The microbial distribution and the methane producing potential of biomass collected from the feeding zone and structured bed of two second stage thermophilic fixed film reactors were assessed. Three levels of food to microorganism ratio of 0.4, 1.0 and 3.0 gCOD per g volatile solids using fermented of two-stage anaerobic digestion and fresh of single-stage anaerobic digestion sugarcane molasses were tested in batch reactors, simulating low to high organic loads. Specific methane production rate values increased as the food to microorganism increased when using fermented molasses, indicating the establishment of efficient methanogenesis at substrate availability levels much higher than the ones considered ideal for single-stage biodigestion at 0.3 to 0.4 gCOD per g volatile solids. The capability of second stage methanogenic systems to withstand organic loads was demonstrated to increase by at least three fold compared to single stage schemes. Success in methane production derived directly from the homogenous establishment in the similar in both feeding zone and structured bed of syntrophic associations between acetogenic as Pelotomaculum, Syntrophothermus, Cloacimonadacea W5, Syntrophomonas and Thermodesulfovibrio acetate oxidizing as Thermoacetogenium, Mesotoga and Pseudothermotoga and hydrogenotrophic methogenic as Methanothermobacter and Methanoculleus genera in replacement to acetoclastic methanogenesis as Methanosaeta genus. Hence, phase separation under thermophilic conditions configures a highly suitable approach to achieve efficient methane production from sugar-rich substrates, because methanogenesis will depend of hydrogenotroph groups that grow faster and are less susceptible to low pH values compared to acetotrophs.