The importance of superoxide anion for Escherichia coli biofilm removal using plasma-activated water

Microbial biofilms cause contaminations in different environmental settings, including pipelines, filters, membranes, food and processing infrastructure. They ultimately pose a major risk to human health and necessitate costly cleaning and repair. Cold plasma, a partially ionised gas, and plasma-activated water (PAW) exhibit powerful disinfectant activity. However, the optimal generating conditions, such as the choice of gas used to produce PAW, remain unclear. Here, a range of different PAWs were generated from argon, nitrogen, air, and oxygen in a plasma bubble spark discharge (BSD) reactor capable of directly treating Escherichia coli (ATCC 25922) biofilms in situ. We measured the reactive oxygen and nitrogen species (RONS) (H₂O₂, NO₃^-, NO₂^-) in PAW and the excited species via optical emission spectroscopy (OES). PAW generated using oxygen (PAW-O₂) was the most effective and completely removed E. coli biofilms on stainless steel surfaces. Confocal microscopy demonstrated that PAW treatment removed most biofilm cells from the surface with only a few dead cells remaining. We demonstrated that intracellular ROS level increases significantly in the PAW-O₂-treated biofilms. Using molecular scavengers, we showed that superoxide anion radical (•O₂^-) played a critical role in the inactivation of E. coli biofilms. We also confirmed the generation of •O₂^- in the PAW-O₂ via electron paramagnetic resonance (EPR) spectrometry. The potential chemical reactions that occurred in PAW were hypothesized via optical emission spectra (OES). Our results demonstrate the importance of input gas and plasma operating conditions to maximise effective RONS production for optimal biofilm removal under real environmental and industry-relevant conditions.