Effect of nanoscale zero valent iron toward bacteria and their response

Nanoscale zero valent iron or nZVI is a reactive iron nanoparticle which has been considered as a promising treatment agent for various contaminants due to its small size and high reactivity. While the successful in-situ environmental application of nZVI has been demonstrated, the increasing use of nZVI possibly leads to the potential environmental impact of nZVI. This research aims to understand the effect of nZVI particularly on environmental bacteria. In this study, Pseudomonas putida were selected as the model bacteria as they are ubiquitous in the environment. Exposure of P. putida to 1.0 g/L of reactive nZVI (R-nZVI) decreased the bacterial viability by three order of magnitude. Bacterial exposure to oxidized nZVI (O-nZVI), a non-toxic form remained in the environment, resulted in one-order of magnitude reduction in cell viability. Proteomic analysis revealed the significant effect of both forms of nZVI on bacterial membrane as suggested by the decreased abundance of membrane-bound proteins and the up-regulation of proteins playing a role in membrane protein folding. Prolonged exposure in the presence of carbon source resulted in the rebound in number of viable cells, suggesting that bacterial cells can adapt themselves to the nZVI-induced damage. According to TEM analysis, nZVI heavily adsorbed onto the bacterial surface and partially localized around the bacterial membrane, fluidizing bacterial membrane. Fatty acid profile analysis showed the significant conversion of cis-isomer to trans-unsaturated fatty acid upon nZVI exposure. The altered membrane composition resulted in the tightly packed bilayer, and more rigid membrane as confirmed by fluorescent anisotropy measurement. It is likely that this membrane rigidification is a bacterial adaptive response to counteract the membrane fluidizing effect of nZVI. Interestingly, repetitive exposures of bacteria to an environmentally relevant concentration of R-nZVI (0.1 g/L) induced the emergence of the small colony variant (SCV) of P. putida exhibiting much smaller colony size and higher persistence to nZVI exposure. Single bacterial exposure to higher concentration of R-nZVI (i.e. 0.5 and 1.0 g/L) also increased the number of this SCV phenotype by approximately ten-fold. It appears that nZVI-induced oxidative stress involves in the emergent SCV phenotype. While most of the SCV phenotype could revert back to normal phenotype in the absence of nZVI, the irreversible SCV phenotype was also detected. Characterization of this irreversible SCV phenotype reveals partial loss of the environmentally relevant traits including swimming motility and biofilm formation. While P. putida F1 is a model strain for toluene degradation study, its irreversible SCV phenotype is slightly more susceptible to toluene and showed four-fold longer lag phase of growth under toluene as sole carbon source, compared to the normal phenotype. Overall, this study unveils the significant effect of nZVI on bacterial membrane (i.e. membrane fluidizing effect) as well as bacterial adaptation responses to the occurred damage.  It demonstrates that the bacterial adaptation should be considered for accurately predicting the toxicity of nZVI. The study on adaptability of other microorganisms is also required. Additionally, nZVI in-situ injection strategies (e.g. single and repetitive injection) should be taken into concern since it may induce the variation in bacterial phenotype.