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Ozonolysis, as a lignocellulosic biomass pretreatment, goes back to 80s; however, in the last years it is becoming widespread again owing to its efficiency and mild operation conditions. Ozone reacts preferably with lignin than carbohydrates, promoting biomass destructuration and delignification, and so the sugar release by enzymatic hydrolysis.The hydrolysate from pretreated biomass has being used as sugars source for secondgeneration fuels production, mainly ethanol, methane and hydrogen. Short-chain carboxylic acids are the main inhibitory compounds generated, being properly removed by water washing. The most common inhibitory compounds reported for other pretreatments, furfural and HMF (5-hydroxymethylfurfural) † , are not found in ozonepretreated hydrolysates. Composition of pretreated biomass and ozone consumption depends on several process parameters: reactor design, moisture content, particle size, pH, reaction time, ozone/air flow and ozone concentration. Additional studies are necessary to clarify process parameters effect and to optimize the process to achieve high yields with economic feasibility.

A L9(3)(4) orthogonal array (OA) experimental design was applied to study the four parameters considered most important in the ozonolysis pretreatment (moisture content, ozone concentration, ozone/oxygen flow and particle size) on ethanol production from sugarcane bagasse (SCB). Statistical analysis highlighted ozone concentration as the highest influence parameter on reaction time and sugars release after enzymatic hydrolysis. The increase on reaction time when decreasing the ozone/oxygen flow resulted in small differences of ozone consumptions. Design optimization for sugars release provided a parameters combination close to the best experimental run, where 77.55% and 56.95% of glucose and xylose yields were obtained, respectively. When optimizing the grams of sugar released by gram of ozone, the highest influence parameter was moisture content, with a maximum yield of 2.98gSUGARS/gO3. In experiments on hydrolysates fermentation, Saccharomyces cerevisiae provided ethanol yields around 80%, while Pichia stipitis was completely inhibited.

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