Plasma-activated water tests show concentration matters for effective bacterial disinfection

In a study published in npj Clean Water, researchers from EPFL investigated how Escherichia coli responds to different concentrations of plasma-activated water (PAW) under nutrient-rich conditions. The team combined conventional colony-forming unit (CFU) counting with nanomotion sensing, a technique that detects nanoscale oscillations produced by metabolically active bacteria.

PAW is produced by exposing water to a low-temperature plasma. During this process, reactive oxygen and nitrogen species are generated and dissolve into the water, resulting in its antimicrobial properties. The technology is being investigated for applications ranging from disinfection and sterilization to agriculture and biomedicine.

A nonmonotonic bacterial response

The researchers exposed E. coli to increasing concentrations of PAW diluted in lysogeny broth, a nutrient-rich growth medium. At the highest concentration, PAW completely inactivated the bacteria, producing a reduction of more than eight orders of magnitude compared with the initial bacterial population.

At intermediate concentrations, however, the response was markedly different. At 62.5% PAW, the number of viable bacteria was approximately twice that measured in the corresponding control condition. Nanomotion measurements independently revealed a similar pattern: bacterial activity increased at intermediate PAW concentrations before dropping sharply at higher concentrations.

"This shows that the interaction between bacteria and plasma-activated water is not simply a matter of increasing inhibition with increasing concentration," says Fabio Avino, a researcher at the Swiss Plasma Center and corresponding author of the study. "At sublethal concentrations, bacteria can display an enhanced physiological response, whereas higher concentrations ultimately lead to their inactivation."

Looking beyond bacterial survival

The combination of the two techniques was particularly important for the study. CFU counting measures the final outcome of bacterial proliferation, while nanomotion sensing provides a real-time measurement of physiological activity. Both approaches independently revealed the same overall biphasic response.

The underlying mechanism remains an open question. The observed response is compatible with a stress-adaptation or hormesis-like effect, in which moderate exposure to reactive species stimulates cellular activity. However, the researchers also point out that PAW can modify the chemical composition of the nutrient medium, for example through oxidation of organic compounds or changes in pH and redox conditions. The present experiments cannot fully distinguish between these effects.

Implications for plasma-based disinfection

The findings highlight the importance of controlling PAW concentration when using plasma-activated water for antimicrobial applications. In complex, nutrient-rich environments, a PAW concentration that is too low could fail to achieve microbial inactivation and may instead produce a transient increase in bacterial activity.

The study also demonstrates the potential of nanomotion sensing as a rapid tool for investigating how microorganisms respond to plasma-generated reactive species. By detecting changes in bacterial physiological activity in real time, the technique could complement conventional culture-based measurements when screening plasma treatment conditions.