Research On The Cytotoxicity Mechanism Of Nanoscale Zero-valent Iron To E.coli

Nanoscale zero-valent iron(nZVI)is a Fe0 particle with the size of 1-100 nm.which has the characteristics of large specific surface area and strong reducibility.In recent decades,nZVI has been intensively studied worldwide for the removal of heavy metals,chlorinated organics and other pollutants in soil and groundwater.On the other hand,with the small size and strong reducibility of nZVI,it may be potentially toxic to surrounding organisms.Microorganisms are the decomposers of the ecosystem,and play important roles in the ecological circulation of elements and energy.Literatures have reported the toxicity of nZVI to microorganisms like bacteria,but the exact bactericidal mechanism of nZVI is still under debate at this moment.Previous toxicology studies are mostly focused on the impact of nZVI size,surface modification and doses,and there is still a knowledge gap regarding the detailed interaction between nZVI and microbial cells.Therefore,in our study,we aim to comprehensively study the key mechanism of the nZVI toxicity toward bacteria from both extracellular and intracellular views,by selecting E.coli as the model organism,besides,parental and isogenic mutant strains of E.coli were adopted to reveal the role of target cellular component or function amid the nZVI toxicity.We first investigate the extracellular bactericidal pathway of nZVI.which is mainly caused by the physical adsorption to interrupt cell membrane,and oxidative damage of the cell through activating oxygen molecules to produce reactive oxygen species(ROSs).Extracellular polymeric substances(EPS)are important macromolecular polymers wrapped the bacterial cells.As a barrier between nZVI and cells,its potential impacts on extracellular toxicity of nZVI cannot be ignored but remained unexplored.We employed parental strains E.coli BW251 13 and mutant strains with the EPS secretion-related.genes knockout,i.e.,E.coli JW2034(less EPS content)and E.coli JW5917(higher EPS content),for comparison.We found that the E.coli strains with higher EPS contents had lower inactivation efficiency under nZVI exposure,and the inactivation efficiency ranked as JW5917<BW25113<JW2034,indicating that EPS could improve the resistance of E.coli to nZVI toxicity.To reveal the underlying mechanism,we investigated the effects of EPS on ROSs-induced oxidative damage and membrane physical damage,which are the main bactericidal pathways of nZVI.Interestingly,the impact of EPS on the toxicity of nZVI are complicated.On one hand,EPS could scavenge ROSs produced by the oxygenation of nZVI,thus the strain JW5917 with higher EPS contents had a significantly stronger quenching effect on ROSs than the strain JW2034 with lower EPS contents.Meanwhile,strain JW5917 had the most significant effect on accelerating the corrosion of nZVI into low-toxic products,which reduced the toxicity of nZVI.On the other hand,it is a surprise to find that EPS accelerated the physical damage of cell membrane,since strain JW5917 showed stronger adsorption of nZVI.and the membrane damage was more serious than other strains.However,membrane damage was not lethal to E.coli JW5917 with higher EPS contents,because cells could get repaired by themselves.Therefore,the influence of EPS on nZVI toxicity is not a straightforward mechanism,but the overall effect of various impacts.and it is concluded that the effect of EPS on ROSs quenching surpass its role in prompting the adsorption of nZVI.Secondly,the intracellular toxicity of nZVI could be related to the intracellular ROSs derivative processes mediated by iron uptake of bacterial cells,but there is no direct evidence available yet in previous studies.Herein,we employed E.coli mutant strains with iron-uptake-related-gene knockout and parental strain for control to study the role of iron uptake.It was demonstrated that mutant strains with excessive iron uptake or intracellular iron chelating protein deficiency were more easily inactivated by nZVI,and strains with DNA oxidative damage repair defects are also more sensitive to nZVI,indicating that the intracellular ROSs reactions induced by iron uptake is an important pathway of intracellular toxicity of nZVI.The Calcein-AM method was used to find the evidence of excessive uptake of ferrous ions by cells.Moreover,the production of intracellular ROSs is directly related to the uptake of ferrous ions under aerobic and anaerobic conditions.We also found that the iron uptake of cells is not only related to the iron dissolution level of nZVI,but more importantly,the damage of the cell membrane caused by adsorption of nZVI particles lead to the increase of the permeability of the cell membrane,thus accelerating the uptake of ferrous ions.It also explains why the bactericidal activity of nZVI is stronger under the anaerobic conditions than that under aerobic conditions,since membrane damage was more serious under anaerobic conditions,leading to more intracellular iron uptake.which has always been ignored in previous studies.Moreover,we chelated the intracellular ferrous ions with 2,2'-bipyridine(BIP)to block the generation of intracellular ROSs.and detected the inactivation efficiency of E.coli,to quantify the contribution of iron uptake induced intracellular ROSs to overall bacterial inactivation.It was found that the contribution of intracellular ROSs to bacterial inactivation was 30?70%and 40?70%respectively under aerobic and anaerobic conditions,indicating that the pathway of cellular damage played an important role in the inactivation of E.coli by nZVI.These findings will contribute to our in-depth understanding of the bactericidal mechanism of nZVI and provide important reference for the assessment of the ecological safety of nZVI deployment amid pollution remediation.