Alcoholic fermentation of released sugars in pretreatment and enzymatic hydrolysis of biomass is a central feature for second generation ethanol (E2G) production. Saccharomyces cerevisiae used industrially in the production of first generation ethanol (E1G) convert sucrose, fructose, and glucose into ethanol. However, these yeasts have no ability to ferment pentose (xylose). Therefore, the present work has focused on E2G production by Scheffersomyces stipitis and Spathaspora passalidarum. The fermentation strategy with high pitch, cell recycle, fed-batch mode, and temperature decrease for each batch were performed in a hydrolyzate obtained from a pretreatment at 130 degrees C with NaOH solution (1.5%w/v) added with 0.15% (w/w) of anthraquinone (AQ) and followed by enzymatic hydrolysis. The process strategy has increased volumetric productivity from 0.35 to 0.38gL(-1)h(-1) (first to third batch) for S. stipitis and from 0.38 to 0.81gL(-1)h(-1) for S. passalidarum (first to fourth batch). Mass balance for the process proposed in this work showed the production of 177.33kg ethanol/ton of sugar cane bagasse for S. passalidarum compared to 124.13kg ethanol/ton of sugar cane bagasse for S. stipitis fermentation. The strategy proposed in this work can be considered as a promising strategy in the production of second generation ethanol. Biotechnol. Bioeng. 2017;114: 2211-2221. (c) 2017 Wiley Periodicals, Inc.