Effect of an ozone pre-treatment on dark fermentation: Influence on the microbiota and pathogen removal

The aim of this study was to investigate the evolution of the microbial community and its relationship with hydrogen production, as well as pathogen reduction during thermophilic dark fermentation using sewage sludge and wine vinasses, under ozone pre-treatment (PT) and non-pre-treated (Control) conditions. Microbial dynamics analysis revealed that PT increased the Eubacteria ratio and shifted the balance between Hydrolytic-Acidogenic Bacteria (HABs) and acetogens. PT enhanced bio-hydrogen production, achieving a 78 % increase in yield and a 90 % improvement in specific hydrogen production compared to control. In both conditions, Eubacteria predominated over Archaea, with a higher proportion of Propionate-Utilizing Acetogens in comparison with Butyrate-Utilizing Acetogens. Hydrolytic activity was 25 % higher in PT, correlating with the highest hydrogen production, reduced non-soluble carbon, and the most active HABs. PT also increased acidification yields by 22 % and microbial activity by 80 %, enhancing hydrogen production primarily through elevated hydrolytic activity. This was associated with a 60 % rise in acid carbon, with acetic and butyric acids comprising 80 % of the total volatile fatty acids. Regarding pathogen reduction, PT reduced Escherichia coli concentrations in the feed by an order of magnitude. In conclusion, PT significantly improved bio-hydrogen production, microbial activity, and process efficiency, while facilitating effective pathogen reduction.