Screening Of Selenium Synthetic Strain And Study On Mechanism And Application Of Selenium Nanoparticles

Selenite is extremely biotoxic,microorganisms capable of transforming toxic selenium oxyanions into non-toxic elemental selenium(Se0)may be considered as an ideal means for the production of selenium nanoparticles(SeNPs).Synthesizing nanoparticles by biological means,which has the advantages of nontoxicity,reproducibility in production,easy scaling-up,well-defined morphology,and uniform particle size,has become a new trend in nanoparticle production and have broad applications in nanotechnology,medical and environmental remedation.Researchers have successfully isolated microorganisms that can reduce sodium selenite to nano-selenium from soil or plant rhizosphere by enrichment culture,and have shown good application prospects.However,the screening of sodium selenite reducing microorganisms from insect gut has not been reported yet.In this study,methods of accumulation cultivation was used,and microorganisms from insect gut were screened in order to isolate new microorganisms with high reduction capacity of sodium selenite,and the mechanism and function of selenium production were studied.The results are as follows:(1)Screening and identification of sodium selenite-reducing microorganisms from insect gut.98 strains were isolated from the intestinal tract of Monochamus alternatus preserved in selenium-enriched soil.After ARADA classification,13 OTUs were obtained.Two strains with high tolerance to sodium selenite were screened out and identified by molecular biology and physiological biochemistry as:Proteus mirabilis YC801 and Alcaligenes faecalis Se03.It is also the first time that these two strains have the ability to red uce sodium selenite to prod uce nano-se lenium.(2)Study on the reduction characteristics of sodium selenite and the mechanism of SeNPs production by Proteus mirabilis YC801.The strain exhibiting high tolerance to selenite(up to 100 mM)was isolated and demonstrated efficient transformation selenite into SeNPs by reducing nearly 100%of 1.0 and 5.0 mM selenite within 42 and 48 h,respectively.Electron microscopy and energy dispersive X-ray analysis demonstrated that the SeNPs were spherical and localized both extracellularly and intracellular,with an average hydrodynamic diameter of 178.3±11.5 nm.In vitro selenite reduction activity assays and Real-time PCR indicated that thioredoxin reductase and similar proteins present in the cytoplasm were likely to be involved in selenite reduction,and that NADPH or NADH served as electron donors.The mRNA expression levels of gshB,B9475 02395,gor,trxA,and cysJ in cells cultured with selenite were not different from those in cells without selenite treatment.Finally,Fourier-transform infrared spectral analysis confirmed the presence of protein and lipid residues on the surfaces of SeNPs.(3)A bacterial strain exhibiting high selenite tolerance and reduction mechanism was isolated from the gut of Monochamus alternatus larvae and identified sas A.faecalis Se03.The isolate exhibited extreme tolerance to selenite(up to 120 mM)when grown aerobically.In the liquid culture medium,it was capable of reducing nearly 100%of 1.0 and 5.0 mM Na2SeO3 within 36 and 42 h,respectively,leading to the formation of selenium nanoparticles(SeNPs).Electron microscopy and energy dispersive X-ray analysis demonstrated that A.faecalis Se03 produced spherical electron-dense SeNPs with an average hydrodynamic diameter of 273.8±16.9 nm,localized in the extracellular and intracellular space.In vitro selenite reduction activity,selenite reduction activity in Se03 cells is localized in the cytoplasmic fraction,but not in the periplasmic,membrane,EPS,supernatant.Furthermore,selenite reduction by the cytoplasmic fraction is an enzymatic process since an electron donor(NADH or NADPH)is required for this reaction to take place.The real-time PCR indicated that proteins such as sulfite reductase and thioredoxin reductase present in the cytoplasm were likely to be involved in selenite reduction.Finally,using Fourier-transform infrared spectrometry,biomacromolecules were detected on the surface of the biogenic SeNPs.(4)The field experiment was carried out using nano-selenium obtained by separating microorganisms in the early stage.The effect of nano-selenium nutrient solution on crop yield and quality was further studied.The results of field experiment showed that the treatment of nano-selenium fertilizer increased the selenium content and further improved the nutritional quality of tomatoes.It is proved that nano-selenium synthesized by microorganisms can be absorbed and transformed by plants,and has the potential to develop into a selenium fortifier.