Study On The Stress Mechanism Of Silver Nanoparticles To Culturability And Cell Viability Of Escherichia Coli

Nanomaterials have been commonly used and released into environment through various ways.Toxic effects caused by nanomaterials have become a focus of attention.Traditional methods for assessing toxicity of nanomaterial were confined to culturable cells,which were unsuitable for cells under viable but non-culturable state?VBNC?.As a typical antibacterial agent,whether silver nanoparticles?AgNPs?could induce cells into VBNC state is still unknown.In addition,researchers have not reached a consensus on the toxic mechanism of AgNPs so far.Most of them make a point that the toxicity is mainly attributed to the release of Ag⁺,while a few people think it related to reactive oxygen species?ROS?.It indicates that further research on toxic mechanism is essential.Therefore,model bacteria Escherichia coli K12 were selected to study toxic effect of AgNPs.Growth assessment,living/dead assessment and cell metabolism assessment were carried out to study the toxic effects of AgNPs.Mechanism was investigated from three aspects:fate of AgNPs,ion release,and intracellular ROS accumulation.Further analysis was conducted by adding antidotes of AgNPs.Main conclusions are as follows:?1?Effect of light and Cl^-concentration on the microbial toxicity of AgNPs.Results of growth curves showed that toxicity of AgNPs increased when NaCl concentration from 10 to50 mM but decreased from 150 to 250 mM.However,data of plate count and growth curve were even different.The number of culturable cells of E.coli K12 with treatment was much smaller than that of CK,but light and high concentration of NaCl increased the number of culturable cells.In addition,SYTO9/PI fluorescent staining method was used to verify the cell viability.The majority of cells were living cells with green fluorescence and maintained cell membrane integrity,indicating that E.coli K12 entered into VBNC state.The detection of ATP production confirmed that cells were still metabolically active.Therefore,AgNPs induced E.coli K12 into the VBNC state,which mediated by light and high concentration of NaCl.?2?The fate of AgNPs influenced its antibacterial toxicity.NaCl treatment made the contact angle of AgNPs decreased,resulting easy contact with hydrophilic surface of cells for higher toxicity.The stability and aggregation degree of AgNPs affected their toxicity.Maximum zeta potential appeared at 50 mM NaCl,suggesting the best dispersibility and solubility of AgNPs.In addition,AgNPs tended to aggregate,and the aggregation degree of AgNPs in dark conditions was higher than that under light irradiation.Finally,the chlorination production of AgNPs was a series of AgCl¹-x-x _x?aq?rather than AgCl_?s?,leading to higher AgNPs toxicity.?3?AgNPs stress mechanism was closely related to both ion release and accumulation of intracellular ROS.The increase of NaCl concentration facilitated the dissolution of AgNPs and light irradiation could slightly promote the oxidative dissolution.Besides,AgNPs treatment significantly increased the intracellular ROS level of E.coli K12,while exogenous addition of NaCl could reduce intracellular ROS production.?4?The dissolved silver and subsequent induced intracellular ROS contributed to AgNPs stress.Ag⁺chelating agents?Na₂S₂O₃ and Na₂S?and ROS scavengers?Vit C and rGSH?not only removed the stress of AgNPs towards E.coli K12,but also reduced intracellular ROS production to CK levels.