Adaptations of Rhodococcus rhodochrous Biofilms to Oxidative Stress Induced by Copper(II) Oxide Nanoparticles

Copper(II) oxide nanoparticles (CuO NPs) are used in different industries and agriculture, thus leading to their release to the environment, which raises concerns about their ecotoxicity and biosafety. The main toxicity mechanism of nanometals is oxidative stress as a result of the formation of reactive oxygen species caused by metal ions released from nanoparticles. Bacterial biofilms are more resistant to physical and chemical factors than are planktonic cells due to the extracellular polymeric matrix (EPM), which performs a protective function. Hydrocarbon-oxidizing bacteria of the genus Rhodococcus, well-known biodegraders of toxic organic pollutants and bioremediation agents, are capable of producing biofilms, which, as we proposed, are more resistant to metal nanoparticles, while the particular adaptation mechanisms have not yet been clarified. In this study, we study the adaptation mechanisms of Rhodococcus rhodochrous IEGM 1363 biofilms to CuO NPs in a wide range of concentrations (0.001-0.1 g/L), including morphological and ultrastructural cell alterations. The results obtained on the long-term dynamics (≤72 h) and localization of EPM structural components, in particular, lipids, polysaccharides, and proteins, indicated their important role in the complex adaptive response of alkanotrophic Rhodococcus to oxidative stress caused by copper nanooxide. The observed changes in the ultrastructure and element composition included binding of CuO nanoparticles by the cell wall to prevent their penetration inside cells and intracellular accumulation of potassium, magnesium, phosphorus, and sulfur in electron-dense inclusions, which may be associated with a metabolic stress reaction. Understanding the mechanisms of interaction between nanometals and Rhodococcus biofilms will contribute to the development of biocatalysts based on immobilized bacterial cells and bioremediation methods.