Effect of Double-Step Steam Explosion Pretreatment in Bioethanol Production from Softwood Franco Cotana & Gianluca Cavalaglio & Mattia Gelosia & Valentina Coccia & Alessandro Petrozzi & Andrea Nicolini Received: 17 January 2014 /Accepted: 7 July 2014 / Published online: 17 July 2014 # Springer Science+Business Media New York 2014 Abstract The study investigated the production of bioethanol from softwood, in particular pine wood chip. The steam explosion pretreatment was largely investigated, evaluating also the potential use of a double-step process to increase ethanol production through the use of both solid and liquid fraction after the pretreatment. The pretreatment tests were carried out at different conditions, determining the composition of solid and liquid fraction and steam explosion efficiency. The enzymatic hydrolysis was carried out with Ctec2 enzyme while the fermentation was carried out using Saccharomyces Cerevisiae yeast “red ethanol”. It was found that the best experimental result was obtained for a single-step pretreated sample (10.6 g of ethanol/100 g of initial biomass dry basis) for a 4.53 severity. The best double-step overall performance was equal to 8.89 g ethanol/100 g of initial biomass dry basis for a 4.27 severity. The enzymatic hydrolysis strongly depended on the severity of the pretreatment while the fermentation efficiency was mainly influenced by the concentration of the inhibitors. The ethanol enhancing potential of a double-step steam explosion could slightly increase the ethanol production compared to single-step potential. Keywords Bioethanol . Softwood . Effect of inhibitors . Steam explosion . Enzymatic hydrolysis . Fermentation Abbreviations SE Steam explosion 5-HMF 5-hydroxymethil furfural NREL National Renewable Energy Laboratory DM Dry matter HPLC High Performance Liquid Chromatography AIR Acid-insoluble residue AIL Acid-insoluble lignin TGA Thermal-gravimetric analysis WIS Water insoluble substrate HY Hydrolysis yield XMG Xylose mannose and galactose Appl Biochem Biotechnol (2014) 174:156–167 DOI 10.1007/s12010-014-1046-4 F. Cotana : G. Cavalaglio (*) : M. Gelosia : V. Coccia : A. Petrozzi : A. Nicolini CIRIAF-Biomass Research Centre, University of Perugia, Via G. Duranti, 06125 Perugia, Italy e-mail: cavalaglio@crbnet.it