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Effect of specific growth rates on metabolic and macromolecular response of bacteria to chronic antimicrobial nanoparticle exposure are studied.

Continuous and batch reactors were used to assess the effect of the exposure of casein-coated silver nanoparticles (AgNPs) on Escherichia coli (E. coli). Additionally, E. coli membrane extracts, membrane permeability and Langmuir film balance assays were used to determine integrity and changes in lipid composition in response to AgNPs exposure. Results showed that batch conditions were not appropriate for the tests due to the production of exopolymeric substances (EPS) during the growth phase. After 5h of contact between AgNPs and the used growth media containing EPS, the nanoparticles increased in size from 86nm to 282nm reducing the stability and thus limiting cell-nanoparticle interactions. AgNPs reduced E. coli growth by 20% at 1mg/L, in terms of Optical Density 670 (OD670), while no effect was detected at 15mg/L. At 50mg/L of AgNPs was not possible to perform the test due to aggregation and sedimentation of the nanoparticles. Membrane extract assays showed that at 1mg/L AgNPs had a greater change in area (-4.4cm(2)) on bacteria compared to 15mg/L (-4.0cm(2)). This area increment suggested that membrane disruption caused by AgNPs had a stabilizing/rigidifying effect where the cells responded by shifting their lipid composition to more unsaturated lipids to counteract membrane rigidification. In chemostats, the constant inflow of fresh media and aeration resulted in less AgNPs aggregation, thus increased the AgNPs-bacteria interactions, in comparison to batch conditions. AgNPs at 1mg/L, 15mg/L, and 50mg/L inhibited the growth (OD670 reduction) by 0%, 11% and 16.3%, respectively. Membrane extracts exposed to 1mg/L, 15mg/L, and 50mg/L of AgNPs required greater changes in area by -0.5cm(2), 2.7cm(2) and 3.6cm(2), respectively, indicating that the bacterial membranes were disrupted and bacteria responded by synthesizing lipids that stabilize or strengthen membranes. This study showed that the chemostat is more appropriate for the testing of nanotoxicological effects when testing bacteria at growing conditions.

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