In this work we suggest a methodology comprising the design and use of cost-effective, sustainable, and environmentally friendly process for biofuel production compatible with the market demands. A new generation biofuel is produced using fatty acids, which were generated from acidogenesis of industrial wastes of bioethanol distilleries, and esterified with selected alcohols by immobilized Candida antarctica Lipase-B. Suitable reactors with significant parameters and conditions were studied through experimental design, and novel esterification processes were suggested; among others, the continuous removal of the produced water was provided. Finally, economically sustainable biofuel production was achieved providing high ester yield (< 97%) along with augmented concentration (3.35 M) in the reaction mixtures at relatively short esterification times, whereas the immobilized lipase maintained over 90% of its initial esterifying ability after reused for ten cycles. (C) 2017 Elsevier Ltd. All rights reserved.