By-products from sugarcane mills have a considerable energy potential, and therefore have been studied aiming to generate biogas emphasising biohydrogen (bioH(2)). Sugarcane molasses, a byproduct from sugar production, are rich in carbohydrates, thus easily biodegraded by anaerobic microorganisms. This study evaluated the production of bioH(2) in unfavorable pH (3.80) using molasses as a feedstock in an anaerobic structured bed reactor (AnSTBR-A) under thermophilic conditions (55 degrees C). The AnSTBR-A operated with an organic loading rate (OLR) of 60 g L-1 d(-1) was able to produce bioH(2) under long-term operation (392 days). The hydrogen yield (HY) was 1.18 mol H-2 mol total carbohydrates(-1). The results highlighted HY variation concomitant with metabolite concentrations. The main role to bioH(2) production in AnSTBR-A was acetate + lactate -> butyric + bioH(2), with a predominance of the organism belonging to the Thermoanaerobacterium genus. (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.