## 3 Results and Discussion  
### Effects of O\({}_{3}\) Concentration and Duration on E. coli O157:H7 Inactivation  
Previous studies have shown that the ozone treatment is effective in pathogen reduction in wastewater or sludge [58; 59; 60]. The current study sought to determine the effects of O3 on the survival populations of _E. coli_ O157:H7 in liquid dairy manure by using different O\({}_{3}\) concentrations and exposure times (Figure 2).  
Figure 2: \(Cont.\)The ozone treatment reduced the amount of _E. coli_ O157: H7, and the reductions increased with the exposure time, particularly at 87.40 or 132.46 mg\(\cdot\)L\({}^{-1}\) (Figure 2). As shown in Figure 2B,C, the pathogen reduction percentages were similar regardless of the initial concentrations of pathogens. To remove the potential interference between the native microbial population and inoculated pathogens, first dairy manure was autoclaved, and then inoculated with _E. coli_ O157: H7 prior to treatment by O\({}_{3}\) (Figure 3). In this condition, the reduction percentages at O\({}_{3}\) concentrations of 87.40 and 132.46 mg\(\cdot\)L\({}^{-1}\) were significantly higher than that of 43.26 mg\(\cdot\)L\({}^{-1}\). O\({}_{3}\) is reported to eliminate bacteria by attacking the glycoproteins and glycolipids in the cell membrane, and the sulfhydryl groups of certain enzymes, therefore, the effects of ozone in complex feedstock such as manure where multiple microorganisms are present depends on the pretreatment of manure [22].  
The results of this study suggest that when potential interference between the native microbial population of manure and inoculated pathogens was reduced by autoclaving the manure, the effects of O\({}_{3}\) were more apparent. Further, pretreatment such as autoclave may have an influence on the structure of the organic contents of manure, which may have allowed increased pathogen reduction [59; 60; 61]. While pretreatment of manure (i.e., inoculation followed by sterilization) was more effective in pathogen removal, the application of pretreatment requires additional cost and facilities. Therefore, for manure management, the application of sterilization could be cost-prohibitive. In this study, we implemented a sterilization process to understand the impacts of ozone on mixed species versus known species, and complex waste material such as manure. To understand the full potential of pretreatment, and its cost-effectiveness in manure treatment, additional studies carrying out pilot-scale implementation are needed.  
Under standard temperature and pressure conditions, the O\({}_{3}\) concentration of 132.46 mg\(\cdot\)L\({}^{-1}\) could be converted to \(\approx\)61,855 ppm, which was significantly higher than the concentrations of O\({}_{3}\) used in previous experiments to remove the pathogens in foods [62; 63; 64; 22; 65; 66], and in liquid organic wastes [21]. Pathogen reductions in foods were considerably higher than those in our study. This indicates that the impacts of ozone may vary depending on the feedstocks, and a higher level of ozone concentrations may  
Figure 2: Change in _E. coli_ levels: (**A**) _E. coli_ O157:H7 counts; (**B**,**C**) reduction percentages in the dairy manure with _E. coli_ inoculation (**B**) or without _E. coli_ inoculation (**C**) under different ozone concentrations and durations (note: the letters above columns showed the differences among different lasting times, which was the same as the next figures; means denoted by a different letter indicate significant differences between treatments (_p_ < 0.05)).  
be needed for waste material. The previous study, while investigating O3 treatments for pathogen reduction, also suggest that the needed ozone concentrations depend on material types [60].