration of cadmium in the water body is not greater than 37.92 mg / L.

[0022] This invention provides a plant S93-3 This strain exhibits remarkable adaptability and remediation capabilities in cadmium-contaminated environments, with its beneficial effects primarily manifested in the following aspects: first, S93-3 The strain exhibits highly efficient removal and immobilization capabilities for cadmium. It can accumulate large quantities of cadmium ions intracellularly through biosorption. At 30 mg / L CdCl2, the maximum Cd removal rate of this strain is 94.87%, and after 7 days of treatment at 10-20 mg / L CdCl2, the maximum Cd removal efficiency is >98%. Under S93-3 treatment conditions, 48% of the Cd is removed through adsorption, and 30% is removed through biotransformation. Simultaneously, under cadmium stress… S93-3 It maintained high photosynthetic activity, which was associated with the transcriptional upregulation of chlorophyll synthesis components (chla and b) and light-gathering complex proteins (Lhca and Lhcb).

[0023] Secondly, strain S93-3 maintained strong physiological activity and tolerance mechanisms under cadmium stress. Studies confirmed that its photosynthetic activity remained at a high level, which was related to the upregulation of chlorophyll synthesis and light-harvesting protein-related genes. Under cadmium stress, S93-3 The strain maintained high photosynthetic activity, which was related to the transcriptional upregulation of chlorophyll synthesis components (chla and b) and light-collecting complex proteins (Lhca and Lhcb). Specifically, through sulfur assimilation and cysteine / methionine metabolic pathways, the strain promoted glutathione (GSH) synthesis, thereby forming a cadmium-plant chelate complex, achieving cadmium detoxification and intracellular fixation. Metabolomics analysis further showed that the accumulation of key metabolites such as aminolevulinic acid helped stabilize porphyrin metabolism and the TCA cycle, enhancing the strain's survival ability in a cadmium environment.

[0024] Furthermore, this strain possesses excellent application potential. Developing it into microbial fertilizer or environmental remediation engineered bacteria and applying it to cadmium-contaminated soil or water can effectively reduce the absorption and translocation of cadmium by crops. Validation experiments in hydroponic rice and lettuce have shown that inoculation... S93-3 It can significantly improve crop growth, reduce oxidative stress, and systematically enhance plant tolerance to cadmium stress by regulating the expression of genes related to cadmium absorption and detoxification in plants.

[0025] therefore, S93-3 The strain provides important microbial resources and application pathways for developing new environmentally friendly and synergistic heavy metal pollution remediation technologies, and has broad prospects in agricultural safety production and ecological environment governance. Attached Figure Description

[0026] Figure 1 For the present invention Coelastrella tenuithecaS93-3 High tolerance to Cd, where A represents different microalgal species under Cd conditions. 2+ Phenotypic characteristics under stress; B represents the IC50 values ​​of different microalgal species under different CdCl2 treatments. 50 Value; C represents the total Cd in the organism and liquid culture medium. 2+ Content; D represents the different extractable chemical forms of Cd in organisms. 2+ ;EG is S93-3 Cells in Cd 2+ SEM analysis under pressure, where CK: control group (cadmium-free); Cd10: 10 mg / L cadmium concentration; Cd35: 35 mg / L cadmium concentration, each experiment was repeated three times; H and I represent the concentrations of cadmium under pressure. 2+ Under coercion S93-3 Cell TEM analysis results; Figure 2 This is a dose-response curve for microalgae exposed to cadmium. The curve represents the IC50 of the cadmium concentration at which growth is inhibited by 50%. 50The value is calculated based on the nonlinear regression curve (n=3); Figure 3 Under cadmium stress S93-3 The efficiency of cadmium absorption; Figure 4 for Coelastrella tenuithecaS93-3 The effect of photosynthesis and antioxidant reactions on Cd 2+ The response to pressure, where A and B are photosynthetic efficiency parameters: Fv / Fm(A) and Y(II)(B); CD represents the response of different Cd values. 2+ Quantitative analysis of Fv / Fm and Y(I) under pressure for 24 hours; EG represents the activity of antioxidant enzymes (SOD, POD, and CAT) at different time points; H represents IC50. 50 Condition Cd 2+ Treatment of GSH content at different time points; IJ is IC 50 Cd at different time points under different conditions 2+ The levels of stress biomarkers (MDA and H2O2) under treatment were measured, and each experiment was repeated three times. P≤0.05, P≤0.01, P≤0.001; Figure 5 To investigate the effects of cadmium stress on plant growth, based on phenotype, a concentration of 10 mg / L of Cd was selected. 2+ To verify S93-3 For Cd 2 + The effect of stress relief on plants; Figure 6 for S93-3 Cd on rice seedlings 2+ The study assessed the mitigation effects of toxicity, with A and B representing phenotypic characteristics of rice plants and roots; C and D representing quantitative analyses of plant height and leaf biomass; E and F representing quantitative analyses of root length and biomass; G representing the determination of Cd content in leaf and root stem organisms; and H and I representing the distribution of soluble Cd chemical forms in leaves and roots. Each experiment was repeated at least three times. P < 0.001. In the CF plot, ad indicates significance at the α = 0.05 level. CK: untreated control. S93-3 : Only treat rice plants S93-3 Cd10: with 10 mg / L Cd 2+ Rice plants treated separately, S93-3 +Cd10: Use S93-3 With 10 mg / L Cd2+ Combined processing methods; Figure 7 for S93-3 Cd on lettuce 2+ The study investigated the mitigation of toxicity, where A and B represent the phenotypes of lettuce stems and roots; C and D represent the quantification of plant height and leaf fresh biomass; E and F represent the quantification of root length and fresh biomass; G represents the cadmium content in leaf and root dry biomass; and H and I represent the distribution of extractable chemical forms of Cd in leaves and roots. Each experiment was repeated at least three times. P≤0.001, ad in the figure indicates a statistically significant difference of P<0.05, CK: untreated control group, S93-3 Used alone S93-3 Rice treated with Cd10: 10 mg / L Cd alone 2+ Processed rice S93-3 +Cd10: S93-3 and 10 mg / LCd 2+ Joint processing; Figure 8 Morphological structure and cross-sectional analysis of rice and lettuce roots, where A and B are the control (CK) and control (C) roots. S93-3 The treated root system exhibited a normal, intact endodermis (ED) and cortex (C); C represents the area under Cd. 2+ Illustration of endothelial and cortical cell degradation and breakdown under stress; D represents the degradation and breakdown of endothelial and cortical cells under Cd. 2+ Under pressure, S93-3 The treated plants maintained the integrity of the cortex and central column (ST); E and H are magnified views of ED and ST. Abbreviations: EX represents the exocortex, EP represents the epidermis, AE represents the air cavities, C represents the cortex, ST represents the central column, and ED represents the endocortex. Figure 9 for S93-3 The mitigation effect of cadmium ion toxicity on the inhibition of photosynthesis in rice seedlings, where A represents the phenotype of maximum efficiency (Fv / Fm) of photosynthetic system II under different treatments; BD represents the quantitative analysis of Y(II), Y(NPQ) and ETR, where Y(II) represents the actual photochemical efficiency, Y(NPQ) represents the non-photochemical quenching, and ETR represents the electron transport rate. Figure 10 for S93-3 The mitigating effect of cadmium ion toxicity on the inhibition of photosynthesis in lettuce, where A represents the phenotype of maximum efficiency (Fv / Fm) of photosynthetic system II under different treatments; BD represents the quantitative analysis of Y(II), Y(NPQ) and ETR; Y(II) represents the actual photochemical efficiency, Y(NPQ) represents the non-photochemical quenching, and ETR represents the electron transport rate. Figure 11 The total cadmium content in the nutrient solution of rice and lettuce under cadmium stress was given. Differences between different letters were statistically significant (P < 0.05). CK: Untreated control group. S93-3 Used alone S93-3 Rice treated with Cd10: 10 mg / L Cd alone 2+ Processed rice S93-3 +Cd10: S93-3 and 10 mg / L Cd 2+ Joint processing; Figure 12 For application S93-3 For Cd 2+ The effects of stress on CAT activity, GSH content, POD activity, SOD activity, MDA content, and H2O2 content in the stems and roots of rice (AF) and lettuce (GL). Different letters indicate statistically significant differences with P < 0.05. Detailed Implementation

[0027] In the description of this invention, terminology is used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly defined.

[0028] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents or instruments used, unless otherwise specified by the manufacturer, are all commercially available reagents and materials; the conditions not specified in the examples are all carried out according to conventional conditions or conditions recommended by the manufacturer. At the same time, the present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all commercially available products in this technical field.

[0029] In the following examples, the modified BG11 solid culture medium formulation is as follows: glucose 18-22 g / L, sodium nitrate 1.3-1.7 g / L, K2HPO4•3H2O 0.03-0.05 g / L, MgSO4•7H2O 0.065-0.085 g / L, CaCl2•2H2O 0.026-0.046 g / L, citric acid 0.005-0.007 g / L, ferric ammonium citrate 0.005-0.007 g / L, EDTA 0.0005-0.0015 g / L, sodium carbonate 0.015-0.025 g / L, A5+Co stock solution 0.8-1.2 mL / L, agar powder 13-17 g / L, pH 6.0-6.5; The A5+Co mother liquor contains the following components: boric acid 0.0027-0.0029 g / L, MnCl2•H2O 0.0017-0.0019 g / L, ZnSO4•7H2O 0.00021-0.00023 g / L, CuSO4•5H2O 0.00007-0.00009 g / L, Na2MoO4•2H2O 0.0003-0.0005 g / L, and Co(NO3)2•6H2O 0.00004-0.00006 g / L.

[0030] The modified BG11 liquid culture medium formula is as follows: glucose 18-22 g / L, sodium nitrate 1.3-1.7 g / L, K2HPO4•3H2O 0.03-0.05 g / L, MgSO4•7H2O 0.065-0.085 g / L, CaCl2•2H2O 0.026-0.046 g / L, citric acid 0.005-0.007 g / L, ferric ammonium citrate 0.005-0.007 g / L, EDTA 0.0005-0.0015 g / L, sodium carbonate 0.015-0.025 g / L, A5+Co stock solution 0.8-1.2 mL / L, pH 6.0-6.5; the A5+Co stock solution contains the following components: boric acid 0.0027-0.0029 g / L, MnCl2•H2O 0.0017-0.0019 g / L, ZnSO4·7H2O 0.00021-0.00023 g / L, CuSO4·5H2O 0.00007-0.00009 g / L, Na2MoO4·2H2O 0.0003-0.0005 g / L, Co(NO3)2•6H2O0.00004-0.00006 g / L.

[0031] 1.1 Determination of Cadmium Tolerance of Different Microalgae Species All representative microalgal species selected for cadmium tolerance analysis were screened from different regions of China and underwent morphological and molecular characterization. The strains were classified as: SZ2 ( Chlorella sorokiniana RW13-10 ( Chlorella variabilis S9-3 Chlorella variabilis ), S93-3 ( Coelastrella tenuitheca S5-2 Parachlorella kessleri ), RW9 ( Parachlorella kessleri ), RM12-2 ( C. vulgaris J3 Auxenochlorella pyrenoidosa L11 Desmodesmus abundans The isolated strains were cultured individually in modified BG11 liquid medium (containing 20 g / L glucose) for 5 days. Cadmium treatment was applied, and appropriate amounts of microalgal cultures with an OD value of 0.5 were inoculated into modified BG11 liquid medium containing 5, 10, 15, 20, 25, 30, and 35 mg / L cadmium chloride (CdCl2), respectively. The cultures were incubated at 28°C and 150 rpm for 7 days. OD values ​​were recorded daily. 680 Biomass and growth status were considered, and the half-maximum inhibitory concentration (IC50) was calculated based on dry biomass. 50 Cadmium-resistant strains were selected for subsequent experiments.

[0032] 1.2 Cd 2+ Let's deal with it. Coelastrella tenuithecaS93-3 TEM and SEM observations Microalgal strains were inoculated into modified BG11 medium containing 0, 10, and 35 mg / L CdCl2, with initial OD... 680 The concentration was 0.5. Cultures were incubated at 28°C and 150 rpm for 5 days. Microalgal cells were harvested by centrifugation, resuspended in sterile water, washed three times via centrifugation, and then fixed overnight at 4°C in 2.5% glutaraldehyde solution (0.1 M PBS buffer, pH 7.5). After centrifugation to remove glutaraldehyde, the samples were dehydrated using a series of fractionated ethanol solutions (50%, 70%, 80%, 90%, 95%, 100%, v / v). Subsequently, the samples were treated twice with tert-butanol for 20 minutes each time to replace the ethanol. Before scanning electron microscopy (SEM) for cell morphology analysis, samples underwent critical point drying and gold sputtering. For transmission electron microscopy (TEM) preparation, dehydrated samples were treated with pure acetone for 20 minutes, embedded in resin, and sectioned into ultrathin sections of 50–60 nm. TEM was used to observe cd-induced intracellular ultrastructural changes.

[0033] 1.3 Determination of Cd content in microalgae and plants Determination of Cd by acid digestion method 2+Content. Specifically, accurately weigh 0.5 g of sample, add 5 mL of concentrated nitric acid (HNO3), and soak overnight in a polytetrafluoroethylene (PTFE) digestion tube at room temperature. Digest the organic matter using a MARS digestion system at 80°C for 2 h, 120°C for 2 h, then raise the temperature to 160°C and hold for 4 h, followed by cooling to room temperature. After digestion, drain the acid using a hot plate, transfer the solution to a 25 mL volumetric flask, dilute to the mark with 1% (v / v) nitric acid solution, and mix thoroughly.

[0034] Cd content was detected using inductively coupled plasma mass spectrometry (ICP-MS).

[0035] 1.4 Cd 2+ Extraction and quantification of extractable chemical forms Chop 1 g of sample and grind into a fine slurry using 20 mL of extraction buffer. Transfer the slurry to a 50 mL centrifuge tube and extract with shaking at 25°C for 18 hours, followed by centrifugation at 5000 g for 10 minutes. Collect the supernatant, resuspend the residue in an equal volume of fresh extractant, and perform three additional extraction cycles at 2-hour intervals under the same conditions. After the final extraction, combine all supernatants into a 150 mL Erlenmeyer flask. Extract the residue sequentially with solvents of increasing polarity to separate different cadmium fractions: 80% ethanol for extraction of inorganic salts (e.g., nitrates, chlorides) and amino acid salts; deionized water for extraction of water-soluble organic salts; 1 mol / L NaCl for extraction of pectin, protein-bound, or adsorbed heavy metals; 2% acetic acid for extraction of water-insoluble heavy metal phosphates; and 0.6 mol / L HCl for extraction of oxalates. The final residue contains residual heavy metals. Quantify the cadmium content in each fraction (supernatant and residue) using ICP-MS.

[0036] 1.5 Assay of antioxidant enzyme activity For microalgae treatment, Coelastrella tenuithecaS93-3 Cultured in modified BG11 medium until OD 680The algal biomass was approximately 4, and then exposed to 20 mg / L CdCl2 for 48 hours. After treatment, algal biomass was harvested by centrifugation at 8000 g for 10 min, and washed twice with sterile deionized water (by resuspension-centrifugation cycle) to thoroughly remove residual supernatant. 0.5 g of microalgal biomass was added to PBS buffer (0.1 M, pH 7.4) and subjected to cryogenic sonication at 100 W for 30 min. The homogenate was centrifuged at 10000 g for 10 min, and the supernatant containing intracellular soluble components was collected for subsequent analysis. In potted treatments, root and shoot tissues (stem and leaf combinations) were harvested after 30 days of CdCl2 exposure and frozen with liquid nitrogen under shaking. In microalgae and plant tissues, SOD, POD, CAT activities, and GSH, H2O2, and MDA contents were quantified according to the kit manufacturer's instructions (CAT No. 2). Each sample was tested in at least three replicates.

[0037] 1.6 Plant Materials and Pot Experiment Design With rice ( Oryza sativa L. cv. Nipponbare ),lettuce( Lactuca sativa L. cv Flandria Using rice seeds as materials, a hydroponic / pot experiment was conducted to test the remediation effect of microalgae on cadmium. Seeds were sterilized with a 1% NaClO solution for 20 min, then thoroughly rinsed with deionized water. Subsequently, the sterilized rice seeds were soaked in 50℃ water for 12 h, placed in germination trays, and stored at 37℃ in the dark to promote germination. After germination, the seeds were grown in Hoglund nutrient solution at 25℃, with a photoperiod of 16 h / 8 h and a relative humidity of 75%. At the four-true-leaf stage, rice plants were treated: (i) the Cd toxicity threshold was determined by adding 0, 5, 10, 15, and 20 mg / L CdCl2 to the nutrient solution, and the optimal Cd remediation effect of microalgae was selected. 2+ Concentration; (ii) by not adding Cd 2+ Control (CK) test for microalgae S93-3 For Cd 2+ Its biological repair capabilities; S93-3 deal with( S93-3 Add only microalgae, OD 680 The value is 1.0; S93-3 and Cd 2+ deal with( S93-3 +Cd10), microalgae addition amount OD 680 The concentration of CdCl2 was 10 mg / L, and the concentration of Cd was 1.0 mg / L. 2+ Treatment (Cd10) involved adding only 10 mg / L of CdCl2.

[0038] For lettuce seed germination, place two seeds in a seedling sponge, soak them in water, and store them in the dark at 25°C. After the cotyledons emerge, separate the seedlings and place them in Hoglund nutrient solution for growth. Once 2-3 leaves have emerged, treat them separately with Cd. 2+ Treatment with microalgae. Each treatment was repeated 3 times, with 12 plants per treatment.

[0039] 1.7 Determination of vegetative growth and physiological and biochemical indicators Root and shoot length, fresh weight, and dry weight were measured. Photosynthetic efficiency indices, including the maximum efficiency of optical system II (Fv / Fm), non-photochemical quenching [Y(NPQ)], actual photochemical efficiency [Y(II)], and relative electron transport rate (rETR) of leaves, were measured using biological instruments. To investigate changes in root microstructure, treated roots were fixed with formaldehyde-acetic acid-ethanol (FAA), double-stained with Safranin O-Fast Green FCF, and frozen sections were then observed under a microscope.

[0040] 1.8 Molecular Detection S93-3 The microalgae strain is deposited at the China Type Culture Collection (CCTCC), isolate number: P202541. Accession number: SRR35180137. C. tenuitheca strain S93-3 The whole genome sequence and the assembled genome of GCA_051903525.1. C. tenuitheca strain S93-3 In Cd 2+ The complete transcription sequences under stress are accessed under accession numbers SRR36385166-SRR36385174, bioproject number PRJNA1378244, and GEO dataset number GSE313091.

[0041] 2 Results 2.1 S93-3 Exhibits high Cd 2+ Tolerance Cd from different microalgae 2+ Based on the growth phenotype of the treatment, SZ2 ( Chlorella sorokiniana S9-3 Chlorella variabilis ), S93-3 ( Coelastrella tenuitheca ), RW9 ( Parachlorella kessleri ) and J3 ( Auxenochlorella pyrenoidosa The addition of 20 mg / L CdCl2 showed Cd... 2+ Tolerance, resistance to chlorophyll degradation and high OD 680 (like Figure 1 (As shown in A). Similarly, based on dry biomass, after 7 days of treatment,S93-3 It exhibits the highest tolerance, IC 50 37.92 mg / L (e.g.) Figure 1 B and Figure 2 (As shown). S9-3 (IC) 50 =30.67 mg / L), J3 (IC 50 =23.77 mg / L), RW9 (IC 50 =13.88 mg / L), SZ2 (IC 50 =12.44 mg / L) and L11 (IC 50 =10.38mg / L) for Cd 2+ It also showed moderate tolerance, while S5-2 showed resistance to Cd. 2+ Extremely sensitive to stress, exhibiting albinism, IC 50 Lowest (1.57 mg / L). Based on these results, using S93-3 Further verification is needed.

[0042] 2.2 Different Cd 2+ Concentration S93-3 Influence of Cd absorption and morphology formation Cd 2+ Poisoning leads to cellular structural abnormalities, ultimately resulting in cell death. SEM analysis shows that... S93-3 Cells at 10 mg / L, Cd 2+ It maintained structural integrity under stress, while at 35 mg / L Cd 2+ Under stress, cells exhibit deformity and cytoplasmic shrinkage. Figure 1 EG). Similarly, TEM analysis showed similar phenotypic characteristics, while at 35 mg / L Cd 2+ Under stress, intracellular structural disorder was observed. Figure 1 H and 1I). Further quantitative analysis showed that, S93-3 The rate of intracellular Cd accumulation increased with increasing CdCl2 addition. The highest Cd content (3.75 g / kg) was observed with the addition of 30 mg / L CdCl2, and the absorption efficiency was 94.87%. Figure 1 C). In liquid culture medium, when 5 mg / L was added, residual unabsorbed Cd... 2+ The concentration was 0.08 mg / L, and it increased to 3.07 mg / L when 35 mg / L was added. Figure 1 C). Cd 2+The absorption efficiency peaked at 98.71% upon supplementation at 10 mg / L, and remained at 98.51% upon supplementation at 15 mg / L. Above this concentration, the efficiency gradually decreased: 96.21% (20 mg / L), 96.52% (25 mg / L), 94.87% (30 mg / L), and 93.29% (35 mg / L). Figure 3 ).

[0043] The chemical form of cadmium (Cd) in plants directly affects its mobility and activity. Ethanol-extracted and water-soluble cadmium exhibited the highest activity, while sodium chloride-extracted and acetic acid-extracted cadmium showed moderate activity, and hydrochloric acid-extracted and residual cadmium showed the lowest activity. To investigate the effects of Cd... 2+ exist S93-3 The bioabsorption and biotransformation mechanisms of Cd were investigated, and the chemically extractable forms after treatment with 15 mg / L CdCl2 for 48 hours were examined. The results showed that the total Cd content reached 1697.30 mg / kg, mainly in the extractable form of NaCl, with a content of 815.80 mg / kg DW, accounting for 48.06% of the total extracted Cd. The extractable forms of CH3COOH, soluble forms of H2O, and residual forms were 519.57 mg / kg, 254.94 mg / kg, and 100.19 mg / kg, respectively, accounting for 30.61%, 15.02%, and 5.90% of the total extracted Cd. In contrast, the extractable forms of HCl and C2H5OH were relatively low, accounting for only 0.34% to 0.06% of the total extracted Cd, respectively. Figure 1 D).

[0044] The results showed that the NaCl extractable and CH3COOH extractable were identified as... S93-3 The main chemical forms of Cd accounted for 78.67%, while HCl-extractable and residual Cd accounted for 15.36%. This represents... S93-3 Key tolerance mechanisms to Cd stress.

[0045] 2.3 Cd 2+ Coercion S93-3 Effects of photosynthesis and antioxidant levels Photosynthesis analysis showed that Cd 2+ Stress inhibited photosynthetic capacity by reducing the Fv / Fm value, while Cd 24 hours later 2+ Y(II) showed no significant difference in exposure ( Figure 4 (AD). Notably, in the experimental group, the Fv / Fm value increased with Cd. 2+ The concentration increases with the increase of Cd. 2+ Stress inhibition S93-3Its enhanced PS activity and ROS scavenging potential enable it to resist Cd. 2+ Causes injury. Follows Cd 2+ IC 50 Following exposure, the activities of antioxidant enzymes initially increased after 24 hours of treatment, then gradually decreased. POD activity peaked at 24 hours, while SOD and CAT activities peaked at 48 hours. Figure 4 EG). GSH content decreased after 24 hours but subsequently increased, indicating its effect on Cd in vacuoles. 2+ The use of sealing ( Figure 4 H).

[0046] Similarly, stress biomarkers, such as malondialdehyde (MDA) and hydrogen peroxide (H2O2) levels, rose sharply at 24 hpi and then gradually decreased. Figure 4 I and J), explanation S93-3 Its ROS removal capability is enhanced.

[0047] 2.4 Cd 2+ Under coercion S93-3 Effects on growth, root structure and photosynthesis of rice and lettuce Cadmium is a highly toxic element harmful to both plant and human health. It is present in soluble form as Cd. 2+ In its extant form, Cd not only directly damages plant vitality but also bioaccumulates in the food chain, posing a threat to human health. Experimental results show that Cd... 2+ Significantly inhibits rice growth ( Figure 5 A). in cd 2+ During the treatment, the plant height was 19.71 cm, the fresh leaf biomass was 0.38 g, the root length was 9.42 cm, and the root biomass was 0.31 g, all significantly lower than the control (CK) (22.13 cm, 1.06 g; 25.38 cm, 0.78 g, respectively). Figure 6 (A and B). And adding... S93-3 It then exhibits a dual function: under normal conditions, S93-3 It not only promoted the growth of rice (plant height, leaf biomass, root length, and root biomass reached 21.46 cm, 0.68 g, 20.58 cm, and 0.94 g, respectively), but also partially alleviated Cd. 2+ Induced coercion. S93-3 In the +Cd group, compared with the Cd group, the plants showed improved indices (20.63 cm, 0.58 g; 17.08 cm, 0.75 g), indicating that... S93-3 As Cd 2+ The potential of toxic biological control agents ( Figure 6F). A similar phenomenon was observed in lettuce. Compared to the control (CK), Cd treatment reduced plant height from 5.82 cm to 4.83 cm, fresh leaf biomass from 8.96 g to 4.82 g, root length from 17.88 cm to 16.88 cm, and fresh root biomass from 2.37 g to 1.26 g. Figure 7 A, B, and 5B). S93-3 The growth-promoting effect was significant, with plant height reaching 7.83 cm, fresh leaf biomass at 16.04 g, root length at 17.67 cm, and root biomass at 3.73 g. Notably, compared to the control (CK), S93-3 The +Cd group performed exceptionally well, with a plant height of 6.65 cm, fresh leaf biomass of 11.79 g, root length of 18.96 cm, and root biomass of 3.04 g. Figure 7 CF).

[0048] S93-3 Not only did it reduce Cd 2+ The toxicity also helps maintain the normal root morphology and structure of rice and lettuce. Figure 8 ).

[0049] Cd 2+ The endoderm and cortical cells of the treated rice and lettuce roots showed severe damage, as well as symptoms of weakened, degenerated and reduced cell structure. S93-3 Groups and S93-3 +Cd group maintains endothelial and cortical cells. Furthermore, Cd 2+ It inhibited photosynthesis in lettuce and rice, and reduced the Y(II), Y(NPQ), and ETR values ​​in rice. Figure 9 The Y(NPQ) value of lettuce decreased ( Figure 10 ),and S93-3 It can not only promote photosynthesis in plants, but also reduce Cd. 2+ Damage to plant photosynthesis.

[0050] 2.5 Application S93-3 Effects on Cd accumulation and extractable chemical speciation in rice and lettuce tissues Cd absorbed from the roots 2+ It is transferred to terrestrial tissues, including leaves, flowers, and seeds, and enters the food chain. At 10 mg / L Cd 2+ Under stress, the total Cd concentrations in the rice stems and roots were 18.35 mg / kg and 235.58 mg / kg, respectively. Figure 6 G). Application S93-3 It significantly reduced Cd accumulation in the tissues of both plant species. Specifically, compared to Cd10 treatment alone, the addition of [Cd10 treatment] significantly reduced [Cd accumulation]. S93-3The Cd concentration in the treated rice stems (0.7 mg / kg) and roots (100.73 mg / kg) decreased by 96.19% and 52.74%, respectively. S93-3 The Cd translocation factor from roots to stems was reduced by 91.15% in the +Cd10 treatment (0.69%) compared to the Cd10 treatment (7.79%). Notably, the residual Cd content in the nutrient solution of the Cd10 group was 398.17 μg / L, while... S93-3 The +Cd10 group had a concentration of 101.48 μg / L, a decrease of 74.51% ( Figure 11 ).

[0051] Analysis of extractable chemical forms of Cd showed that, compared with the Cd10 group, S93-3 In the +Cd10 group, all Cd forms, except for the HCl-extractable fraction, were significantly reduced. S93-3 In the +Cd10 treatment, the dominant Cd species in leaves was NaCl-extractable (80.34%), followed by H2O-extractable (4.73%), CH3COOH-extractable (4.59%), C2H5OH-extractable (4.31%), residual (4.02%), and HCl-extractable (2.01%). In contrast, the Cd10 group showed that residual (42.88%) and NaCl-extractable (34.28%) Cd were the main forms. Figure 6 I). In the rice root system, S93-3 In the +Cd10 group, residual Cd (48.13%) and NaCl-extractable Cd (35.82%) were dominant, while in the Cd10 group, the proportions of residual Cd (40.55%) and NaCl-extractable Cd (28.60%) were lower, but the proportion of H2O-soluble Cd was higher (18.89%). Figure 6 I).

[0052] Under 10 mg / L Cd stress, the Cd content in the aboveground parts and roots of lettuce in group Cd10 was 6.89 mg / kg and 11.41 mg / kg, respectively; while compared with group Cd10, S93-3 The Cd accumulation in the aboveground parts of the +Cd10 group was reduced (4.04 mg / kg, a decrease of 41.36%), but the accumulation in the roots was increased (15.98 mg / kg, an increase of 40.05%). Figure 7 G). S93-3 The residual Cd content in the nutrient solution of the +Cd10 group (164.34 μg / L) was significantly lower than that of the Cd10 group (541.88 μg / L). Figure 11 ).

[0053] Further analysis of extractable chemical speciation showed that S93-3The CH3COOH extractable and residual Cd in lettuce leaves from the +Cd10 group were significantly lower than those from the Cd10 group. S93-3 In the +Cd10 treatment, NaCl-extractable Cd was dominant (78.94%), while the proportion of NaCl-extractable Cd in the Cd10 group was lower (49.49%), and the proportion of remaining Cd was also lower (23.55%), totaling 73.04%. Figure 7 H). In the root system, the distribution of Cd morphology differs among different treatments. S93-3 The abundance order of the +Cd10 group was: NaCl extractable (36.66%), > residual (23.60%), > C2H5OH extractable (23.40%), > CH3COOH extractable (9.05%), > H2O- extractable (6.83%), > HCl extractable (0.46%). In contrast, the Cd10 group showed a different pattern: NaCl extractable (41.99%) > C2H5OH extractable (27.55%) > residual (15.90%) > CH3COOH extractable (7.56%) > H2O- extractable (6.68%) > HCl extractable (0.32%). Figure 7 I).

[0054] 2.6 Application S93-3 For Cd 2+ Effects of plant oxidative stress and antioxidant system under stress Under normal environmental conditions, plants generally maintain a balance between the production and scavenging of reactive oxygen species. At 10 mg / L Cd... 2+ Under stress, the activities of SOD, POD, and CAT in rice roots significantly decreased to 6.17%, 82.51%, and 32.69% of the control (CK), respectively. In the aboveground parts, SOD and CAT decreased by 56.14% and 47.46% compared to the CK, respectively, while POD activity unexpectedly increased to 242.40%. Figure 12 (AD). Compared with the control (CK), Cd treatment induced different oxidative damage patterns: H2O2 increased by 47.73% and MDA increased by 89.34% in the aerial parts; H2O2 increased by 617.31% and MDA increased by 177.60% in the roots. It is noteworthy that... S93-3 Common application ( S93-3 +Cd10 significantly mitigated these effects, reducing H2O2 and MDA in the aboveground parts by 30.14% and 12.76%, respectively, with even more pronounced reductions in H2O2 and MDA in the roots (H2O2 reduced by 81.67%, MDA reduced by 64.57%). Figure 12 E, F). GSH content exhibited tissue-specific regulation. Although both stems and roots treated with Cd10 showed significant GSH accumulation compared to the control (CK), S93-3+Cd10 maintained a relatively high level of GSH in the stem, but led to a decrease in GSH content in the root. Figure 12 B) indicates that there are differences in glutathione metabolism between aboveground and underground tissues.

[0055] The same oxidative stress response pattern was also observed in lettuce. (10 mg / L Cd) 2+ Treatment significantly increased the levels of H2O2 and MDA in both the aboveground and belowground parts of lettuce. Compared with the control (CK), Cd significantly increased the levels of H2O2 and MDA. 2+ Treatment induced H2O2 and MDA increases of 63.16% and 116.19% in the stems and 86.09% and 463.61% in the roots, respectively. Notably, compared to Cd treatment alone, S93-3 The combined application of pesticides offset these effects: H2O2 and MDA levels in the aboveground parts decreased by 28.62% and 26.08%, respectively, while the reductions in the underground parts were even greater (H2O2 decreased by 40.77%, and MDA decreased by 72.40%). Figure 12 K, L). Therefore, compared with the CK group, 10 mg / L Cd 2+ It significantly inhibited the activities of SOD (0.45 times), POD (0.36 times), and CAT (0.62 times) in the aboveground parts, while significantly reducing the contents of POD (0.36 times), CAT (0.18 times), and GSH (0.19 times) in the roots. However, application... S93-3 It significantly improved the antioxidant defense system of both the above-ground and below-ground parts of lettuce under Cd exposure. For example: in Cd... 2+ Under coercion, S93-3 Treatment increased the activities of SOD, POD, and CAT in the aboveground parts by 2.57 times, 1.59 times, and 1.11 times, respectively, and the GSH content was higher than that of Cd alone. 2+ Increased to 1.76 times. In the underground portion, compared to the Cd10 group, S93-3 The enhancement in the +Cd10 group was also significant (SOD: 1.71 times; POD: 1.94 times; CAT: 2.29 times; GSH: 1.52 times), indicating that... S93-3 It plays a powerful role in restoring redox homeostasis. Figure 12 GJ).

[0056] Table 1

[0057] It should be noted that the specific embodiments described above are exemplary, and those skilled in the art can devise various solutions inspired by the disclosure of this invention. These solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents.

Claims

1. Thin-walled monostellar algae ( Coelastrella tenuitheca ) S93-3 Its application in reducing cadmium concentration in the environment is characterized by, The thin-walled monostellate algae S93-3 It was deposited at the China Center for Type Culture Collection on November 28, 2025, with accession number CCTCC NO: P202541.

2. The application according to claim 1, characterized in that, The thin-walled monostellate algae S93-3 The ITS sequence is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The thin-walled monostar algae S93-3 It can be mixed with other excipients permitted by the product to prepare a formulation for application.

4. The application according to claim 3, characterized in that, The concentration of cadmium in the environment is no greater than 37.92 mg / L.

5. Thin-walled monostellate algae S93-3 Applications in alleviating cadmium toxicity in plants under cadmium stress, or applications in preparing products for alleviating cadmium toxicity in plants under cadmium stress, characterized by... The thin-walled monostellate algae S93-3 It was deposited at the China Center for Type Culture Collection on November 28, 2025, with accession number CCTCC NO: P202541.

6. Thin-walled monostellate algae S93-3 Applications in promoting plant biomass increase under cadmium stress, or applications in preparing products that promote plant biomass increase under cadmium stress, characterized by... The thin-walled monostellate algae S93-3 It was deposited at the China Center for Type Culture Collection on November 28, 2025, with accession number CCTCC NO: P202541.

7. A method for mitigating cadmium toxicity in plants under cadmium stress, characterized in that, Using the thin-walled monostellar algae of claim 1 S93-3 Plants were treated with bacterial suspensions.

8. The method for alleviating cadmium toxicity in plants under cadmium stress according to claim 7, characterized in that, The plants being treated are watered plants.

9. The method for alleviating cadmium toxicity in plants under cadmium stress according to claim 7, characterized in that, The step of treating plants with the bacterial suspension includes: Thin-walled monostar algae S93-3 OD produced by strain 600 The seed culture was prepared at a concentration of 0.5, and then inoculated into cadmium-contaminated samples and cultured at 25-37°C for 1-7 days.

10. The method for mitigating cadmium toxicity in plants under cadmium stress according to claim 7, characterized in that, The plant in question is either rice or lettuce.

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