Bactericidal Activity of Bulk Nanobubbles through Active Oxygen Species Generation.

We investigated the bactericidal activity of bulk nanobubbles (NBs) using E. coli, a model bacterium. Bulk NBs were produced by forcing gas through a porous alumina membrane with an ordered arrangement of nanoscale straight holes in contact with water. NBs with different gas contents, including CO2, O2, and N2, were generated and evaluated for their bactericidal effects. The survival rate of E. coli was significantly reduced in a suspension of CO2-containing NB (CO2-NB water). The N2-NB water demonstrated a small amount of bactericidal behavior, but its impact was not as significant as that of CO2-NB water. When E. coli was retained in O2-NB water, the survival rate was even higher than that in pure water (PW). We investigated the generation of reactive oxygen species (ROS) in NB suspensions by electron spin resonance spectroscopy. The main ROS generated in the NB water were hydroxyl radicals and OH·, and the production of ROS was the strongest in CO2-NB water, which was consistent with the results of the bactericidal effect measurements. We assumed that NB mediated by ROS would exhibit bactericidal behavior and proposed a kinetic model to explain the retention time variation of the survival rate. The results calculated based on the proposed model matched closely with the experimental results.

Radical generation and bactericidal activity of nanobubbles produced by ultrasonic irradiation of carbonated water.

Our previous study showed that nanobubbles (NBs) encapsulating CO2 gas have bactericidal activity due to reactive oxygen species (ROS) (Yamaguchi et al., 2020). Here, we report that bulk NBs encapsulating CO2 can be efficiently generated by ultrasonically irradiating carbonated water using a piezoelectric transducer with a frequency of 1.7 MHz. The generated NBs were less than 100 nm in size and had a lifetime of 500 h. Furthermore, generation of ROS in the NB suspension was investigated using electron spin resonance spectroscopy and fluorescence spectrometry. The main ROS was found to be the hydroxyl radical, which is consistent with our previous observations. The bactericidal activity lasted for at least one week. Furthermore, a mist generated by atomizing the NB suspension with ultrasonic waves was confirmed to have the same bactericidal activity as the suspension itself. We believe that the strong, persistent bactericidal activity and radical generation phenomenon are unique to NBs produced by ultrasonic irradiation of carbonated water. We propose that entrapped CO2 molecules strongly interact with water at the NB interface to weaken the interface, and high-pressure CO2 gas erupts from this weakened interface to generate ROS with bactericidal activity.