Subcellular mechanism of microbial inactivation during water disinfection by cold atmospheric-pressure plasma

Although the identification of effective reactive oxygen species (ROS) generated by plasma has been extensively studied, yet the subcellular mechanism of microbial inactivation has never been clearly elucidated in plasma disinfection processes. In this study, subcellular mechanism of yeast cell inactivation during plasma-liquid interaction was revealed in terms of comprehensive factors including cell morphology, membrane permeability, lipid peroxidation, membrane potential, intracellular redox homeostasis (intracellular ROS and H2O2, and antioxidant system (SOD, CAT and GSH)), intracellular ionic equilibrium (intracellular H+ and K+) and energy metabolism (mitochondrial membrane potential, intracellular Ca2+ and ATP level). The ROS analysis show that ·OH, 1O2, ·O2−and H2O2 were generated in this plasma-liquid interaction system and ·O2−served as the precursor of 1O2. Additionally, the solution pH was reduced. Plasma can effectively inactivate yeast cells mainly via apoptosis by damaging cell membrane, intracellular redox and ion homeostasis and energy metabolism as well as causing DNA fragmentation. ROS scavengers (l-His, d-Man and SOD) and pH buffer (phosphate buffer solution, PBS) were employed to investigate the role of five antimicrobial factors (·OH, 1O2, ·O2−, H2O2 and low pH) in plasma sterilization. Results show that they have different influences on the aforementioned cell physiological activities. The ·OH and 1O2 contributed most to the yeast inactivation. The ·OH mainly attacked cell membrane and increased cell membrane permeability. The disturb of cell energy metabolism was mainly attributed to 1O2. The damage of cell membrane as well as extracellular low pH could break the intracellular ionic equilibrium and further reduce cell membrane potential. The remarkable increase of intracellular H2O2 was mainly due to the influx of extracellular H2O2 via destroyed cell membrane, which played a little role in yeast inactivation during 10-min plasma treatment. These findings provide comprehensive insights into the antimicrobial mechanism of plasma, which can promote the development of plasma as an alternative water disinfection strategy.