The combined application of ultrasound and UVC have the potential to control mature Listeria monocytogenes and Bacillus subtilis biofilms

The present study aims to determine the bactericidal and disintegrating effect of ultraviolet type C treatments combined with ultrasound against biofilms of Listeria monocytogenes and Bacillus subtilis formed at different incubation periods (i.e. 24, 48, and 72 h). Treatments applied are UVC doses ranging from 60 to 360 mJ/cm ² (5–30 s) and US doses of 5–15 min, depending on the maturity of biofilms and the microorganism. Initially, we investigated the effects of UVC on planktonic cells, revealing a significantly higher lethality in L. monocytogenes compared to B. subtilis (P < 0.05). Moreover, B. subtilis demonstrated greater resistance in both its spore and vegetative forms (P < 0.05). The application of UVC on biofilms demonstrates that, as the more structured and robust the biofilm is, the more difficult it is for UVC to penetrate the biofilm structure and reach the cells that conform them, which is why higher lethality is observed in the shortest formation period (i.e. 24 h). In addition, as the UVC dose increases, microorganisms show a higher resistance to the treatment, thus showing a greater reduction in the microbial population at lower doses. The single application of ultrasound (US) manages to reduce the cell load between 1 and 4 log CFU/cm ², depending on the microorganism and exposure time. The results obtained after the combination of both technologies achieve higher lethality, 5.2 log reductions at the lowest doses (i.e. applying 5 min of US and 5 s of UVC). Furthermore, the maximum reduction possible for these experiments (≥5.6 log) is achieved in any treatment applying a 15-s UVC exposure time. Significant differences are observed (P < 0.05) as the maturity stage increases ([+Let] 24 ^a > 48 ^b > 72 ^c hours). The combination of these two technologies demonstrates highly promising outcomes across all examined bacterial structures, including spores and vegetative biofilms.