Research On A Selenite-reducing Bacterium Stenotrophomonas Sp. EGS12

Remediation of selenium contamination is one of environmental protection fields,and technologies are mainly including physical methods,chemical reduction and bioremediation.Bio-reduction of toxic selenium oxides to low-toxic elemental selenium by microorganisms is one of methods in bioremediation of selenite and selenate.As the heavy metals and antibiotics included in Se-contaminated environment,the applicated strains concluded in bioremediation are called for the ability to withstand adverse conditions and the environmental adaptability,these characteristics are not achieved by most of studied selenite-reducing bacteria and they are therefore not the good candidates for bioremediation of Se-contaminated environment.Meanwhile,elemental selenium nanoparticles?SeNPs?are applicated widely in medicine,nanobiosensors,optoelectronics industry and environmental protection.Advantages of biogenic synthesis of SeNPs lie in the high reducing rate,high production,low cost and product SeNPs easy to purified.As mentioned above,it is necessary to screen a selenite reducing strain that has characteristics of high reducing rate,high resistant to selenite and broad resistant to heavy metals and antibiotics.With the Na₂SeO₃ complemented medium and low temperature culturing method,a selenite reducing strain named EGS12 that can tolerates 83-100 mmol/L of selenite was isolated from high-Se soil collected from Yutangba,Enshi city,Hubei province.This strain can reduce 39.2%2 mmol/L Na₂SeO₃ to red elemental selenium in 48 h.By morphological,physiochemical and 16S rDNA sequence alignment results,strain EGS12 is belonging to Proteobacteria,Gamaproteobacteria,Xanthomonadales,Xanthomonadaceae,Stenotrophomonas,and systematic name is Stenotrophomonas sp.EGS12.The elemental selenium nanoparticles reduced from selenite by strain EGS12 and sized 100-200 nm was confirmed by transmission electron microscopy and Energy Dispersive X-ray Spectrometer?EDX?analysis.Stenotrophomonas sp.EGS12 exhibits innate resistant to multiple toxic metals,such as Pb?NO₃?₂,NiCl₂,CdCl₂,ZnSO₄,CuSO₄,CoCl₂,HgCl₂,AgNO₃,Fe₂?NO₃?₃,MnSO₄,as well as 60 mmol/L AsO₄^3-.Furthermore,this bacterium tolerates high levels?50-2000?g/mL?of various antimicrobial agents,tetracycline,ampicillin,erythromycin,chloramphenicol,kanamycin,gentamycin and spectinomycin.Optimum growth temperature and pH are 37?and 8.0.Stenotrophomonas sp.EGS12 can reduced 2 mmol/L selenite to red elemental selenium nanoparticles completely in 4 days,which is the top reducing rate in the present isolated selenite reducing strains.On the basis of above characteristics,we considered Stenotrophomonas sp.EGS12 as the ideal candidates for the bioremediation of Se-contaminated environment,and this strain has potential to be chosen for producing the selenium nanoparticles as its ability of biogenic synthesis of SeNPs.The conditions of selenite reduction were optimized by single-factor test and response surface methodology,and the optimum contions for Stenotrophomonas sp.EGS12 reducing selenite to elemental selenium are pH 9.0,temperature 35?and rotation rate 130 r/min.Results of response surface analysis showed that pH,temperature and rotation rate influence the selenite reducing rate independently,and the interactions of three factors were faintly.Therefore,the processing of selenite reduction by Stenotrophomonas sp.EGS12 can be controlled easily.Purification and composition analysis of red elemental SeNPs produced by Stenotrophomonas sp.EGS12 were conducted.SeNPs were spherical and well-distributed particles observed by scanning electron microscopy and the size was 100-200 nm,same as the observations of transmission electron microscopy.SeNPs were composed of five elements,C,Se,O,P and S as analyzed by EDX,and the content of Se was 32.64%.The improvement of purity of SeNPs is necessary while Stenotrophomonas sp.EGS12 is used to producing SeNPs.Mechanisms of selenite reduction in Stenotrophomonas sp.EGS12 were supposed by the experimental results.The selenite reducing site in bacterial cell was located in the cytoplasm as cell protein fractioned experiment and in vivo activity tests.Na₂SeO₃ was transported into the cell by nitrate transporter,because the reduction was inhibited completely by nitrate transporter inhibitor 2,4-dinitrophenol.In biochemical analysis for selenite reductases seeking,glutathione and KNO₃ can increase the reducing rate,and glutathione inhibitor BSO and nitrate reductase inhibitor tungsten can inhibit the reduction significantly.These phenomenon indicate that the reduction of selenite in strain EGS12 was conducted by thiol-mediated reductases and nitrate reductase.Amplification products of several thiol-mediated reductases genes from genome of Stenotrophomonas sp.EGS12sustained the above hypothesis partly.In addition,genes coded for efflux pump and multidrug transporter were also amplified and they are suspected to be responsible for broad spectrum of heavy metal tolerance and antibiotics resistance.