# Effect of Ozonized Water against Pathogenic Bacteria and Filamentous Fungi on Stainless Steel
## 3 Results and Discussion
### Filamentous Fungi  
On filamentous fungi, a concentration of ozone equal to 8.5 mg L\({}^{-1}\) was applied since preliminary tests at the concentrations used against bacteria did not prove effective at all in inactivating fungal conidia. The tested strains were not affected by aqueous ozone if inoculated as a multi-layer of cells, with the exception of _H. burtonii_ strains that proved subjected to a partial inactivation when acidified ozonized water was used. Dissimilarly, on single layers of cells, a gradual decrease was observed for all strains belonging to _P. nortactium_ or _H. burtonii_. A statistically significant difference within the same fungal species was observed comparing data obtained testing either not-acidified or acidified ozonated water. On multi-layered cells, differences were observed only for _Hyphopichia_ strains at any treatment time, with a marked decrease in survivors registered when citric acid was added to ozonized water. Differently, on single-layered cells, the observed statistical differences seemed randomized: they wereregistered at 8 min for _A. brasiliensis_, at 6 min for _P. nordicum_ 45, or from 4 to 8 min for _P. nordicum_ 1169, and from 2 to 8 min for _Hymphoichia_ strains. When the multi-layered cells were tested in both liquid matrices, the _A. brasiliensis_ and _P. nordicum_ strains did not reach even 1.0 LCR after the longest treatment time. Conversely, for _H. burtonii_ strains, the results differed when not-acidified and acidified ozonated water were tested: from 1.0 to 1.1 LCR after 10 min in the former case or more than 5.0 LCR after 6-10 min in the latter case were reached, depending on the isolate considered. When the single-layered cells were tested, _A. brasiliensis_ inactivation kinetics did not change compared to the multi-layered spatial arrangement. On the contrary, strain- and species-specificity was observed for the tested _Hymphoichia_ and _Penicillium_ strains. _P. nordicum_ isolates underwent from 2.9 to 4.7+ LCR in not-acidified water and from 3.8 to 4.40 LCR in acidified water. Similarly, _H. burtonii_ strains were subjected to a minimum of 3.4 LCR just after 6 min of treatment up to 5.0+ LCR after 10 min in not-acidified water and to 3.8-4.4 LCR in acidified water. When compared with the other tested strains, _Aspergillus brasiliensis_ showed the greatest resistance to the sanitizing agent applied whenever arranged in a multi-layer or in a single layer. It yet resulted in a point of reference when not-ionizing radiation such as the UVC light [34; 46] or chemicals such as the ozone itself [35] have to be tested. This was probably due to the great amounts of melanin and melanin-like pigments in its outer cellular wall [51; 52]. Furthermore, it must be taken into account that, like other filamentous fungi in the genus _Aspergillus_, it possesses good amounts of ROS-scavenging enzymes such as superoxide dismutases (SOD) and catalases (CAT) [53]. SOD proved critical to cell survival following ozone exposure by eliminating the superoxide radicals and thereby preventing the formation of hydroxyl radicals, whereas CAT proved necessary for protection against ozone exposure [54]. Therefore, the comprehension of melanin and of major ROS-scavenging enzymes could be responsible for its higher robustness against ozonized water. With regard to the literature, few papers have dealt with the effect of ozonized water on filamentous fungi either in static or dynamic conditions, and none of them reported tests on metallic or plastic supports. In static conditions, [PERSON] et al. [55] tested filamentous fungi inoculated in ozonized water and registered a great variability in fungal response. All the tested strains (_Fusarium solani_, _Trichoderma vriide_, and _Aspergillus calidoustus_) were reduced from one to three Logs after 10 min of treatment at 8 ppm, with the exception of _P. spinulosum_ that did not prove affected by aqueous ozone.