Anaerobic digestion is a viable process to recover bioenergy through sugarcane vinasse in the form of methane. However, interruptions in the anaerobic reactors operation during the sugarcane off-season is clearly a challenge to the anaerobic systems’ performance, which potentially negatively compromise the energy balance in a sugarcane biorefinery. Glycerol, a by-product of biodiesel production, is a viable option for vinasse replacement in the off-season period. Anaerobic co-digestion of these wastewaters can promote improvements in operational conditions and enhance energy recovery. Thus, this study evaluated the exchange of substrates, vinasse and glycerol, in an anaerobic structured bed reactor (AnSTBR), using glycerol as substrate in off-season and vinasse in the following harvest season, in mono- and co-digestion processes. The AnSTBR operation showed promising results regarding the organic matter removal (higher than 80%, in terms of chemical oxygen demand) and methane yields (higher than 225 NmLCH4.g−1CODrem). The methane produced, for a cycle of harvest, off-season and harvest again, can improve approximately 17.5% the energy generation in the sugarcane biorefinery, using glycerol as substrate, instead of shutting down the anaerobic reactor in off-season, resulting in a positive economic balance. Furthermore, the operational strategy of gradually exchanging substrates ensured the system stability throughout the operation.
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