Cadmium-reduced selenium-enriched crop cultivation conditioner, cultivation substrate and application

Abstract

The application discloses a kind of rich selenium crops of reducing cadmium cultivation conditioning agent, cultivation matrix and application, belong to microalgae rich selenium and its application technical field.The conditioning agent is prepared by the following steps: S1, mesoporous silica is dispersed in Fe 3+ Solution reaction obtains mesoporous silica carrier;S2, take the algal liquid of the polycentrum pruniforme in logarithmic growth phase, adjust OD 680 To 0.5, add sodium selenite solution, light culture, centrifugal collection microalgae;S3, the microalgae obtained in step S2 is mixed with the mesoporous silica carrier obtained in step S1 according to mass ratio 10:1~5:1, add deionized water, oscillation reaction under 25 DEG C, avoid light condition, obtain the culture algal liquid;S4, the culture algal liquid is separated to obtain selenium-rich microalgae.The application has the beneficial effect that the crops cultivated by the rich selenium crops of reducing cadmium cultivation conditioning agent are not only rich in selenium but also low in cadmium content, grow well, and have good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of selenium-rich microalgae and its application. More specifically, the present application relates to a cadmium-reducing selenium-rich crop cultivation conditioner, a cultivation substrate and an application. BACKGROUND

[0002] With the continuous deterioration of environmental quality, heavy metal pollution has attracted public attention. Cadmium is a toxic non-essential transition metal, which is distributed in soil, air and water, especially in soil and water, and is easily absorbed and accumulated in plants, posing a serious threat to human and animal health. It has been listed as a class 1 human carcinogen. Cadmium is transmitted through food chain and food web and accumulated in plants and animals, with a half-life of about 25-30 years, directly threatening food production, crop safety and drinking water safety, and ultimately endangering human health. Cadmium exposure is associated with a variety of diseases such as prostate cancer, lung cancer, testicular cancer, kidney dysfunction, rhinitis, pulmonary emphysema and bone fracture. In addition, cadmium can cause chronic inflammation of the lungs, pulmonary fibrosis, pulmonary emphysema and lung tumors, and reduce male fertility, resulting in a decrease in sperm count and a decrease in motility.

[0003] At present, the methods for treating soil cadmium pollution include physical, chemical, biological and combined remediation technology. Physical remediation technology can transfer or separate pollutants by physical means, but it has high cost and can easily damage the soil structure. Chemical remediation technology uses chemical reagents to change the form of pollutants, reduce their toxicity or mobility, but it may introduce new chemical substances and pose potential risks. In wastewater treatment, physical, chemical and biological methods are also used to treat cadmium-contaminated wastewater. Biological remediation technology has a wide range of applications, low cost, simple operation process and no secondary pollution, and has broad prospects in cadmium pollution control. Microalgae have high growth rate, high surface area and small individual size, and have strong heavy metal adsorption capacity. Studies have shown that microalgae have much higher adsorption and accumulation capacity for heavy metals than other organisms, and can efficiently adsorb cadmium ions to reduce the cadmium content in the environment. In addition, wastewater ecosystems are rich in organic carbon, nitrogen and phosphorus required for microalgae growth, and microalgae can not only effectively purify wastewater during growth and metabolism, but also significantly reduce the chemical oxygen demand, total nitrogen and total phosphorus in wastewater. Therefore, using microalgae to treat cadmium-containing wastewater can achieve simultaneous reduction of multiple pollutants, thereby improving the efficiency of treatment. Compared with other remediation technologies, microalgae have environmental friendliness and economic feasibility in treating cadmium pollution, and are expected to play a more important role in the field of 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 ten-thousandth of the total body weight, it is one of the important elements for maintaining health. Selenium has outstanding antioxidant properties and is a cofactor for many antioxidant enzymes, such as GSH-Px, SOD, and CAT. These enzymes play a crucial role in resisting cell damage caused by free radicals and lipid peroxides. The detoxification effect of selenium in plants has been confirmed. Although selenium is not essential for higher plants, numerous studies have confirmed that different forms and appropriate concentrations of selenium have a positive impact on plant growth and improve tolerance to abiotic stresses. Selenium not only promotes plant growth and development but also enhances their tolerance to heavy metal stress and improves their adaptability to environmental stress. Selenium alleviates cadmium stress in plants by improving photosynthesis, regulating the balance of mineral nutrition, enhancing the antioxidant capacity, and reducing cadmium content in plant cells.

[0005] While selenium and microalgae are both effective in reducing cadmium absorption by plants, their impact on reducing the total amount of cadmium in soil remains limited, posing a challenge to the long-term remediation of cadmium pollution in soil. In practical production, how to produce low-cadmium, selenium-enriched foods in areas with high cadmium levels remains a pressing issue. Currently, there are few technologies and methods for selenium enrichment in plants to reduce cadmium, especially methods for simultaneously reducing cadmium in plants and enriching crops with selenium, which cannot be widely applied in agricultural production. Future research should focus on developing new methods and technologies that can both enrich selenium and reduce cadmium, providing technical support for the production of healthy, safe, and high-quality selenium-enriched products in cadmium-contaminated soils. Summary of the Invention

[0006] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0007] Another objective of this invention is to provide a cadmium-reducing and selenium-enriched crop cultivation conditioner, cultivation substrate, and application. Crops cultivated using this cadmium-reducing and selenium-enriched crop cultivation conditioner are not only rich in selenium but also have low cadmium content and grow well, showing promising application prospects.

[0008] In order to achieve these objectives and other advantages according to the present invention, a cadmium-reducing and selenium-enriched crop cultivation conditioner is provided, comprising Scenedesmus polysaccharide and sodium selenite;

[0009] The conditioner is prepared through the following steps:

[0010] S1, Disperse mesoporous silica in a Fe-containing medium 3+ In the solution, under the protection of an inert atmosphere, a reducing agent is added to carry out the reaction to obtain a mesoporous silica support;

[0011] S2. Take the algal solution of *Scenedesmus multipinnatifida* 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, light culture for 4 days, centrifugal collection of microalgae;

[0012] S3, the microalgae obtained in step S2 is mixed with the mesoporous silica carrier obtained in step S1 at a mass ratio of 10:1-5:1, deionized water is added, and after oscillation reaction at 25°C in the dark for 30 minutes, the culture algal liquid is obtained;

[0013] S4, the selenium-rich microalgae is obtained by separating the culture algal liquid, and the selenium-rich microalgae liquid is obtained by mixing the selenium-rich microalgae with deionized water, which is the conditioning agent.

[0014] Preferably, step S1 specifically comprises the following steps:

[0015] S11, vacuum drying mesoporous silica at 150°C for 2 hours to remove adsorbed water in the pores, obtaining pretreated mesoporous silica;

[0016] S12, dispersing the pretreated mesoporous silica in a 0.1-0.3 mol / L FeCl3 solution prepared by deoxygenated deionized water at 1:10-1:20 w / v, stirring at 200-400 rpm under nitrogen / argon protection for 1-2 hours, obtaining a reaction liquid;

[0017] S13, maintaining an inert atmosphere, adding 0.5-1.5 mol / L NaBH4 solution dropwise to the reaction liquid in S12, dropwise adding speed 1-2 mL / min, controlling the molar ratio of NaBH4 to Fe 3+ Molar ratio of 3:1-5:1, reaction temperature 25-35°C, continuous stirring until no bubbles are generated;

[0018] S14, after the reaction is completed, washing the precipitate 3 times by centrifugation with deoxygenated ethanol, centrifugation parameters: 8000 rpm, 10 min; then placing the precipitate in a vacuum dryer at 40°C for 12 hours, obtaining the mesoporous silica carrier.

[0019] Preferably, step S4 specifically comprises the following steps:

[0020] S41, adding 0.001%-0.005% w / v food-grade foaming agent and 0.01% w / v trehalose to the culture algal liquid;

[0021] S42, introducing low-pressure microbubbles, the parameters of the low-pressure microbubbles being specifically: pressure 0.05-0.1 MPa, microbubble diameter 50-200 μm, gas flow 0.1-0.3 L / min, lasting for 5-10 minutes, so that the algal cells are enriched in the foam layer;

[0022] S43, collecting the foam layer, rinsing 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-rich microalgae.

[0023] Preferably, the food-grade foaming agent in step S41 is a composite foaming agent composed of sophorolipids and rhamnolipids at a mass ratio of 3-5:1, and the total concentration of the composite foaming agent is 0.0015%-0.0025% w / v.

[0024] Step S41 specifically comprises: first, pre-mixing the composite foaming agent and oligomeric trehalose at 40-45 DEG C to form a micellar complex, and then adding the algal liquid.

[0025] The application also provides a cultivation substrate comprising the selenium-rich crop cultivation conditioner, and the cultivation substrate is in the form of a cultivation liquid or a cultivation soil.

[0026] Preferably, the cultivation substrate is a cultivation soil, and the cultivation soil comprises a substrate soil, water and a selenium-rich microalgae liquid.

[0027] The application also provides an application of the selenium-rich crop cultivation conditioner in cultivating selenium-rich crops.

[0028] The application at least has the following beneficial effects:

[0029] The application provides a selenium-rich crop cultivation conditioner, a cultivation substrate and an application, and the selenium-rich crop cultivated by using the selenium-rich crop cultivation conditioner is not only rich in selenium but also has a low cadmium content and grows well, thus having a good application prospect.

[0030] Other advantages, objects and features of the application will be partly embodied in the following description, and will be partly understood by those skilled in the art through research and practice of the application. DETAILED DESCRIPTION

[0031] Figure 1 Fig. A is a microscope image of an isolated algal strain in the screening and identification process of cadmium-resistant algae species, and Fig. B is a plate expansion of the target algal species screened;

[0032] Figure 2 Fig. A is a TUFA-based SC-Z8 phylogenetic tree, and Fig. B is an RBCL-based SC-Z8 phylogenetic tree;

[0033] Figure 3The tolerance of Chlamydomonas reinhardtii (Cr-wt) (A), Chlorella pyrenoidosa (xl-12) (B), Chlorella sorokiniana (dp3) (C) and Scenedesmus dimorphus (SC-Z8) (D) to cadmium;

[0034] Figure 4 The growth phenotype of rice treated with selenium-rich microalgae SC-Z8;

[0035] Figure 5 The plant height (A) and dry weight (B) of rice treated with selenium-rich microalgae SC-Z8;

[0036] Figure 6 The cadmium (A) and selenium content (B) of rice treated with selenium-rich microalgae SC-Z8;

[0037] Figure 7 The growth phenotype of rice treated with selenium-rich microalgae SC-Z8;

[0038] Figure 8 The plant height (A) and dry weight (B) of rice treated with selenium-rich microalgae SC-Z8;

[0039] Figure 9 The cadmium (A) and selenium content (B) of rice treated with selenium-rich microalgae SC-Z8;

[0040] Figure 10 The growth phenotype of lettuce treated with selenium-rich microalgae SC-Z8;

[0041] Figure 11 The dry weight of lettuce treated with selenium-rich microalgae SC-Z8;

[0042] Figure 12 The cadmium (A) and selenium content (B) of lettuce treated with selenium-rich microalgae SC-Z8;

[0043] Figure 13 The total cadmium content of rice treated with selenium-rich microalgae prepared by different methods;

[0044] Figure 14 The total selenium content of rice treated with selenium-rich microalgae prepared by different methods. DETAILED DESCRIPTION

[0045] The present application will be further described in conjunction with the following examples, so that those skilled in the art can implement the present application according to the description.

[0046] The present application is based on the following design principles: first, screening of cadmium-resistant algae species, then comparing the tolerance test of the screened algae species with other algae species, verifying that the screened algae species have significant cadmium resistance, then combining the screened algae species with selenium-containing substances (such as sodium selenite), testing the combined use effect, and obtaining that the combination of the screened algae species and selenium can greatly reduce the cadmium content of the plant and greatly improve the selenium content of the plant, and the two have synergistic effect, which is much better than the effect or additive effect of single use. See the following.

[0047] <Screening and identification of cadmium-resistant algae species>

[0048] Samples were collected from lakes and rice planting soils in Tianfu New Area, Chengdu, Sichuan. The collected water and soil samples were diluted by appropriate multiples, i.e. original solution, 10 times, 100 times and 1000 times. 200 μL of liquid with different dilution concentrations was drawn by a pipette and spread on BG11 plate medium, and then cultured in an artificial climate box. When green single colonies grew, single colonies were picked up for resuspension and microscopic examination for preliminary screening of target algae species. The target algae species isolated and purified were observed under a microscope. The morphological characteristics (A figure in Figure 1 ) were analyzed, and it was found that the algae strain was green, the cells were oval, and there were one or 2-4 linearly arranged cells per pole, with one spine per pole. The algae strain obtained by morphological screening was preliminarily identified as Scenedesmus, named SC-Z8, and plate propagation was carried out (B figure in Figure 1 ).

[0049] The species relationship of SC-Z8 algae strain was identified from the molecular level. The Elongation factor Tu ( TUFA ) and RuBis-Co ( RBCL ) genes were selected to design primers for PCR amplification and sequencing of the products. The sequences obtained by sequencing were compared with homologous sequences on the NCBI website, and the base alignment of the sequences obtained by sequencing and homologous sequences was performed using MEGE11 software. Based on the aligned sequence file, the Neighbor-Joining Method was used to construct a phylogenetic tree. The reliability of the topological structure of the phylogenetic tree was calculated by Bootstrap for 1000 times of iteration, and finally an evolutionary tree graph was generated, as shown in Figure 2 . The results showed that the isolated algae species SC-Z8 and Desmodesmusabundans algal species in the Scenedesmaceae family clustered into a branch (A figure in Figure 2 ). Based on the RBCL sequence with higher variation degree, the isolated algae species SC-Z8 formed a separate branch, but had the closest relationship with Desmodesmusabundans branch (B figure in Figure 2 ). According to the above results, it is determined that the isolated algae species SC-Z8 in this experiment is a member of the Scenedesmaceae family.Desmodesmus Scenedesmus polysacchariformis ( Desmodesmusabundans ).

[0050] <Comparison of Cadmium Tolerance Among Different Algal Species>

[0051] Take algal solution in the stable growth phase, initial OD 680 After adjusting to 3.0, different concentrations of cadmium ions were added, and the algal solution was observed at 0d, 2d, 4d, and 6d. See details below. Figure 3 , Figure 3 The tolerance of Chlamydomonas reinhardtii (Cr-wt), Chlorella proteoglycans (xl-12), Chlorella sorokinosa (dp3), and Scenedesmus multipinnatifida (SC-Z8) to cadmium was investigated.

[0052] from Figure 3 It can be seen from this that in Chlamydomonas reinhardtii (Cr-wt) ( Figure 3 In A), by day 2, under 40 mg / L cadmium ion treatment, the algal solution turned yellow, and the growth of *Chlamydomonas reinhardtii* was significantly inhibited, even resulting in death, indicating its relatively weak tolerance to cadmium. *Chlorella proteoglycans* (xl-12) ( Figure 3 In (B), by day 6, under a cadmium ion treatment of 70 mg / L, the algal 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. *Chlorella sorokinense* (dp3) Figure 3 In C), by day 6, under 400 mg / L cadmium ion treatment, the algal solution turned yellow, indicating that *Chlorella sorokinense* can maintain relatively good growth under low cadmium concentrations, but its growth will be affected to some extent under high cadmium stress. *Scenedesmus multipinnatifida* (SC-Z8) Figure 3 In D), by day 6, the color change of the algal solution remained relatively slow under the cadmium ion treatment condition of 1300 mg / L. This result shows that *Scenedesmus multipinnatifida* may have a strong cadmium tolerance and can maintain a relatively stable state in a high cadmium concentration environment. Therefore, *Scenedesmus multipinnatifida* was selected as the algal species for subsequent experiments.

[0053] <Effects of combined application of microalgae and selenium on rice growth and cadmium / selenium accumulation in rice under cadmium stress>

[0054] To investigate the effects of combined application of microalgae (SC-Z8) and selenium on rice growth and cadmium / selenium accumulation under cadmium stress, a hydroponic rice experiment was conducted.

[0055] 1. Experimental Methods

[0056] The experimental method specifically includes the following steps:

[0057] Step 1: Rice seed treatment:

[0058] Rice seeds were soaked in 2% sodium hypochlorite for 10 min, then washed with distilled water for 6 times, and then soaked in tap water in the dark and placed in a constant temperature incubator at 30°C for 24 h, with water changed every 6 h. After the seeds turned white, they were transferred to an artificial climate chamber with a temperature setting of 28°C during the day and 25°C at night, 14h / 10h (light / dark), light intensity of 300 μmol·m −2 −1 After 7 days of tap water culture, seedlings with consistent growth were selected for hydroponics test. The hydroponics test nutrient solution was Hoagland's nutrient solution, and the composition of Hoagland's nutrient solution was: 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, 6.81 mg / L FeSO4, and the pH was adjusted to 7.0 before use. The test rice was grown on the cotton.

[0059] Step two, preparation of SC-Z8 algal liquid in the stable growth phase:

[0060] SC-Z8 P. tricornutum was cultured in BG11 liquid medium at 28°C, 150 rpm, 100 μmol·m -2 1 light intensity until it reached the stable growth phase, which was the SC-Z8 algal liquid in the stable growth phase.

[0061] Step three, preparation of conditioner:

[0062] SC-Z8 P. tricornutum algal liquid in the stable growth phase was mixed with sodium selenite to obtain the conditioner, and the mass fraction of microalgae in the algal liquid was 8%.

[0063] Step four, experimental group setting:

[0064] A total of 8 experimental groups were set up, with 5 parallels in each experimental group, for a total of 40 bottles randomly arranged. In each parallel, the hydroponics device was a transparent plastic hydroponics bottle, and 400 mL of Hoagland's nutrient solution was added to each bottle. In the experimental groups involving the addition of conditioner and SC-Z8 algal liquid, the addition amount of conditioner in each bottle was 40 mL, and the addition amount of SC-Z8 algal liquid in each bottle was 40 mL. The 8 experimental groups were:

[0065] ​​(1) Blank control group: only Hoggland nutrient solution is used, without adding any other substances;

[0066] (2) 3 mg / L cadmium treatment group: cadmium chloride is added to Hoggland nutrient solution, so that the concentration of cadmium chloride in the nutrient solution is 3 mg / L;

[0067] (3) 3 mg / L cadmium + 1 mg / L selenium treatment group: cadmium chloride and sodium selenite are added to Hoggland nutrient solution, so that the concentration of cadmium chloride in the nutrient solution is 3 mg / L and the concentration of sodium selenite is 1 mg / L.

[0068] (4) 3 mg / L cadmium + 5 mg / L selenium treatment group: 3 mg / L cadmium chloride and sodium selenite are added to Hoggland nutrient solution, so that the concentration of cadmium chloride in the nutrient solution is 3 mg / L and the concentration of sodium selenite is 5 mg / L;

[0069] (5) 3 mg / L cadmium + algae treatment group: cadmium chloride and SC-Z8 algal liquid are added to Hoggland nutrient solution, so that the concentration of cadmium chloride in the nutrient solution is 3 mg / L;

[0070] (6) 3 mg / L cadmium + 1 mg / L selenium + algae treatment group: cadmium chloride and conditioner are added to Hoggland nutrient solution, so that the concentration of cadmium chloride in the nutrient solution is 3 mg / L and the concentration of sodium selenite is 1 mg / L;

[0071] (7) 3 mg / L cadmium + 5 mg / L selenium + algae treatment group: 3 mg / L cadmium chloride and conditioner are added to Hoggland nutrient solution, so that the concentration of cadmium chloride in the nutrient solution is 3 mg / L and the concentration of sodium selenite is 5 mg / L;

[0072] (8) Algae treatment group: SC-Z8 algal liquid is added to Hoggland nutrient solution.

[0073] Step four, index determination

[0074] The plant height, aboveground dry weight, cadmium content and selenium content of rice after 30 days of water culture were determined, and the results are shown in Figure 4 , Figure 5 , Figure 6 . Among them:

[0075] The plant height determination method is to measure the plant height of rice at harvest time using a tape measure, and the plant height of 5 rice plants is measured and averaged.

[0076] The determination method of aboveground dry weight is to rinse the aboveground part of harvested rice with deionized water for 3 times, absorb the surface water, quickly put into liquid nitrogen, and then put the sample into a freeze dryer, and bake until the water is removed, and reach a constant weight, which is the dry biomass of rice, that is, the dry weight of rice aboveground part.

[0077] The methods for determining the cadmium and selenium content in the aboveground parts were as follows: The dry weight of each sample was weighed, and all samples were pulverized and mixed using a high-speed pulverizer for the determination of total cadmium and total selenium content. After digestion, the total cadmium content in the samples was determined according to Method I of Chinese National Standard GB 5009.268-2016, "National Food Safety Standard - Determination of Multiple Elements in Food," and the total cadmium concentration was determined using inductively coupled plasma mass spectrometry. The total selenium content was determined according to Chinese National Standard GB 5009.93-2017, "National Food Safety Standard - Determination of Selenium in Food," and was measured using an atomic fluorescence spectrophotometer.

[0078] 2. Analysis of Experimental Results

[0079] Figure 4 The image shows the growth phenotypes of rice treated with selenium and algae. Compared with the blank control group, the plants in the 3 mg / L cadmium treatment group were significantly dwarfed, with plant height and aboveground dry weight reduced by 47.69% and 38.99%, respectively. Figure 5 (A and B) indicate that cadmium stress significantly inhibited rice growth. Compared to the 3 mg / L cadmium treatment group, the plant height of the 3 mg / L cadmium + 1 mg / L selenium treatment group increased by 10.95%, while the plant height of the 3 mg / L cadmium + algae and 3 mg / L cadmium + 1 mg / L selenium + algae treatment groups increased by 28.94% and 57.35%, respectively, with dry weight increases of 15.66% and 57.28%. Furthermore, compared to the blank control group, the dry weight of the algae-only treatment group increased by 27.41%. The experimental results show that the combined treatment of microalgae and selenium can effectively alleviate the inhibitory effect of cadmium on rice growth, with the 1 mg / L selenium + algae combination showing the best effect, while the effect of high concentrations of selenium (Se, 5 mg / L) is limited due to potential toxicity.

[0080] Figure 6 A represents the cadmium content in the aboveground parts of the microalgae SC-Z8 and selenium co-treatment. Compared with the 3 mg / L cadmium treatment, the cadmium content in the 3 mg / L cadmium + algae treatment was not significantly different (p>0.05), which may be because the cell activity of most algal cells decreased at a cadmium concentration of 3 mg / L, thus limiting their adsorption capacity. However, the cadmium content in the 3 mg / L cadmium + 1 mg / L selenium and 3 mg / L cadmium + 5 mg / L selenium treatment groups decreased by 41.43% and 59.10%, respectively, indicating that selenium can inhibit cadmium accumulation in rice. Compared with the 3 mg / L cadmium treatment, the cadmium content in the 3 mg / L cadmium + 1 mg / L selenium + algae and 3 mg / L cadmium + 5 mg / L selenium + algae treatment groups decreased by 53.59% and 63.83%, respectively. This indicates that the addition of selenium not only inhibits cadmium accumulation but may also improve algal cell activity and enhance algal adsorption capacity by alleviating the stress of cadmium on algae. However, the combination of low-concentration selenium (1 mg / L) and algae has a better effect on rice growth. Figure 6B is the selenium content of the aboveground part of rice, compared with the blank control group, the addition of exogenous selenium significantly improves the total selenium content of rice plants. Therefore, the joint addition of selenium and polyknot grid algae can reduce the inhibition of cadmium on rice, reduce the accumulation of cadmium in rice, and increase the enrichment of selenium in rice.

[0081] <Effect of selenium-rich microalgae on the growth of rice and the reduction of cadmium in cadmium stress environment>

[0082] 1. Experimental method

[0083] The experimental method specifically includes the following steps:

[0084] Step 1, rice seed treatment:

[0085] After the rice seeds were soaked in 2% sodium hypochlorite for 10 minutes and washed 6 times with distilled water, the seeds were soaked in tap water in the dark and placed in a constant temperature incubator at 30°C for 24 hours, with water changed every 6 hours. After the seeds turned white, they were transferred to an artificial climate chamber with a temperature setting of 28°C during the day and 25°C at night, 14h / 10h (light / dark), light intensity of 300μmol·m −2 ·s −1 -2, and humidity of 60%. After 7 days of tap water culture, uniform seedlings were selected for hydroponics experiment. The hydroponics experiment nutrient solution was Hogland nutrient solution, and the composition of the Hogland nutrient solution was: 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, 6.81 mg / L FeSO4, and the pH was adjusted to 7.0 before use. The test rice was grown on the cotton.

[0086] Step 2, preparation of microalgae solution:

[0087] Preparation method: select the logarithmic growth phase of polyknot grid algae SC-Z8 algal solution, adjust the initial OD680 to 0.5, add 10mg / mL sodium selenite solution, make the selenium concentration in the algal solution 0mg / L, 1mg / L and 5mg / L respectively, after 4 days of culture, centrifuge at 1000rpm for 5min, collect the precipitate as selenium-rich microalgae, and resuspend with pure water to obtain selenium-rich microalgae solution. The operation of resuspension with pure water is as follows: mix the collected precipitate with pure water to obtain selenium-rich microalgae solution, and the mass fraction of selenium-rich microalgae in the selenium-rich microalgae solution is 8%;

[0088] When 10 mg / mL sodium selenite solution is added to make the selenium concentration in the algal liquid 1 mg / L, the prepared selenium-rich microalgae liquid is 1 mg / L selenium-rich microalgae liquid.

[0089] When 10 mg / mL sodium selenite solution is added to make the selenium concentration in the algal liquid 1 mg / L, the prepared selenium-rich microalgae liquid is 1 mg / L selenium-rich microalgae liquid.

[0090] When 10 mg / mL sodium selenite solution is added to make the selenium concentration in the algal liquid 5 mg / L, the prepared selenium-rich microalgae liquid is 5 mg / L selenium-rich microalgae liquid.

[0091] Step three, experimental group setting:

[0092] A total of 8 experimental groups, 5 parallel for each experimental group, a total of 40 bottles randomly arranged. In each parallel, the water culture device is a transparent plastic water culture bottle, 400 mL of Hoagland nutrient solution is added to each bottle, and the following involves the addition of microalgae liquid (control microalgae liquid, 1 mg / L selenium-rich microalgae liquid, 5 mg / L selenium-rich microalgae liquid), the addition amount in each bottle is 40 mL. The 8 experimental groups are as follows:

[0093] (1) Blank control group: only Hoagland nutrient solution is used, without adding any other substances;

[0094] (2) 3 mg / L cadmium treatment group: add cadmium chloride to the nutrient solution to make the concentration of cadmium chloride in the nutrient solution 3 mg / L;

[0095] (3) 3 mg / L cadmium + algae treatment group: add cadmium chloride and control microalgae liquid to the nutrient solution to make the concentration of cadmium chloride in the nutrient solution 3 mg / L;

[0096] (4) 3 mg / L cadmium + 1 mg / L selenium-rich algae: add 3 mg / L cadmium chloride and 1 mg / L selenium-rich microalgae liquid to the nutrient solution to make the concentration of cadmium chloride in the nutrient solution 3 mg / L;

[0097] (5) 3 mg / L cadmium + 5 mg / L selenium-rich algae: add 3 mg / L cadmium chloride and 5 mg / L selenium-rich microalgae liquid to the nutrient solution to make the concentration of cadmium chloride in the nutrient solution 3 mg / L;

[0098] (6) Algae treatment group: add control microalgae liquid to the nutrient solution;

[0099] (7) 1 mg / L selenium-rich algae treatment group: add 1 mg / L selenium-rich microalgae liquid to the nutrient solution;

[0100] (8) 5 mg / L selenium-rich algae treatment group: add 5 mg / L selenium-rich microalgae liquid to the nutrient solution.

[0101] Step four, the plant height, dry weight of aboveground, cadmium content of rice after 30 days of hydroponics were determined, the results are shown in Figure 7~9 .

[0102] 2. Analysis of experimental results

[0103] Figure 7 Figure A is the growth phenotype of rice treated with selenium-rich microalgae. Compared with the cadmium treatment group, the growth status of rice plants in the 3mg / L cadmium + 1mg / L selenium-rich algae and 3mg / L cadmium + 5mg / L selenium-rich algae treatment groups was improved, and the dry weight increased by 25.61% and 29%, respectively, and was significantly higher than that of the cadmium treatment group (p<0.05) Figure 8 Compared with the blank control group, the growth status of rice leaves in the 1mg / L and 5mg / L selenium-rich algae treatment groups was good, and the dry weight increased by 20% and 29.44%, respectively, indicating that selenium-rich algae treatment had a positive effect on rice growth.

[0104] Figure 9 Figure A is the cadmium content of rice treated with selenium-rich microalgae. Compared with the cadmium treatment group, the cadmium content of the 3mg / L cadmium + 1mg / L selenium-rich algae and 3mg / L cadmium + 5mg / L selenium-rich algae treatment groups decreased by 19.71% and 22.19%, respectively. Figure 9 Figure B is the selenium content of rice aboveground. Compared with the blank control group, the selenium content of rice cultured with 5mg / L selenium-rich algae was significantly enriched. The cadmium content determination showed that selenium-rich microalgae could reduce the accumulation of cadmium in rice by enhancing the adsorption or accumulation of cadmium by algae.

[0105] Effect of selenium-rich microalgae on the growth of lettuce and the reduction of cadmium and selenium in cadmium stress environment

[0106] 1. Experimental method:

[0107] The experimental method specifically includes the following steps:

[0108] Step one, preparation of microalgae liquid:

[0109] Preparation method: select the logarithmic growth phase of Scenedesmus dimorphus SC-Z8 algal liquid, adjust the initial OD680 to 0.5, add 10mg / mL sodium selenite solution, so that the selenium concentration in the algal liquid is 0mg / L, 1mg / L and 5mg / L, respectively. After 4 days of culture, centrifuge at 1000rpm for 5min to collect the precipitate, which is selenium-rich microalgae, and resuspend with pure water to obtain selenium-rich microalgae liquid. The operation of resuspension with pure water is as follows: mix the collected precipitate with pure water to obtain selenium-rich microalgae liquid, and the mass fraction of selenium-rich microalgae in the selenium-rich microalgae liquid is 8%;

[0110] When 10 mg / mL sodium selenite solution is added to make the selenium concentration in the algal liquid 1 mg / L, the prepared selenium-rich microalgae liquid is 1 mg / L selenium-rich microalgae liquid.

[0111] When 10 mg / mL sodium selenite solution is added to make the selenium concentration in the algal liquid 1 mg / L, the prepared selenium-rich microalgae liquid is 1 mg / L selenium-rich microalgae liquid.

[0112] When 10 mg / mL sodium selenite solution is added to make the selenium concentration in the algal liquid 5 mg / L, the prepared selenium-rich microalgae liquid is 5 mg / L selenium-rich microalgae liquid.

[0113] Step two, preparation of cultivation soil and setting of experimental groups

[0114] A total of 8 experimental groups were set up, each with 5 replicates, and the cultivation soil of each experimental group was prepared as follows:

[0115] The substrate soil (Pindstrup, Denmark) was mixed with vermiculite and stirred uniformly to obtain treated substrate soil. 700 g of the treated substrate soil was weighed, 1300 mL of tap water was measured, and microalgae liquid was added to the tap water to obtain a mixed liquid. The mixed liquid was poured into the treated substrate soil, stirred uniformly, and then divided into five pots for potting (5 replicates). In the experimental groups involving the addition of microalgae liquid (control microalgae liquid, 1 mg / L selenium-rich microalgae liquid, 5 mg / L selenium-rich microalgae liquid), the addition amount of microalgae liquid was 200 mL.

[0116] The specific settings of the 8 experimental groups are as follows:

[0117] (1) Blank control group: only treated substrate soil, without adding any other substances;

[0118] (2) 3 mg / L cadmium treatment group: 10 mg / mL cadmium chloride solution was added to the tap water to make the cadmium ion concentration in the tap water 3 mg / L;

[0119] (3) 3 mg / L cadmium + algae: 10 mg / mL cadmium chloride and control microalgae liquid were added to the tap water to make the cadmium ion concentration in the tap water 3 mg / L;

[0120] (4) 3 mg / L cadmium + 1 mg / L selenium-rich algae: 10 mg / mL cadmium chloride solution and 1 mg / L selenium-rich microalgae liquid were added to the tap water to make the cadmium ion concentration in the tap water 3 mg / L;

[0121] (5) 3 mg / L cadmium + 5 mg / L selenium-rich algae: 10 mg / mL cadmium chloride solution and 5 mg / L selenium-rich microalgae liquid were added to the tap water to make the cadmium ion concentration in the tap water 3 mg / L;

[0122] (6) Algae: tap water added with control microalgae liquid;

[0123] (7) 1 mg / L selenium-rich algae: tap water added with 1 mg / L selenium-rich microalgae liquid;

[0124] (8) 5 mg / L selenium-rich algae: tap water added with 5 mg / L selenium-rich microalgae liquid.

[0125] Step three, planting of lettuce

[0126] Take an appropriate amount of lettuce seeds and evenly spread them on the cultivation soil. Place the pots in a constant temperature room at 20°C, 14h / 10h (light / dark), light intensity of 150 μmol / m 2 / s, and when the seedlings grow, select 5 seedlings with consistent growth and remove the rest. When the seedlings grow up, select 5 pots with consistent growth and remove one. Each pot is watered with 500 mL of tap water every 5 days, and 50 mL of corresponding microalgae liquid is added on the 15th day. A total of 40 pots are randomly arranged, and the plants are harvested after 30 days of treatment.

[0127] 2. Experimental results

[0128] The dry weight, cadmium content and selenium content of the plants harvested after 30 days of treatment were determined, and the specific results are shown in Figure 10~12 .

[0129] Figure 10 The growth phenotype of lettuce under different microalgae and selenium treatment conditions is 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 3 mg / L cadmium treatment group, the dry weight of lettuce in the 3 mg / L cadmium + algae, 3 mg / L cadmium + 1 mg / L selenium-rich algae, and 3 mg / L cadmium + 5 mg / L selenium-rich algae treatment groups increased by 15.53%, 38.45%, and 41.10%, respectively, which was significantly higher than that of the cadmium treatment group (P<0.05). p <0.05>( Figure 11 Compared with the blank control group, the growth of lettuce in the algae treatment group was similar to that of the control group, but the dry weight of lettuce in the 1 mg / L and 5 mg / L selenium-rich algae treatment groups increased by 15.18% and 19.29%. This indicates that selenium-rich algae treatment has a positive effect on lettuce growth and does not produce adverse effects. Phenotypic observation and dry weight show that cadmium stress has an inhibitory effect on lettuce growth, and the addition of selenium-rich algae can alleviate the inhibitory effect of cadmium stress on lettuce growth to varying degrees.

[0130] Figure 12A is the cadmium content of the above-ground part of the treated lettuce on the 30th day. Compared with the cadmium treatment, the cadmium content of the 3 mg / L cadmium + algae, 3 mg / L cadmium + 1 mg / L selenium-rich algae, and 3 mg / L cadmium + 5 mg / L selenium-rich algae treatments decreased by 7.06%, 14.04%, and 18.74%, respectively, all with significant differences. Figure 12 B is the selenium content of the above-ground part of the treated lettuce on the 30th day. Similar to the results of selenium-rich microalgae cultivation in rice, compared with the blank control group, the selenium content of the lettuce cultivated with 5 mg / L selenium-rich algae was significantly enriched.

[0131] <Comparison experiment of different preparation methods of selenium-rich microalgae>

[0132] 1. Experimental method

[0133] The experimental method specifically includes the following steps:

[0134] Step 1: Experimental group setting

[0135] A total of three experimental groups are set up, as follows:

[0136] Experimental group (1):

[0137] The preparation of selenium-rich microalgae is as follows: select the algal liquid of the logarithmic growth phase of Polysiphonia fastigiata, adjust the initial OD 680 to 0.5, add 10 mg / mL sodium selenite solution, prepare an algal liquid with a sodium selenite concentration of 1 mg / L, cultivate for 4 days to obtain a cultured algal liquid, and separate the cultured algal liquid to obtain selenium-rich microalgae. The specific method for separating the cultured algal liquid to obtain selenium-rich microalgae includes the following steps:

[0138] S41, mix the compound foaming agent and trehalose at 45°C to form a micellar complex, then add it to the algal liquid after 4 days of cultivation. The compound foaming agent is a compound foaming agent composed of sophorolipid and rhamnolipid at a mass ratio of 4:1. The addition amount of the compound foaming agent is 0.002% w / v, and the addition amount of trehalose is 0.01% w / v.

[0139] S42, introduce low-pressure microbubbles. The parameters of the low-pressure microbubbles are as follows: pressure 0.08 MPa, microbubble diameter 100 μm, gas flow rate 0.2 L / min, and duration 10 minutes. The algal cells are enriched in the foam layer.

[0140] S43, collect the foam layer, rinse the foam with an isotonic solution containing 1.0% NaCl and 0.01% trehalose by mass fraction, and obtain the selenium-rich microalgae.

[0141] Experimental group (2):

[0142] The preparation of selenium-rich microalgae includes the following steps:

[0143] S1, preparation of mesoporous silica carrier, specifically comprising the following steps:

[0144] S11, vacuum drying the mesoporous silica at 150°C for 2 hours to remove the adsorbed water in the pores, to obtain pretreated mesoporous silica;

[0145] S12, dispersing the pretreated mesoporous silica in a 0.2 mol / L FeCl3 solution prepared by 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;

[0146] S13, maintaining an inert atmosphere, adding 1 mol / L NaBH4 solution dropwise to the reaction solution in S12, the dropwise rate is 2 mL / min, and the molar ratio of NaBH4 to Fe 3+ is 4:1, the reaction temperature is 30°C, and the stirring is continued until no bubbles are generated;

[0147] S14, after the reaction is completed, washing the precipitate by centrifugation with deoxygenated ethanol for 3 times, the centrifugation parameters are 8000 rpm and 10 min, and then placing the precipitate in a vacuum dryer at 40°C for 12 hours, to obtain a mesoporous silica carrier loaded with nano zero-valent iron.

[0148] S2, taking the algal liquid of the polyergonum in the logarithmic growth phase, adjusting the OD 680 to 0.5, adding 10 mg / mL sodium selenite solution to a final concentration of 1 mg / L, and culturing under light for 4 days, and collecting the microalgae by centrifugation;

[0149] S3, mixing the microalgae obtained in step S2 with the mesoporous silica carrier obtained in step S1 at a mass ratio of 8:1, adding deionized water, and oscillating the reaction under light-free conditions at 25°C for 30 minutes, to obtain the culture algal liquid.

[0150] S4, placing the culture algal liquid in a centrifuge at 1000 rpm for 5 min, and collecting the precipitate to obtain the selenium-rich microalgae.

[0151] Experimental group (3):

[0152] The selenium-rich microalgae are prepared as follows: selecting the algal liquid of the polyergonum in the logarithmic growth phase, adjusting the initial OD 680 to 0.5, adding 10 mg / mL sodium selenite solution to prepare an algal liquid with a sodium selenite concentration of 1 mg / L, culturing for 4 days, and collecting the precipitate after centrifugation at 1000 rpm for 5 min to obtain the selenium-rich microalgae.

[0153] Step two, index determination

[0154] 1) Determination of the viable cell rate and cell debris rate of selenium-rich microalgae

[0155] The method is FDA-PI double staining fluorescence microscopy counting (ISO 20688:2018).

[0156] Determine the viable cell rate and cell debris rate of selenium-rich microalgae separated from experimental group (1) and experimental group (3). The specific results are shown in Table 1.

[0157] 2) Determination of total selenium retention rate in selenium-rich microalgae

[0158] Method: GB 5009.93-2017 (ICP-MS).

[0159] Determine the total selenium retention rate in selenium-rich microalgae separated from experimental group (1) and experimental group (3). The specific results are shown in Table 2.

[0160] 3) Determination of cadmium reduction and selenium enrichment effect

[0161] Method: same as the experimental method in <Effect of selenium-rich microalgae in cadmium stress environment on rice growth and cadmium reduction and selenium enrichment>. Among them:

[0162] The treatment method of experimental group (1) is: 3mg / L cadmium + 1mg / L selenium-rich microalgae solution prepared by experimental group (1);

[0163] The treatment method of experimental group (2) is: 3mg / L cadmium + 1mg / L selenium-rich microalgae solution prepared by experimental group (2);

[0164] The treatment method of experimental group (3) is: 3mg / L cadmium + 1mg / L selenium-rich microalgae solution prepared by experimental group (3).

[0165] Determine the total cadmium content and total selenium content of the aboveground part of the rice harvested from experimental group (1), experimental group (2), and experimental group (3). The specific results are shown in Figure 13~Figure 14 .

[0166] 2, Analysis of experimental results

[0167] 1) Determination results of the viable cell rate and cell debris rate of selenium-rich microalgae

[0168] Table 1 Determination results of the viable cell rate and cell debris rate of selenium-rich microalgae obtained by the separation method of experimental group (1) and experimental group (3)

[0169] Experimental Example Viable cell rate (%) Cell debris rate (%) Experimental Group (1) 98.2 0.8 Experimental Group (3) 80.3 19.7

[0170] 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 98.2%, indicating that the low-pressure microbubble combined with trehalose method can effectively avoid mechanical damage.

[0171] 2) Results of total selenium retention rate determination in selenium-enriched microalgae

[0172] Table 2. Results of total selenium retention rate in selenium-enriched microalgae obtained by the separation methods in experimental groups (1) and (3).

[0173] Experimental Example Total selenium retention rate (%) Experimental Group (1) 92.5 Experimental Group (3) 75.8

[0174] As can be seen from Table 2, compared with the centrifugation method of experimental group (3), the separation method of experimental group (1) obtained microalgae with a total selenium retention rate of up to 92.5%, indicating that the low-pressure microbubbles combined with trehalose can effectively maintain the stability of intracellular selenium morphology, thereby making selenium well enriched in microalgae.

[0175] 3) Cadmium-reducing and selenium-enriching effects

[0176] from Figure 13~Figure 14 As can be seen from the results, compared with the centrifugation 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 mesoporous silica can reduce cadmium ions in the cultivation substrate, thus achieving the effect of reducing cadmium. Moreover, mesoporous silica can adsorb free cadmium and selenium, reducing biological toxicity. In addition, the combination of microalgae and mesoporous silica carrier allows the carrier to adsorb cadmium ions in the cultivation substrate, protecting algal cells. At the same time, microalgae can encapsulate the carrier, delaying the oxidation of nano-zero valent iron. The two complement each other and have a synergistic effect.

[0177] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their 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 polysacchariformis and sodium selenite; The conditioner is prepared through the following steps: S1, Disperse mesoporous silica in a Fe-containing medium 3+ In the solution, under the protection of an inert atmosphere, a reducing agent is added to carry out the reaction to obtain a mesoporous silica support; S2. Take the algal solution of *Scenedesmus multipinnatifida* in the logarithmic growth phase and adjust the OD... 680 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. Mix 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, add deionized water, and shake and react for 30 minutes at 25°C in the dark to obtain the culture algae solution. S4. Separate the cultured algae solution to obtain selenium-enriched microalgae, mix the selenium-enriched microalgae with deionized water to obtain the selenium-enriched microalgae solution, which is the conditioner; Step S4 specifically includes the following steps: S41. Add 0.0015%-0.0025% w / v of food-grade foaming agent and 0.01% w / v of trehalose to the cultured algae solution; the food-grade foaming agent is a composite foaming agent composed of sophorolipid and rhamnolipin in a mass ratio of 3-5:

1. First, premix the composite foaming agent with oligo-trehalose at 40-45℃ to form a micelle complex, and then add it to the algae solution; S42. Introduce low-pressure microbubbles. The specific parameters of the low-pressure microbubbles are: pressure 0.05–0.1 MPa, microbubble diameter 50~200 μm, gas flow rate 0.1–0.3 L / min, for 5–10 minutes, so that algal cells are enriched in the foam layer. S43. Collect the foam layer and rinse the foam with an isotonic solution containing 0.8%-1.0% NaCl and 0.01% trehalose to obtain the selenium-enriched microalgae.

2. The cadmium-reducing and selenium-enriched crop cultivation conditioner as described in claim 1, characterized in that, Step S1 specifically includes the following steps: S11. Vacuum dry the mesoporous silica at 150°C for 2 hours to remove the adsorbed water in the pores and obtain the pretreated mesoporous silica. S12. Disperse the pretreated mesoporous silica in a 0.1–0.3 mol / L FeCl3 solution prepared with deoxygenated deionized water at a ratio of 1:10–1:20 w / v, and stir at 200–400 rpm for 1–2 hours under nitrogen / argon protection to obtain the 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 dropping rate of 1-2 mL / min, controlling the reaction between NaBH4 and Fe. 3+ The molar ratio is 3:1-5:1, the reaction temperature is 25-35°C, and the mixture is stirred continuously until no more bubbles are generated. S14. After the reaction is complete, the sample is washed three times by centrifugation with deoxyethanol. The precipitate is collected and centrifuged at 8000 rpm for 10 min. The precipitate is then placed in a vacuum dryer at 40°C for 12 hours to obtain the mesoporous silica support.

3. A cultivation substrate, characterized in that, The cultivation substrate includes the cadmium-reducing and selenium-enriched crop cultivation conditioner as described in any one of claims 1 to 2, wherein the cultivation substrate is in the form of cultivation liquid or cultivation soil.

4. The cultivation substrate as described in claim 3, characterized in that, The cultivation substrate is cultivation soil, which includes substrate soil, water, and selenium-enriched microalgae solution. The cultivation soil is prepared by mixing the selenium-enriched microalgae solution with water evenly and then mixing it with the substrate soil evenly. The mass fraction of the selenium-enriched microalgae solution in the cultivation soil is 5-10%.

5. The application of the cadmium-reducing and selenium-enriched crop cultivation conditioner as described in any one of claims 1 to 2 in the cultivation of cadmium-reducing and selenium-enriched crops.

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