Biosynthesis And Characterization Of Silver Nanoparticles By Supernatant From The Arctic Bacterium Paracoccus Sp. Arc7-R13

Silver nanoparticles?AgNPs?are widely used in chemical sensors,catalysts,drug delivery and batteries due to its good electrical conductivity,antibacterial properties and optical properties.Compared with traditional chemical and physical synthesis approaches,biological methods have attracted more and more attentions due to their environmental protections,rapidity,and simple operation.The biosynthesis of silver nanoparticles requires the simultaneous action of reducing agents and protective agents.Meanwhile,the distinct environment such as high dissolved oxygen,ultraviolet radiation and low temperatures in the Arctic Ocean causes oxidative stress to be the main factor affecting the survival of microorganisms,indicating that there might exist various antioxidative strains.It is of great research value to synthesize silver nanoparticles by using the culture supernatant of antioxidative strains from the Arctic.In this paper,19 antioxidative strains were isolated from 30 Arctic Ocean sediments by using Zobell 2216E medium supplemented with 4 mmol/L H₂O₂ as the screening medium.Molecular identification and phylogenetic analysis were also carried out on the basis of 16S rRNA gene,the results showed that these isolates belonged to 4 classes,11 genera,and 12 species respectively:?-Proteobactria accunted for the major.At the genus level,it included Pseudoalteromonas,Colwellia,Psychrobacter,Paracoccus,Arthrobacter,Kocuria,Haloperacillus,Bacillus,Planomicrobium,and Chryseoglobus.The results of H₂O₂ resistance test showed that Arc7-R13 and Arc7-D had stronger H₂O₂ tolerance.The bacterial culture with diluting rate of 10³ and 10⁴ could grow on the plates with 5mmol/L H₂O₂.Complete genome sequencing and functional annotation were performed on the strain Arc7-R13 and Arc7-D.The whole genome of the strain Arc7-R13 includes one circular chromosome?3,231,929 bp?and eight plasmids?41,701-223,650 bp?,with an average G+C content of 67.45 mol%,and three 16S rRNA operons,49 tRNA genes and 16 gene islands.Among the COG annotations,50 genes were related to oxidative activity.289 genes affiliated with redox processes and another 78 genes associated with redox activity were identified by GO annotation.The complete genome of strain Arc7-D comprises of one circular chromosome?4,305,442 bp?,with an average G+C content of 37.67 mol%,consisiting six 16S rRNA operons?five as 16s-23s-5s and one as 16s-23s-5s-5s?,77 tRNA genes and 10 gene islands.There were 36 and 19 genes associated with antioxidative activity in the COG and GO annotations,respectively.By KEGG analysis,Arc7-R13 and Arc7-D have three metabolic pathways?peroxisome pathway,oxidative phosphorylation pathway and glutathione metabolic pathway?related to oxidative stress adaptation.The supernatant of 19 isolates were mixed with same value of AgNO₃ solution,and the mixtures were kept at 1200 Lx visible light illumination for 48 h.The formation of silver nanoparticles was confirmed by UV–Vis spectrum on the spectrophotometer?UV-vis?in range of 300–700 nm.The synthesized AgNPs by using supernatant of Paracoccus sp.showed a characteristic peak at approximate 420nm,and the color of the reaction liquid changed from light yellow to dark brown,which further confirmed the accumulation of AgNPs.The optimum final concentration of AgNO₃,temperature and pH for the AgNPs synthesis were 4 mmol/L,37°C and 7 respectively.The AgNPs synthesized by strain Arc7-R13 were spherical and ellipsoidal in shape observed with TEM.The size of AgNPs ranged from 2 nm to 20 nm with average size of 6.9 nm.The FTIR results showed that the aromatic compounds,alcohols,phenols and proteins might act as reducing and protedting agents in the formation of silver nanoparticles.The results of X-ray diffraction showed the AgNPs were face-centered cubic crystal.The synthesized AgNPs were tested for the antimicrobial activity by using standard agar-well diffusion method.The results showed that the AgNPs had inhibitory effects on the four strains tested,including Escherichia.coli,Staphylococcus.aureus,Pseudomonas.aeruginosa and Bacillus.subtilis.The MIC of AgNPs against the four test strains were also determined by gradient dilution methods,and the AgNPs concentrations were 60?g/mL,30?g/mL,40?g/mL,and 30?g/mL,respectively.This study successfully synthesized AgNPs using Arctic marine microbes,which can provide new ideas for the development and utilization of polar microbes as a special species.