Conditioner for cadmium-reducing selenium-rich crop cultivation, cultivation medium and application

Abstract

The invention discloses a conditioner for cadmium-reducing selenium-rich crop cultivation, a cultivation medium and application, and belongs to the technical field of microalgae selenium enrichment and application thereof. The conditioner is prepared by the following steps: S1, dispersing mesoporous silica into a solution containing Fe < 3 + > to react to obtain a mesoporous silica carrier; s2, taking an algae solution of scenedesmus spinosus in a logarithmic phase, adjusting OD680 to 0.5, adding a sodium selenite solution, carrying out illumination culture, and centrifugally collecting microalgae; s3, mixing the microalgae obtained in the step S2 and the mesoporous silica carrier obtained in the step S1 according to a mass ratio of 10: 1-5: 1, adding deionized water, and carrying out an oscillatory reaction at 25 DEG C under a dark condition to obtain a culture algae solution; and S4, separating the culture algae liquid to obtain the selenium-rich microalgae. The conditioner has the beneficial effects that crops cultivated by utilizing the conditioner for cadmium-reducing selenium-rich crop cultivation are rich in selenium, low in cadmium content and good in growth, and have a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of microalgae selenium enrichment and its application, and more specifically, to a cadmium-reduced selenium-enriched crop cultivation conditioner, a cultivation substrate and its application. Background Art

[0002] As environmental quality continues to deteriorate, heavy metal pollution has aroused widespread public concern. Cadmium is a toxic, non-essential transition metal found in soil, air, and water. It is particularly mobile in soil and water, easily absorbed and accumulated in plants, posing a serious threat to human and animal health. It has been classified as a Class 1 human carcinogen. Cadmium is transmitted through the food chain and food web and accumulates in plants and animals, with a half-life of approximately 25-30 years. It directly threatens food production, crop safety, and drinking water safety, ultimately endangering human health. Cadmium exposure is associated with a variety of diseases, including prostate cancer, lung cancer, testicular cancer, renal dysfunction, rhinitis, emphysema, and fractures. Furthermore, cadmium can cause chronic lung inflammation, pulmonary fibrosis, emphysema, and lung tumors, and reduce male fertility, leading to decreased sperm count and motility.

[0003] Currently, methods for remediating cadmium contamination in soil include physical, chemical, biological, and combined remediation technologies that integrate multiple approaches. Physical remediation techniques transfer or separate pollutants through physical means. While highly efficient, they are costly and can damage soil structure. Chemical remediation techniques use chemical reagents to modify the form of pollutants, reducing their toxicity or mobility. However, this may introduce new chemicals, posing potential risks. In wastewater treatment, physical, chemical, and biological methods are also used to treat cadmium-contaminated wastewater. Bioremediation holds great promise for cadmium pollution control due to its wide applicability, low cost, simple operation, and lack of secondary pollution. Microalgae, with their high growth rate, large surface area, and small size, possess a strong capacity for heavy metal adsorption. Research has shown that microalgae have a far superior capacity for heavy metal adsorption and accumulation than other organisms, effectively absorbing cadmium ions and reducing cadmium levels in the environment. Furthermore, wastewater ecosystems are rich in the organic carbon, nitrogen, and phosphorus required for microalgae growth. During their growth and metabolism, microalgae not only effectively purify wastewater but also significantly reduce chemical oxygen demand, total nitrogen, and total phosphorus in the wastewater. Therefore, using microalgae to treat cadmium-containing wastewater can simultaneously reduce multiple pollutants, thereby improving treatment efficiency. Compared to other remediation technologies, microalgae treatment of cadmium pollution is both environmentally friendly and economically feasible, and is expected to play a more important role in cadmium pollution control in the future.

[0004] Selenium is an essential trace element for human growth and development. Although it accounts for less than one hundred thousandth of a person's total body weight, it is a crucial element for maintaining health. Selenium possesses remarkable antioxidant properties and is a cofactor for numerous antioxidant enzymes, such as GSH-Px, SOD, and CAT. These enzymes play a crucial role in defending against cellular damage caused by free radicals and lipid peroxides. Selenium's detoxification role in plants has been demonstrated. Although selenium is not essential for higher plants, numerous studies have demonstrated that various forms and appropriate concentrations of selenium have positive effects on plant growth and tolerance to abiotic stress. Selenium not only promotes plant growth and development but also enhances tolerance to heavy metal stress and improves plant adaptability to environmental stress. Selenium alleviates cadmium stress in plants by improving photosynthesis, regulating mineral balance in cadmium-stressed plants, enhancing antioxidant capacity, and reducing cadmium levels in plant cells.

[0005] Although selenium and microalgae are both effective in reducing cadmium absorption by plants, their effects on reducing the total amount of cadmium in the soil are still limited, which makes the long-term management of soil cadmium pollution a challenge. In actual production, how to produce low-cadmium selenium-rich foods in areas with high cadmium content remains a practical problem that needs to be solved urgently. At present, there are still few technologies and methods for plant selenium enrichment and cadmium reduction, especially there are few methods and technologies for achieving selenium enrichment in crops while reducing cadmium in plants, which cannot be widely used in agricultural production. In the future, it is necessary to further explore new methods and technologies that can both enrich selenium and reduce cadmium, and provide technical support for the production of healthy, safe, and high-quality selenium-rich products in cadmium-contaminated soils. Summary of the Invention

[0006] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0007] Another object of the present invention is to provide a conditioner, a cultivation substrate and an application for the cultivation of cadmium-reduced and selenium-rich crops. The crops cultivated using the conditioner for the cultivation of cadmium-reduced and selenium-rich crops are not only selenium-rich but also have a low cadmium content and grow well, and have good application prospects.

[0008] In order to achieve these purposes and other advantages according to the present invention, a cadmium-reducing and selenium-enriched crop cultivation conditioner is provided, comprising Scenedesmus multispinosa and sodium selenite; The conditioning agent is prepared by the following steps: S1, disperse mesoporous silica in Fe-containing 3+ In the solution, under the protection of an inert atmosphere, a reducing agent is added to react to obtain a mesoporous silica carrier; S2. Take the algae solution of Scenedesmus multispinosa in the logarithmic growth phase and adjust the OD 680to 0.5, add 10 mg / mL sodium selenite solution to a final concentration of 1-5 mg / L, culture under light for 4 days, and collect the microalgae by centrifugation; S3, mixing the microalgae obtained in step S2 with the mesoporous silica carrier obtained in step S1 at a mass ratio of 10:1 to 5:1, adding deionized water, and shaking the mixture at 25° C. in the dark for 30 minutes to obtain the algae culture solution; S4. Separating the cultured algae liquid to obtain selenium-rich microalgae, and mixing the selenium-rich microalgae with deionized water to obtain the selenium-rich microalgae liquid, which is the conditioning agent.

[0009] Preferably, step S1 specifically includes the following steps: S11, vacuum drying the mesoporous silica at 150° C. for 2 hours to remove adsorbed water in the pores to obtain pretreated mesoporous silica; S12, dispersing the pretreated mesoporous silica in a 0.1-0.3 mol / L FeCl3 solution prepared in deoxygenated deionized water at a ratio of 1:10-1:20 w / v, stirring at 200-400 rpm for 1-2 hours under nitrogen / argon protection to obtain a reaction solution; S13, maintaining an inert atmosphere, add 0.5-1.5 mol / L NaBH4 solution dropwise to the reaction solution in S12 at a rate of 1-2 mL / min to control the NaBH4 and Fe 3+ The molar ratio is 3:1-5:1, the reaction temperature is 25-35°C, and stirring is continued until no bubbles are generated; S14. After the reaction is completed, the mixture is washed three times by centrifugation with deoxyethanol, and the precipitate is taken. The centrifugation parameters are: 8000 rpm, 10 min; and the precipitate is placed at 40°C for vacuum drying for 12 hours to obtain the mesoporous silica carrier.

[0010] Preferably, step S4 specifically includes the following steps: S41, adding 0.001%-0.005% w / v of a food-grade foaming agent and 0.01% w / v of trehalose to the cultured algae solution; S42, introducing low-pressure microbubbles, with the following parameters: pressure of 0.05–0.1 MPa, microbubble diameter of 50–200 μm, gas flow rate of 0.1–0.3 L / min, for 5–10 minutes, to allow algal cells to accumulate in the foam layer; S43, collecting the foam layer, and washing the foam with an isotonic solution containing 0.8%-1.0% by mass of NaCl and 0.01% by mass of trehalose to obtain the selenium-enriched microalgae.

[0011] Preferably, the food-grade foaming agent in step S41 is a composite foaming agent composed of sophorolipids and rhamnolipids in a mass ratio of 3-5:1, and the total concentration of the composite foaming agent is 0.0015%-0.0025% w / v; Step S41 specifically comprises: premixing the composite foaming agent and oligosaccharide at 40-45° C. to form a micelle complex, and then adding the mixture into the algae solution.

[0012] The present invention also provides a cultivation matrix, comprising the cadmium-reduced and selenium-enriched crop cultivation conditioner, wherein the cultivation matrix is ​​in the form of a cultivation solution or a cultivation soil.

[0013] Preferably, the cultivation matrix is ​​cultivation soil, which includes matrix soil, water, and selenium-rich microalgae liquid. The cultivation soil is prepared by uniformly mixing the selenium-rich microalgae liquid with water and then with the matrix soil. The mass fraction of the selenium-rich microalgae liquid in the cultivation soil is 5-10%.

[0014] The present invention also provides an application of a cadmium-reduced and selenium-enriched crop cultivation conditioner in the cultivation of cadmium-reduced and selenium-enriched crops.

[0015] The present invention has at least the following beneficial effects: The present invention provides a conditioner for cultivating cadmium-reduced and selenium-enriched crops, a cultivation substrate and applications thereof. Crops cultivated with the conditioner for cultivating cadmium-reduced and selenium-enriched crops are not only selenium-rich but also have a low cadmium content and grow well, thus having good application prospects.

[0016] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure A in the middle shows the microscopic examination of the algae strains isolated during the screening and identification of cadmium-resistant algae species, and Figure B shows the plate propagation of the target algae species obtained through screening; Figure 2 Phylogenetic tree of SC-Z8 constructed based on TUFA (Figure A) and RBCL (Figure B) sequences; Figure 3 Tolerance of Chlamydomonas reinhardtii (Cr-wt) (A), Chlorella pyrenoidosa (xl-12) (B), Chlorella sorokinensis (dp3) (C), and Scenedesmus polyspinosa (SC-Z8) (D) to cadmium. Figure 4 This is the growth phenotype of rice co-treated with microalgae SC-Z8 and selenium; Figure 5 The results of plant height (A) and dry weight (B) of the aboveground part of rice after co-treatment with selenium and algae; Figure 6The results of cadmium (A) and selenium (B) content in the aboveground part of rice treated with selenium and algae; Figure 7 This is the growth phenotype of rice treated with selenium-enriched microalgae; Figure 8 Plant height (A) and dry weight (B) of the aboveground part of rice treated with selenium-enriched microalgae; Figure 9 Cadmium (A) and selenium (B) contents in the aboveground parts of rice treated with selenium-enriched microalgae; Figure 10 This is the growth phenotype diagram of lettuce treated with selenium-enriched microalgae; Figure 11 The figure shows the measurement results of the dry weight of the aboveground part of lettuce treated with selenium-enriched microalgae; Figure 12 Cadmium (A) and selenium (B) contents in the aerial parts of lettuce treated with selenium-enriched microalgae; Figure 13 The figure shows the results of total cadmium content determination in the aboveground part of rice after the rice culture solution was treated with selenium-enriched microalgae prepared by different preparation methods; Figure 14 This figure shows the results of determining the total selenium content in the aboveground part of rice after the rice culture solution was treated with selenium-rich microalgae prepared by different preparation methods. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0019] This invention is based on the following design principles: first, algae species are screened for cadmium tolerance. These selected species are then compared with other species in tolerance testing to verify their significant cadmium tolerance. The selected species are then combined with a selenium-containing substance (such as sodium selenite) for testing. The results show that the combination of these selected species and selenium significantly reduces cadmium levels in plants and significantly increases selenium levels. This synergistic effect is far superior to either the effects of using them separately or in combination. Details are described below.

[0020] <Screening and Identification of Cadmium-Resistant Algae Species> Samples were collected from lakes and rice-growing soil in Tianfu New District, Chengdu, Sichuan. The collected water and soil samples were diluted appropriately, namely, the original solution, 10 times, 100 times, and 1000 times. 200 μL of the liquid of different dilution concentrations was pipetted onto BG11 plate culture medium and inverted in an artificial climate chamber for culture. After a green single clone grew, the single clone was picked, resuspended, and examined under a microscope to preliminarily screen the target algae species. The isolated and purified target algae species were observed under a microscope. Their morphological characteristics ( Figure 1(Figure A in the figure) The algae strain was found to be green, with oval cells, isolated cells or 2-4 cells arranged linearly, with a thorn at each pole. The strain obtained by morphological screening was initially identified as Scenedesmus, named SC-Z8, and propagated on plates ( Figure 1 B).

[0021] The species relationship of SC-Z8 algae strain was identified at the molecular level. Elongation factor Tu ( TUFA )and RuBis-Co ( RBCL ) gene, PCR amplification was performed using primers designed for the gene, and the product was sequenced. The sequenced sequences were compared using blast comparison on the NCBI website to obtain homologous sequences. The sequenced sequences were aligned with the homologous sequences using MEGE11 software. The neighbor-joining method was used to construct a phylogenetic tree based on the aligned sequence files. The reliability of the topological structure of the phylogenetic tree was calculated using the bootstrap method 1000 times, and the final evolutionary tree diagram was generated, as shown in the figure below. Figure 2 The results showed that the isolated algae species SC-Z8 was different from those in the Polyspinous Scenedesmus family. Desmodesmusabundans The algae species were clustered into one branch ( Figure 2 A). Based on the construction of RBCL sequence with higher degree of variation, the isolated algae species SC-Z8 formed a separate branch, but Desmodesmusabundans The branch relationship is closest ( Figure 2 B). Based on the above results, it was determined that the algae species SC-Z8 isolated in this experiment belonged to the genus Desmodesmus of the family Scenedesmus polyspinosa ( Desmodesmus ) of Scenedesmus multispinosa ( Desmodesmusabundans ).

[0022] Comparison of cadmium tolerance among different algae species Take the algae solution in the stable growth period, the initial OD 680 After adjusting to 3.0, add different concentrations of cadmium ions and observe the algae solution at 0d, 2d, 4d, and 6d. Figure 3 , Figure 3 The tolerance of Chlamydomonas reinhardtii (Cr-wt), Chlorella pyrenoidosa (xl-12), Chlorella sorokinensis (dp3) and Scenedesmus polyspinosa (SC-Z8) to cadmium.

[0023] from Figure 3 It can be seen that in Chlamydomonas reinhardtii (Cr-wt) ( Figure 3 In A), by the second day, under the condition of 40mg / L cadmium ion treatment, the color of the algae solution turned yellow, and the growth of Chlamydomonas reinhardtii was significantly inhibited, and even death occurred, indicating that its tolerance to cadmium was relatively weak. Figure 3In B), on the 6th day, under the condition of 70mg / L cadmium ion treatment, the color of the algae solution turned yellow, indicating that Chlorella pyrenoidosa has a certain degree of tolerance to cadmium and can maintain a relatively stable growth state within a certain cadmium concentration range. Figure 3 C), by the 6th day, under the condition of 400 mg / L cadmium ion treatment, the color of the algae solution turned yellow, which shows that Chlorella sorokinensis can maintain a relatively good growth trend under low concentration cadmium environment, but its growth will be affected to a certain extent under high concentration cadmium stress. Figure 3 D), by the 6th day, the color change of the algae liquid was still relatively slow under the condition of 1300 mg / L cadmium ion treatment. This result shows that Scenedesmus multispinosa may have a strong cadmium tolerance and can maintain a relatively stable state in a high cadmium concentration environment. Therefore, the subsequent experiments selected Scenedesmus multispinosa as the research species.

[0024] Effects of Combined Application of Microalgae and Selenium on Rice Growth and Cadmium / Selenium Accumulation in Cadmium-Stressed Environments To investigate the effects of combined application of microalgae (SC-Z8) and selenium on rice growth and cadmium / selenium accumulation in rice under cadmium stress, a rice hydroponic experiment was conducted.

[0025] 1. Experimental methods The experimental method specifically includes the following steps: Step 1: Rice seed treatment: Rice seeds were sterilized by soaking in 2% sodium hypochlorite for 10 min, then washed six times with distilled water. The seeds were then immersed in tap water in a 30°C constant temperature incubator for 24 h in the dark. The water was changed every 6 h until the seeds turned white. The seeds were then transferred to an artificial climate chamber with a temperature of 28°C during the day and 25°C at night, with a 14h / 10h (light / dark) cycle and a light intensity of 300 μmol·m −2 ·s −1 The humidity was 60%. After 7 days of cultivation in tap water, seedlings with consistent growth were selected for hydroponic culture experiments. The nutrient solution used for the rice hydroponic culture experiments was Hoagland's solution, which consists of: 393.81 mg / L Ca(NO₃)₂, 136.09 mg / L KH₂PO₄, 303.30 mg / L KNO₃, 120.37 mg / L MgSO₄, 29.22 mg / L NaCl, 1.42 mg / L H₃BO₃, 0.58 mg / L MnCl₂, 0.06 mg / L ZnSO₄, 0.025 mg / L CuSO₄, 0.004 mg / L H₂MoO₄, and 6.81 mg / L FeSO₄. The pH was adjusted to 7.0 before use. The test rice was grown on cotton.

[0026] Step 2: Preparation of SC-Z8 algae solution in the stable growth phase: SC-Z8 Scenedesmus polyspinosa was cultured in BG11 liquid medium at 28°C, 150 rpm, and 100 μmol·m -2 ·s- 1 The SC-Z8 algae solution is cultured under light conditions until the growth stable period, which is the SC-Z8 algae solution in the growth stable period.

[0027] Step 3: Preparation of conditioning agent: The conditioning agent is obtained by uniformly mixing algae liquid of SC-Z8 Scenedesmus polyspinosa in a stable growth phase and sodium selenite, wherein the mass fraction of microalgae in the algae liquid is 8%.

[0028] Step 4. Experimental group settings: A total of 8 experimental groups were set up, with 5 replicates in each experimental group, for a total of 40 bottles randomly arranged. In each replicate, the hydroponic device was a transparent plastic hydroponic bottle, and 400mL of Hoagland nutrient solution was added to each bottle. In the experimental groups involving the addition of conditioning agents and SC-Z8 algae solution, the amount of conditioning agent added to each bottle was 40mL, and the amount of SC-Z8 algae solution added to each bottle was 40mL. The 8 experimental groups were: (1) Blank control group: only Hoagland nutrient solution was used without adding any other substances; (2) 3 mg / L cadmium treatment group: Cadmium chloride was added to the Hoagland nutrient solution to make the concentration of cadmium chloride in the nutrient solution 3 mg / L; (3) 3 mg / L cadmium + 1 mg / L selenium treatment group: Cadmium chloride and sodium selenite were added to the Hoagland nutrient solution so that the concentration of cadmium chloride in the nutrient solution was 3 mg / L and the concentration of sodium selenite was 1 mg / L.

[0029] (4) 3 mg / L cadmium + 5 mg / L selenium treatment group: 3 mg / L cadmium chloride and sodium selenite were added to the Hoagland nutrient solution, so that the concentration of cadmium chloride in the nutrient solution was 3 mg / L and the concentration of sodium selenite was 5 mg / L; (5) 3 mg / L cadmium + algae treatment group: cadmium chloride and SC-Z8 algae solution were added to the Hoagland nutrient solution to make the cadmium chloride concentration in the nutrient solution 3 mg / L; (6) 3 mg / L cadmium + 1 mg / L selenium + algae treatment group: cadmium chloride and conditioner were added to the Hoagland nutrient solution so that the concentration of cadmium chloride in the nutrient solution was 3 mg / L and the concentration of sodium selenite was 1 mg / L; (7) 3 mg / L cadmium + 5 mg / L selenium + algae treatment group: 3 mg / L cadmium chloride and conditioner were added to the Hoagland nutrient solution, so that the concentration of cadmium chloride in the nutrient solution was 3 mg / L and the concentration of sodium selenite was 5 mg / L; (8) Algae treatment group: SC-Z8 algae solution was added to Hoagland nutrient solution.

[0030] Step 4: Index determination The plant height, aboveground dry weight, aboveground cadmium content and selenium content of rice were measured after 30 days of hydroponics. Figure 4 、 Figure 5 、 Figure 6 .in: The method for determining plant height is as follows: use a tape measure to measure the plant height of rice at harvest, and take the average value of the plant heights of 5 rice plants.

[0031] The method for determining the dry weight of the aboveground part is as follows: rinse the aboveground part of the harvested rice with deionized water three times, absorb the surface moisture, quickly freeze it in liquid nitrogen, and then place the sample in a freeze dryer and dry it until the moisture is removed and a constant weight is reached. This is the biomass of the rice after drying, that is, the dry weight of the aboveground part of the rice.

[0032] Cadmium and selenium content in aboveground parts was determined by weighing the dry weight and pulverizing all samples using a high-speed grinder for uniform mixing. Total cadmium and selenium content were determined after digestion. Total cadmium content was determined according to the first method of the Chinese National Standard GB5009.268-2016, "National Food Safety Standard - Determination of Multiple Elements in Food," and total cadmium concentration was determined using inductively coupled plasma-mass spectrometry. Total selenium content was determined according to the Chinese National Standard GB 5009.93-2017, "National Food Safety Standard - Determination of Selenium in Food," and was measured by atomic fluorescence spectrometry.

[0033] 2. Analysis of experimental results Figure 4 The figure shows the growth phenotype of rice treated with selenium and algae. Compared with the blank control group, the plants in the 3mg / L cadmium treatment group were significantly dwarfed, with plant height and aboveground dry weight reduced by 47.69% and 38.99%, respectively ( Figure 5 Figures A and B indicate that cadmium stress significantly inhibits rice growth. Compared to the 3 mg / L cadmium treatment, the 3 mg / L cadmium + 1 mg / L selenium treatment increased plant height by 10.95%. The 3 mg / L cadmium + algae and 3 mg / L cadmium + 1 mg / L selenium + algae treatments increased plant height by 28.94% and 57.35%, respectively, and dry weight increased by 15.66% and 57.28%. Furthermore, compared to the blank control, the algae treatment alone increased dry weight by 27.41%. These results indicate that combined treatment with microalgae and selenium effectively mitigates the inhibitory effects of cadmium on rice growth, with the 1 mg / L selenium + algae combination achieving the most effective effect. However, the potential toxicity of high-concentration selenium (5 mg / L) limited its effectiveness.

[0034] Figure 6A represents the cadmium content in the aboveground part of microalgae SC-Z8 treated with selenium. Compared with the 3 mg / L cadmium treatment, the 3 mg / L cadmium + algae treatment showed no significant difference in cadmium content (p>0.05). This may be due to the reduced cell viability of most algal cells at 3 mg / L cadmium, resulting in limited adsorption capacity. However, cadmium content decreased by 41.43% and 59.10% in the 3 mg / L cadmium + 1 mg / L selenium and 3 mg / L cadmium + 5 mg / L selenium treatments, respectively, indicating that selenium can inhibit cadmium accumulation in rice. Compared with the 3 mg / L cadmium treatment, cadmium content decreased by 53.59% and 63.83% in the 3 mg / L cadmium + 1 mg / L selenium + algae and 3 mg / L cadmium + 5 mg / L selenium + algae treatments, respectively. This suggests that selenium not only inhibits cadmium accumulation but also enhances algal cell activity and adsorption capacity by alleviating cadmium stress on the algae. The combination of low-concentration selenium (1 mg / L) and algae has a greater effect on rice growth. Figure 6 B represents the selenium content in the aboveground part of the rice. Compared with the blank control, the addition of exogenous selenium significantly increased the total selenium content of the rice plants. Therefore, the co-addition of selenium and Scenedesmus polyspinosa can alleviate the inhibitory effects of cadmium on rice growth, reduce cadmium accumulation in rice, and increase selenium enrichment in rice.

[0035] Effects of Selenium-Enriched Microalgae on Rice Growth and Cadmium-Reducing and Selenium-Enriching in Cadmium-Stressed Environments 1. Experimental methods The experimental method specifically includes the following steps: Step 1: Rice seed treatment: Rice seeds were sterilized by soaking in 2% sodium hypochlorite for 10 min, then washed six times with distilled water. The seeds were then immersed in tap water in a 30°C constant temperature incubator for 24 h in the dark. The water was changed every 6 h until the seeds turned white. The seeds were then transferred to an artificial climate chamber with a temperature of 28°C during the day and 25°C at night, with a 14h / 10h (light / dark) cycle and a light intensity of 300 μmol·m −2 ·s −1 The humidity was 60%. After 7 days of cultivation in tap water, seedlings with consistent growth were selected for hydroponic culture experiments. The nutrient solution used for the rice hydroponic culture experiments was Hoagland's solution, which consists of: 393.81 mg / L Ca(NO₃)₂, 136.09 mg / L KH₂PO₄, 303.30 mg / L KNO₃, 120.37 mg / L MgSO₄, 29.22 mg / L NaCl, 1.42 mg / L H₃BO₃, 0.58 mg / L MnCl₂, 0.06 mg / L ZnSO₄, 0.025 mg / L CuSO₄, 0.004 mg / L H₂MoO₄, and 6.81 mg / L FeSO₄. The pH was adjusted to 7.0 before use. The test rice was grown on cotton.

[0036] Step 2: Preparation of microalgae solution: Preparation method: select Scenedesmus multispinosa SC-Z8 algae liquid in the logarithmic growth phase, adjust its initial OD680 to 0.5, add 10 mg / mL sodium selenite solution to adjust the selenium concentration in the algae liquid to 0 mg / L, 1 mg / L and 5 mg / L respectively, culture for 4 days, centrifuge at 1000 rpm for 5 minutes, collect the precipitate (selenium-enriched microalgae), and resuspend it in purified water to obtain a selenium-enriched microalgae liquid. The resuspension operation with purified water is specifically as follows: mix the collected precipitate with purified water to obtain a selenium-enriched microalgae liquid, and the mass fraction of selenium-enriched microalgae in the selenium-enriched microalgae liquid is 8%; Among them, when 10 mg / mL sodium selenite solution is added, the selenium concentration in the algae liquid is 0 mg / L, which means that no 10 mg / mL sodium selenite solution is added, and the selenium-rich microalgae liquid prepared at this time is the control microalgae liquid; When 10 mg / mL sodium selenite solution is added to make the selenium concentration in the algae liquid 1 mg / L, the prepared selenium-rich microalgae liquid is 1 mg / L selenium-rich microalgae liquid; When 10 mg / mL sodium selenite solution is added to make the selenium concentration in the algae liquid 5 mg / L, the prepared selenium-rich microalgae liquid is a 5 mg / L selenium-rich microalgae liquid.

[0037] Step 3. Experimental group settings: A total of 8 experimental groups were set up, with 5 replicates in each experimental group, for a total of 40 bottles randomly arranged. In each replicate, the hydroponic apparatus consisted of transparent plastic bottles, each containing 400 mL of Hoagland's nutrient solution. The following microalgae solutions (control microalgae solution, 1 mg / L selenium-enriched microalgae solution, and 5 mg / L selenium-enriched microalgae solution) were added to each bottle, with a volume of 40 mL added to each bottle. The 8 experimental groups were: (1) Blank control group: only Hoagland nutrient solution was used without adding any other substances; (2) 3 mg / L cadmium treatment group: cadmium chloride was added to the nutrient solution so that the concentration of cadmium chloride in the nutrient solution was 3 mg / L; (3) 3 mg / L cadmium + algae treatment group: cadmium chloride and control microalgae solution were added to the nutrient solution to make the cadmium chloride concentration in the nutrient solution 3 mg / L; (4) 3 mg / L cadmium + 1 mg / L selenium-enriched algae: 3 mg / L cadmium chloride and 1 mg / L selenium-enriched microalgae solution were added to the nutrient solution to make the cadmium chloride concentration in the nutrient solution 3 mg / L; (5) 3 mg / L cadmium + 5 mg / L selenium-enriched algae: 3 mg / L cadmium chloride and 5 mg / L selenium-enriched microalgae solution were added to the nutrient solution to make the cadmium chloride concentration in the nutrient solution 3 mg / L; (6) Algae treatment group: control microalgae solution was added to the nutrient solution; (7) 1 mg / L selenium-enriched algae treatment group: 1 mg / L selenium-enriched microalgae solution was added to the nutrient solution; (8) 5 mg / L selenium-enriched algae treatment group: 5 mg / L selenium-enriched microalgae solution was added to the nutrient solution.

[0038] Step 4: Determine the plant height, aboveground dry weight, and aboveground cadmium content of rice after 30 days of hydroponics. Figures 7-9 .

[0039] 2. Analysis of experimental results Figure 7 This is a growth phenotype diagram of rice treated with selenium-enriched microalgae. Compared with the cadmium treatment group, the rice plant growth condition improved in the 3mg / L cadmium + 1mg / L selenium-enriched microalgae and 3mg / L cadmium + 5mg / L selenium-enriched microalgae treatment groups, with dry weight increased by 25.61% and 29%, respectively, and both were significantly higher than the cadmium treatment group (p<0.05) ( Figure 8 Compared with the blank control group, the rice leaves in the 1 mg / L and 5 mg / L selenium-enriched algae treatment groups grew well, and their dry weight increased by 20% and 29.44%, respectively, indicating that selenium-enriched algae treatment has a positive effect on rice growth.

[0040] Figure 9 A is the cadmium content in the aboveground part of rice treated with selenium-enriched microalgae. Compared with the cadmium treatment, the cadmium contents in the 3 mg / L cadmium + 1 mg / L selenium-enriched algae and 3 mg / L cadmium + 5 mg / L selenium-enriched algae treatments decreased by 19.71% and 22.19%, respectively. Figure 9 B represents the selenium content in the aboveground part of rice. Compared with the blank control, the selenium content in rice cultured with 5 mg / L selenium-enriched algae was significantly enriched. Cadmium content measurements showed that selenium-enriched microalgae may reduce cadmium accumulation in rice by enhancing cadmium adsorption or accumulation in the algae.

[0041] Effects of Selenium-Enriched Microalgae on Lettuce Growth and Cadmium-Reduction and Selenium-Enrichment in Cadmium-Stressed Environments 1. Experimental methods: The experimental method specifically includes the following steps: Step 1: Preparation of microalgae solution: Preparation method: select Scenedesmus multispinosa SC-Z8 algae liquid in the logarithmic growth phase, adjust its initial OD680 to 0.5, add 10 mg / mL sodium selenite solution to adjust the selenium concentration in the algae liquid to 0 mg / L, 1 mg / L and 5 mg / L respectively, culture for 4 days, centrifuge at 1000 rpm for 5 minutes, collect the precipitate (selenium-enriched microalgae), and resuspend it in purified water to obtain a selenium-enriched microalgae liquid. The resuspension operation with purified water is specifically as follows: mix the collected precipitate with purified water to obtain a selenium-enriched microalgae liquid, and the mass fraction of selenium-enriched microalgae in the selenium-enriched microalgae liquid is 8%; Among them, when 10 mg / mL sodium selenite solution is added, the selenium concentration in the algae liquid is 0 mg / L, which means that no 10 mg / mL sodium selenite solution is added, and the selenium-rich microalgae liquid prepared at this time is the control microalgae liquid; When 10 mg / mL sodium selenite solution is added to make the selenium concentration in the algae liquid 1 mg / L, the prepared selenium-rich microalgae liquid is 1 mg / L selenium-rich microalgae liquid; When 10 mg / mL sodium selenite solution is added to make the selenium concentration in the algae liquid 5 mg / L, the prepared selenium-rich microalgae liquid is a 5 mg / L selenium-rich microalgae liquid.

[0042] Step 2: Preparation of cultivation soil and experimental group setting A total of 8 experimental groups were set up, with 5 replicates in each experimental group. The cultivation soil of each experimental group was prepared as follows: Matrix soil (Pindstrup, Denmark) was mixed with vermiculite and stirred to obtain the treated matrix soil. 700 g of the treated matrix soil was weighed, and 1300 mL of tap water was measured. The microalgae solution was added to the tap water to obtain a mixture. This mixture was poured into the treated matrix soil, stirred evenly, and then distributed to five potted plants (five replicates). In the following experimental groups involving the addition of microalgae solution (control microalgae solution, 1 mg / L selenium-enriched microalgae solution, and 5 mg / L selenium-enriched microalgae solution), the amount of microalgae solution added was 200 mL.

[0043] The settings of the 8 experimental groups are as follows: (1) Blank control group: only the matrix soil was treated without adding any other substances; (2) 3 mg / L cadmium treatment group: 10 mg / mL cadmium chloride solution was added to tap water to make the cadmium ion concentration in tap water 3 mg / L; (3) 3 mg / L cadmium + algae: 10 mg / mL cadmium chloride and control microalgae solution were added to tap water to make the cadmium ion concentration in tap water 3 mg / L; (4) 3 mg / L cadmium + 1 mg / L selenium-rich algae: 10 mg / mL cadmium chloride solution and 1 mg / L selenium-rich microalgae solution were added to tap water to make the cadmium ion concentration in the tap water 3 mg / L; (5) 3 mg / L cadmium + 5 mg / L selenium-rich algae: 10 mg / mL cadmium chloride solution and 5 mg / L selenium-rich microalgae solution were added to tap water to make the cadmium ion concentration in the tap water 3 mg / L; (6) Algae: Add control microalgae solution to tap water; (7) 1 mg / L selenium-enriched algae: add 1 mg / L selenium-enriched microalgae solution to tap water; (8) 5mg / L selenium-rich algae: Add 5mg / L selenium-rich microalgae liquid to tap water.

[0044] Step 3: Planting lettuce Take an appropriate amount of lettuce seeds and spread them evenly on the cultivation soil. Place the potted plants in a constant temperature room at 20°C, 14h / 10h (light / dark), and the light intensity is 150μmol / m 2 After the seedlings emerge, the treatment is continued until five seedlings of uniform growth remain. Once the seedlings have grown, select five pots of uniformly growing plants and continue until one remains. Each pot is watered with 500 mL of tap water every five days, followed by an additional 50 mL of the corresponding microalgae solution on the 15th day. A total of 40 pots are randomly arranged and the plants are harvested after 30 days of treatment.

[0045] 2. Experimental results The dry weight, cadmium content and selenium content of the aboveground parts of the plants harvested 30 days after treatment were determined. Figures 10-12 .

[0046] Figure 10 The growth phenotypes of lettuce under different microalgae and selenium treatment conditions were shown. Compared with the blank control group, the growth of lettuce in the cadmium treatment group was inhibited to a certain extent. Compared with the 3mg / L cadmium treatment group, the dry weight of lettuce plants in the 3mg / L cadmium + algae, 3mg / L cadmium + 1mg / L selenium-enriched algae, and 3mg / L cadmium + 5mg / L selenium-enriched algae treatment groups increased by 15.53%, 38.45%, and 41.10%, respectively, which were significantly higher than those in the cadmium treatment group ( p <0.05) ( Figure 11 Compared to the blank control, the lettuce growth in the algae-only treatment group was similar to that of the control group. However, the lettuce dry weight increased by 15.18% and 19.29% in the 1 mg / L and 5 mg / L selenium-enriched algae treatments, respectively. This indicates that selenium-enriched algae treatment has a positive effect on lettuce growth and has no adverse effects. Phenotypic observations and dry weight measurements indicate that cadmium stress inhibits lettuce growth, and the addition of selenium-enriched algae can alleviate this inhibitory effect to varying degrees.

[0047] Figure 12 A is the cadmium content in the aboveground part of the escarole on the 30th day after treatment. Compared with the cadmium treatment, the cadmium contents in the treatments of 3 mg / L cadmium + algae, 3 mg / L cadmium + 1 mg / L selenium-enriched algae, and 3 mg / L cadmium + 5 mg / L selenium-enriched algae decreased by 7.06%, 14.04%, and 18.74%, respectively, all showing significant differences. Figure 12 B is the selenium content in the aboveground part of lettuce on the 30th day of treatment, which is similar to the results of selenium-enriched microalgae culture in rice. Compared with the blank control group, the selenium content in lettuce cultured with 5 mg / L selenium-enriched algae was significantly enriched.

[0048] Comparative experiment on different preparation methods of selenium-rich microalgae 1. Experimental methods The experimental method specifically includes the following steps: Step 1: Experimental group setup There are three experimental groups, as follows: Experimental group (1): The preparation of selenium-rich microalgae is as follows: select the algae solution of Scenedesmus multispinosa in the logarithmic growth phase, and adjust its initial OD 680 The pH value was adjusted to 0.5, and a 10 mg / mL sodium selenite solution was added to prepare an algae solution with a sodium selenite concentration of 1 mg / L. After culturing for 4 days, a cultured algae solution was obtained, and the cultured algae solution was separated to obtain selenium-enriched microalgae. The specific method for separating the cultured algae solution to obtain selenium-enriched microalgae includes the following steps: S41. Premixing a composite foaming agent with trehalose at 45° C. to form a micellar complex, and then adding the mixture to the algal solution after culturing for 4 days, wherein the composite foaming agent is a composite foaming agent composed of sophorolipid and rhamnolipid in a mass ratio of 4:1, the amount of the composite foaming agent added is 0.002% w / v, and the amount of the trehalose added is 0.01% w / v; S42, introducing low-pressure microbubbles, wherein the parameters of the low-pressure microbubbles are: pressure of 0.08 MPa, microbubble diameter of 100 μm, gas flow rate of 0.2 L / min, for 10 minutes, so that the algae cells are enriched in the foam layer; S43, collecting the foam layer, and washing the foam with an isotonic solution containing 1.0% by mass of NaCl and 0.01% by mass of trehalose to obtain the selenium-enriched microalgae.

[0049] Experimental group (2): The preparation of selenium-rich microalgae includes the following steps: S1. Preparation of mesoporous silica carrier, specifically comprising the following steps: S11, vacuum drying the mesoporous silica at 150° C. for 2 hours to remove adsorbed water in the pores to obtain pretreated mesoporous silica; S12, dispersing the pretreated mesoporous silica in a 0.2 mol / L FeCl3 solution prepared in deoxygenated deionized water at a ratio of 1:15 w / v, stirring at 300 rpm for 2 hours under nitrogen / argon protection to obtain a reaction solution; S13, maintaining an inert atmosphere, add 1 mol / L NaBH4 solution dropwise to the reaction solution in S12 at a rate of 2 mL / min to control the NaBH4 and Fe 3+ The molar ratio was 4:1, the reaction temperature was 30°C, and stirring was continued until no bubbles were generated; S14. After the reaction is completed, the mixture is washed three times by centrifugation with deoxyethanol, and the precipitate is taken. The centrifugation parameters are: 8000 rpm, 10 min; and the precipitate is placed in a vacuum dryer at 40°C for 12 hours to obtain a mesoporous silica carrier loaded with nano-zero-valent iron.

[0050] S2. Take the algae solution of Scenedesmus multispinosa in the logarithmic growth phase and adjust the OD 680 to 0.5, add 10 mg / mL sodium selenite solution to a final concentration of 1 mg / L, culture under light for 4 days, and collect the microalgae by centrifugation; S3. The microalgae obtained in step S2 and the mesoporous silica carrier obtained in step S1 were mixed in a mass ratio of 8:1, deionized water was added, and the mixture was shaken and reacted at 25° C. in the dark for 30 minutes to obtain the cultured algae solution.

[0051] S4. Place the cultured algae solution at 1000 rpm, centrifuge for 5 minutes, and collect the precipitate, which is the selenium-rich microalgae.

[0052] Experimental group (3): The preparation of selenium-rich microalgae is as follows: select the algae solution of Scenedesmus multispinosa in the logarithmic growth phase, and adjust its initial OD 680 Adjust to 0.5, add 10 mg / mL sodium selenite solution to prepare an algae solution with a sodium selenite concentration of 1 mg / L. After culturing for 4 days, obtain the cultured algae solution. Place the cultured algae solution at 1000 rpm and centrifuge for 5 minutes. Collect the precipitate, which is the selenium-rich microalgae.

[0053] Step 2: Index determination 1) Determination of viable cell rate and cell fragmentation rate of selenium-enriched microalgae The method is FDA-PI double staining and fluorescence microscopy counting (ISO 20688:2018).

[0054] The viable cell rate and cell fragmentation rate of the selenium-enriched microalgae isolated from the experimental group (1) and the experimental group (3) were determined. The specific results are shown in Table 1.

[0055] 2) Determination of total selenium retention rate in selenium-rich microalgae Method: GB 5009.93-2017 (ICP-MS).

[0056] The total selenium retention rates of the selenium-rich microalgae isolated from the experimental groups (1) and (3) were determined. The specific results are shown in Table 2.

[0057] 3) Determination of cadmium reduction and selenium enrichment effects Method: The same experimental method as in "Effects of Selenium-enriched Microalgae on Rice Growth and Cadmium-Reduction and Selenium-Enrichment in Cadmium-Stressed Environment". The treatment method of experimental group (1) was: 3 mg / L cadmium + 1 mg / L selenium-enriched microalgae solution prepared in experimental group (1); The treatment method of experimental group (2) was: 3 mg / L cadmium + 1 mg / L selenium-enriched microalgae solution prepared in experimental group (2); The treatment method of experimental group (3) was: 3 mg / L cadmium + 1 mg / L selenium-rich microalgae liquid prepared in experimental group (3).

[0058] The total cadmium and selenium contents in the aboveground parts of rice harvested from experimental group (1), experimental group (2), and experimental group (3) were determined. The specific results are shown in Figure 13-14 .

[0059] 2. Analysis of experimental results 1) Results of viable cell rate and cell fragmentation rate determination of selenium-enriched microalgae Table 1 Results of viable cell rate and cell fragmentation rate of selenium-rich microalgae obtained by the separation methods of experimental group (1) and experimental group (3) Experimental example Viable cell rate (%) Cell fragmentation rate (%) Experimental group (1) 98.2 0.8 Experimental group (3) 80.3 19.7 As can be seen from Table 1, compared with the centrifugal separation method of experimental group (3), the separation method of experimental group (1) obtained microalgae with a viable cell rate of up to 98.2%, indicating that the low-pressure microbubbles combined with trehalose can effectively avoid mechanical damage.

[0060] 2) Determination of total selenium retention rate in selenium-rich microalgae Table 2 Determination results of total selenium retention rate in selenium-rich microalgae obtained by the separation methods of experimental group (1) and experimental group (3) Experimental example Total selenium retention rate (%) Experimental group (1) 92.5 Experimental group (3) 75.8 As can be seen from Table 2, compared with the centrifugal separation method of experimental group (3), the total selenium retention rate of microalgae obtained by the separation method of experimental group (1) was as high as 92.5%, indicating that the low-pressure microbubbles combined with trehalose can effectively maintain the stability of the intracellular selenium form, thereby allowing selenium to be fully enriched in the microalgae.

[0061] 3) Cadmium reduction and selenium enrichment effect from Figure 13-14 It can be seen that compared with the centrifugal separation method of experimental group (3), the low-pressure microbubble combined with trehalose method of experimental group (1) can further improve the cadmium reduction and selenium enrichment effect of rice. At the same time, the addition of mesoporous silica in experimental group (2) greatly improves the cadmium reduction and selenium enrichment effect of rice. This is because the nano zero-valent iron in the mesoporous silica can reduce the cadmium ions in the cultivation matrix, thereby reducing cadmium, and mesoporous silica can adsorb free cadmium and selenium, reducing biological toxicity. In addition, microalgae are combined with mesoporous silica carriers, and the carriers can adsorb cadmium ions in the cultivation matrix to protect algae cells. At the same time, microalgae can wrap the carriers to delay the oxidation of nano zero-valent iron. The two complement each other and have synergistic effects.

[0062] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A cadmium-reducing and selenium-enriched crop cultivation conditioner, characterized in that: including Scenedesmus polyspinosa and sodium selenite; The conditioning agent is prepared by the following steps: S1, disperse mesoporous silica in Fe-containing 3+ In the solution, under the protection of an inert atmosphere, a reducing agent is added to react to obtain a mesoporous silica carrier; S2. Take the algae solution of Scenedesmus multispinosa in the logarithmic growth phase and adjust the OD 680 to 0.5, add 10 mg / mL sodium selenite solution to a final concentration of 1-5 mg / L, culture under light for 4 days, and collect the microalgae by centrifugation; S3, mixing the microalgae obtained in step S2 with the mesoporous silica carrier obtained in step S1 at a mass ratio of 10:1 to 5:1, adding deionized water, and shaking the mixture at 25° C. in the dark for 30 minutes to obtain the algae culture solution; S4. Separating the cultured algae liquid to obtain selenium-rich microalgae, and mixing the selenium-rich microalgae with deionized water to obtain the selenium-rich microalgae liquid, which is the conditioning agent.

2. The cadmium-reducing and selenium-rich crop cultivation conditioner according to claim 1, wherein Step S1 specifically includes the following steps: S11, vacuum drying the mesoporous silica at 150° C. for 2 hours to remove adsorbed water in the pores to obtain pretreated mesoporous silica; S12, dispersing the pretreated mesoporous silica in a 0.1-0.3 mol / L FeCl3 solution prepared in deoxygenated deionized water at a ratio of 1:10-1:20 w / v, stirring at 200-400 rpm for 1-2 hours under nitrogen / argon protection to obtain a reaction solution; S13, maintaining an inert atmosphere, add 0.5-1.5 mol / L NaBH4 solution dropwise to the reaction solution in S12 at a rate of 1-2 mL / min to control the NaBH4 and Fe 3+ The molar ratio is 3:1-5:1, the reaction temperature is 25-35°C, and stirring is continued until no bubbles are generated; S14. After the reaction is completed, the mixture is washed three times by centrifugation with deoxyethanol, and the precipitate is taken. The centrifugation parameters are: 8000 rpm, 10 min; and the precipitate is placed at 40°C for vacuum drying for 12 hours to obtain the mesoporous silica carrier.

3. The cadmium-reducing and selenium-rich crop cultivation conditioner according to claim 1, wherein Step S4 specifically includes the following steps: S41, adding 0.001%-0.005% w / v of a food-grade foaming agent and 0.01% w / v of trehalose to the cultured algae solution; S42, introducing low-pressure microbubbles, with the following parameters: pressure of 0.05–0.1 MPa, microbubble diameter of 50–200 μm, gas flow rate of 0.1–0.3 L / min, for 5–10 minutes, to allow algal cells to accumulate in the foam layer; S43, collecting the foam layer, and washing the foam with an isotonic solution containing 0.8%-1.0% by mass of NaCl and 0.01% by mass of trehalose to obtain the selenium-enriched microalgae.

4. The cadmium-reducing and selenium-rich crop cultivation conditioner according to claim 3, characterized in that The food-grade foaming agent in step S41 is a composite foaming agent composed of sophorolipids and rhamnolipids in a mass ratio of 3-5:1, and the total concentration of the composite foaming agent is 0.0015%-0.0025% w / v; Step S41 specifically comprises: premixing the composite foaming agent and oligomeric trehalose at 40-45° C. to form a micelle complex, and then adding the mixture into the algae solution.

5. A cultivation medium, characterized in that The present invention comprises the cadmium-reduced and selenium-enriched crop cultivation conditioner according to any one of claims 1 to 4, wherein the cultivation substrate is in the form of a cultivation solution or a cultivation soil.

6. The cultivation substrate according to claim 5, wherein The cultivation matrix is ​​cultivation soil, which includes matrix soil, water, and selenium-rich microalgae liquid. The cultivation soil is prepared by uniformly mixing the selenium-rich microalgae liquid with water and then with the matrix soil. The mass fraction of the selenium-rich microalgae liquid in the cultivation soil is 5-10%.

7. Use of the cadmium-reduced and selenium-rich crop cultivation conditioner according to any one of claims 1 to 4 in cultivating cadmium-reduced and selenium-rich crops.

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