Kinetic, metabolic and macromolecular response of bacteria to chronic nanoparticle exposure in continuous culture

Nanoparticles with antimicrobial properties are used in thousands of nano-enabled consumer products. Therefore, it is important to understand the response mechanisms of bacteria that are exposed to these nanoparticles at different conditions. Moreover, it is necessary to evaluate possible microbial adaptation mechanisms. In our study, Escherichia coli K-12 MG1655 (E. coli) were grown continuously in bioreactors at two specific growth rates (0.1 h^-1 and 0.2 h^-1) and then exposed to chronic concentrations of casein-coated silver nanoparticles (AgNPs) [1 mg L^-1] for about 180 generations. After initiating the injection of AgNPs, the results showed a change in growth kinetic parameters between non-exposed and exposed systems. Maximum yield (Y_max) decreased by 33%, while the maintenance coefficient (m_s) increased by 52%. This evidence was indicating the versatility of the culture to growth in the exposed conditions and even the ability to achieve a new stationary state. However, the adaptation was achieved at a metabolic cost. Comparing the concentration and composition of extra-cellular substances that were produced showed differences between the control and exposed conditions, and also between the exposed systems in the two growth conditions. In the AgNPs-exposed bioreactor (EB) growing at 0.1 h^-1, AgNPs-ES complexes showed that the ratio of the area representing β-sheets to the area representing α-helix proteins was 2.4, which implies the formation of a protein corona, while at an exposed growth rate of 0.2 h^-1 this ratio was <1, indicating no protein corona. Transcriptomic results showed gene regulation in response to AgNPs exposure as a function of the specific growth rate. Batch exposure tests using the resultant cultures for each condition showed a lower inhibitory effect for the AgNPs on EB at 0.1 h^-1 than on control bacteria (CB) at 0.1 h^-1 in terms of membrane permeation and reactive oxygen species generation. Overall, our study showed that culture growth conditions significantly affects bacterial response to nanoparticle exposure. Therefore, these growth parameters should be determined and reported when performing toxicological tests.