Mechanisms Of Simultaneous Reduction Of Metalloids Selenite And Tellurite In Bacillus Sp.Y3

The metalloids selenium(Se)and tellurium(Te)both belong to Group 16 of the periodic table.They have been a threat to human health for a long time.Due to their high photoconductivity and thermal conductivities,the demand for Se and Te has increased in recent years.Se is an essential element for humans and animals when taken in small amounts of up to 40??g/day,while it is toxic in excessive amounts(>400??g/day).Te is toxic to all microorganisms,even in small amounts as low as 1?g/m L.Se and Te have high redox activities in the environment and exist in different oxidation states.Microbial reduction of selenite[Se(?)]and tellurite[Te(?)]to Se(0)and Te(0),respectively,is a detoxified mechanism and such process provides potential application in environmental metalloid bioremediation.To study the microbial Se(?)and Te(?)reduction mechanisms,different Se(?)and Te(?)resistant bacterial strains were isolated from the wastewater of a pig farm.Bacillus spp.,Sphigonomas spp,Enterobacter spp.and Methylobacterium spp.were the major Se(?)and Te(?)resistant and reduction bacteria.After comparing their growth and reduction ability a bacterial isolate Y3 was further selected and identified by 16S r RNA gene analysis,genomic analysis,biophysical and biochemical tests and fatty acid analysis.Bacillus sp.Y3 was strictly aerobic,has rod-shaped cells and colonies on R2A plates are white,round,smooth,moist.The strain can grow at 4–50°C and p H 5–9 with an optimum growth temperature of 28°C and p H were 7.0,respectively.Strain Y3 had the highest 16S r RNA gene similarities with type strains of Bacillus altitudinis DSM 21631^T(99.2%)and Bacillus xiamenensis HYC-10^T(99.1%).The phylogenetic results based on genomic data was similar to that of 16S r RNA gene.Biophysical and biochemical tests and fatty acid analysis showed that strain Y3 had certain differences compared to B.altitudinis DSM 21631^T(99.2%)and B.xiamenensis HYC-10^T.However,after these comparison analyses,we still cannot distinguish strain Y3 into specie level.Thus,we named strain Y3 as Bacillus sp.Y3.Bacillus sp.Y3 had a high tolerance with minimum inhibitory concentrations(MICs)of250 m M for Se(?)and 0.7 m M for Te(?)respectively.The bacterium Y3 had almost the same reduction efficiencies for Se(?)and Te(?)individually or combined,and the reduction products were Se(0)and Te(0),respectively.During aerobic cultivation this bacterium showed a high reduction efficiency of 100%and 90%for Se(?)and Te(?),respectively.Moreover,when the Bacillus sp.Y3 was cultured in the presence of Se(?)and Te(?)together it reduced both metalloids at the same time.The SEM or Ultra-thin TEM analysis showed the metalloids reduction products were Se(0)nano particles(Bio-Se NPs)or Te(0)nano-rods,for the reduction of Se(?)and Te(?),respectively.The Se(0)nano-particles were found on the bacterial surface or outside of the bacterial cells,while the production of Te nano rods were inside the bacterial cells.Proteomic and genomics analysis revealed that the putative thioredoxin disulfide reductase(Trx R)had an increased expression level of 18.2-fold,compared with the strain without the addition of the metalloids and sulfate metabolic and energy generation pathway proteins were also significantly upregulated after the addition of Se(?)and Te(?).q RT-PCR also showed increased Trx R transcription levels of 15.8-fold,in the presence of Se(?)and Te(?).Compared to a wild-type E.coli strain,the Trx R-overexpressed E.coli strain showed higher resistance levels and reduction efficiencies.Additionally,the Trx R protein showed in vitro Se(?)and Te(?)reduction activities when NADPH or NADH was present.NADPH was a more appropriate electron donor compared to NADH.When NADPH was used as the electron donor,the optimum conditions for enzyme activity assay were p H 8.0and a temperature of 37 ^oC.The Km values of Te(?)and Se(?)reductions were 16.31 m M and 2.91 m M,respectively,and the Vmax values of Te(?)and Se(?)reductions were 12.23?M min^-1 mg^-1 and 11.20?M min^-1 mg^-1,respectively.In order to further investigate of application potential of the Bio-Se NPs produced by strain Y3,the Bio-Se NPs were extracted and purified from the strain Y3 cells cultivated with Se(VI).The Scanning Electron Microscope(SEM),X Ray Diffraction(XRD),Fourier Transform Infrared Spectroscopy(FTIR)and Dynamic Light Scattering(DLS)analysis showed that the Bio-Se NPs are round shaped and have a diameter from 100-274 nm and are composed of Se(0)nano-particles and other organic compounds.Later,the Bio-Se NPs were applied to test its adsorption abilities of different dyes.It showed that the Bio-Se NPs produced by strain Y3 could efficiently adsorb and remove three types of dyes congo red,methylene blue and safranine T.Further it was observed that the Bio-Se NPs changed the polarity under acidic and basic conditions,under acidic conditions they had a positive charge and could adsorb cationic dyes such as congo red,while under basic conditions they had a negative charge and could adsorb positively charged dyes such as methylene blue and safranine T.Adsorption kinetics,isothermal adsorption and adsorption thermodynamics analyses revealed the maximum adsorption capacity of the Bio-Se NPs for congo red,methylene blue and safranine T were 1607.5 mg/g,1640.9 mg/g and 2053.1 mg/g,respectively.In conclusion,the results discovered a new Bacillus strain with the simultaneously Se(?)and Te(?)reduction ability.The results of genomics,proteomics,q RT-PCR,gene expression analysis and enzymatical analysis showed that the Se(?)and Te(?)reductions of strain Y3are related to the sulfate reduction pathway and that thioredoxin-disulfide reductase Trx R is the new found enzyme associated with bacterial Te(?)resistance and reduction using NADPH as the electronic donor.The discovery of Trx R enriches the bacterial Se(?)and Te(?)resistance and reduction mechanisms.In application part,the Se(?)reduction produce Bio-Se NPs from Bacillus sp.Y3 could efficiently adsorb different dyes.Based on the results of their study,Bacillus sp.Y3 also has the potential for bioremediation of selenium and tellurium contamination.