| Selenium(Se),as an essential micronutrient,has important physiological functions for humans and animals.Th dietary guidelines for Chinese residents recommended that the intake of Se is about 50?g·d-1for an adult.More than 65%of agricultural land belongs to low-selenium soil in China,so it is difficult to achieve the recommended intake of Se by eating natural crops with low Se content for human.Therefore,organic Se-enriched crops cultivated by artificial Se application have been become the main source of human dietary selenium supplement.However,a small amount of exogenous inorganic Se was used by plants during the cultivation process of Se-enriched crops.Long-term accumulation of unused Se polluted the soil and groundwater environment,and it also polluted surface water sources and poisoned aquatic animals with rainwater.In addition,residual inorganic Se on the surface of crops would cause the toxicity to human through the food chains.Therefore,it is necessary to explore how to reduce the biological toxicity and environmental risks caused by the application of inorganic Se fertilizer.In this study,the biomass of Se-enriched algae was used as a raw material to prepare the fertilizer of organic Se containing amino acid(SAF).Then SAF was applied to leek seedlings instead of inorganic Se fertilizer(SF),in order to efficiently produce organic Se-enriched products and reduce environmental risks.In the first stage,the effects of exogenous inorganic Se on the growth of what and Se enrichment ability of C.pyrenoidosa were explored.The feasibility and optimal cultivation conditions of C.pyrenoidosa as the bio-carrier for enriching organic Se were determined.Then the biomass of Se-enriched algae was harvested by the green and efficient composite flocculant,and the obtained biomass was prepared as the SAF.In the second stage,the effects of SAF application on the growth,physiological characteristics,Se content,heavy metals contents of leek seedlings were explored and the Se content and fractions in soil environment after SAF application were also explored.The main results are shown as follows:(1)Sodium selenite had dual influences on the growth of C.pyrenoidosa during different growth phases(exponential phase,late exponential phase and early stationary phase).The cells of C.pyrenoidosa in exponential phase could be exposed to high doses of Se,which should be used as the bio-carrier for enriching organic Se.The EbC50 value for selenite in C.pyrenoidosa cultures cultivated for 72h and 96h were 15.37mg·L-1 and 12.01 mg·L-1,respectively.The low concentrations of selenite(?5 mg·L-1)could promote the accumulation of algae biomass by the increasing of the cell antioxidant enzyme activity and inhibition of the lipid peroxidation.However,the accumulation of excessive Se in cells damaged the chloroplast structure and produced oxidative when cells were exposed to higher concentration of selenite(>5 mg·L-1),which inhibited the growth of C.pyrenoidosa.Furthermore,the decrease of intracellular organic Se content under higher Se concentration.Therefore,the optimal cultivation conditions for enriching selenium in C.pyrenoidosa in exponential phase were determined as 2 mg·L-1 sodium selenite and 5-day cultivation.The intracellular total Se content was 92.72±6.43?g·g-1 DW in which 78%of total Se existed as organic Se.(2)Clay minerals were modified with acidic chitosan in order to improve the flocculation performance of clay minerals for flocculating Se-enriched algae.The flocculation efficiency(FE)of modified composite flocculants(CMB,CMD and CMZ)was improved by 45-50%compared to that of unmodified clay minerals(bentonite,diatomite and zeolite).The results showed that the surface of clay minerals without modification have negatively charged,whereas the surface of modified composite flocculants have positively charged.The variation of electrical property reduced the repulsive force between the flocculant and the negatively-charged algal cells,which made algal cells more easily destabilized.Moreover,the surfaces of composite flocculants contained many cavities,which favours the adsorption of algal cells.Among the composite flocculants,CMD showed the highest FE(96%)under the conditions of the mass ratio set as 1:6(chitosan:diatomite),the dosage set as 0.2 g·L-1,the p H set as 8.2 and the sedimentation time set as 120 min.(3)The optimal hydrolysis conditions for preparing SAF were determined as solid-liquid ratio of 1:5,acid concentration of 5 mol·L-1 and reaction time of 12 h.Under this condition,the hydrolysis rate of protein in algae reached 63%.Moreover,the optimal conditions for preparing another organic Se-fertilizer(amino acid chelated Se fertilizer,ASF)were determined as the chelating reaction temperature of 60?,the p H of6.1 and the reaction time of 30 min.Compared with the ASF,the SAF has higher amino acid content(4.76g·100 m L-1)and organic Se content(64.73%of total Se)with the same total Se content.Therefore,the application of SAF shows the better utilization value and prospect.(4)The SAF,SF and the amino acid fertilizer(AF)were respectively applied at roots or leaves of leek seedlings,in order to determine the effects of SAF on the growth and physiological characteristics of leek seedlings.The results showed that root application of low concentration SAF(Se?1 mg·kg-1,amino acid?5.54 g·kg-1)were more favorable for regulating the growth,increasing the chlorophyll content,the soluble sugar and vitamin C contents,enhancing the antioxidant property and improving nutritional value of leek seedlings.However,the root application of high concentration SAF,SF and AF damaged the structure of root cells and inhibited the transportation of nutrients and water from root to shoot.Furthermore,the accumulation of excessive Se in leaves affected the transfer of photosynthetic electrons and caused the disorder of carbohydrate metabolism of leek seedlings.(5)The application of Se fertilizers(SAF and SF)significantly enhanced the bioaccumulation of Se in leek seedlings.The leaves could promote the accumulation of Se in the shoot of leeks when foliar application of Se fertilizers.The root application of Se fertilizer could not only strengthen the Se accumulation in root of leeks,but also improved the organic Se content in shoot of leeks.The results showed that organic Se(SAF)might be preferentially absorbed and accumulated by leeks compared with inorganic Se(SF).Therefore,the root application of SAF instead of SF was more conducive to the organic Se content(62.46-96.07%of the total Se)in shoot of leeks.Moreover,the contents of organic matter-bound Se(14.37-54.09%of total Se)in the soil significantly increased after root application of the SAF,which was beneficial for reducing the environmental risks caused by the loss of highly toxic soluble Se.(6)In the Cd,Cr and Pb co-contaminated soil,the biomass and chlorophyll content of leeks were significantly decreased compared with the blank control.However,the foliar application of SAF and SF(?1 mg·L-1)reduced the effects of heavy metal stress on the growth and chlorophyll synthesis of leeks through the antagonism between Se and heavy metals.At the same time,the contents of Cr,Cd and Pb in the shoot of leeks respectively decreased by 22.40-72.70%,28.93-84.78%and 20.17-91.82%after application of Se fertilizer.Comparing to SF,SAF treatment(1 mg·kg-1)showed the obvious antagonism accumulation of heavy metals in leeks.After the SAF treatment,the higher Se cocent in leeks affected the contents of heavy metals by changing the antioxidant property,and the application of nitrogen nutrition affected the absorption processes of nutrients and heavy metals from root to shoot of leeks.In addition,Se treatments(both SAF and SF)might change the characteristics of microbial communities in the rhizosphere soil environment by affecting the migration process of heavy metals from non-rhizosphere to rhizosphere,especially for the SAF treatment.After the SAF treatment,the enrichment of Bacteroidetes,Actinobacteria and Gemmatimonadetes were beneficial for adsorbing and complexing heavy metals in the soil and promoting the decomposition of soil organic matter,which could reduce the effects of heavy metal stress on the growth of leek seedings. |
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