Maintenance energy plays an important role both in basic kinetic studies and in process development. Numerous studies have been devoted to the maintenance concept in various microbial fields but very few in biological wastewater treatment. Using a fermenter coupled to a mass spectrometer, we investigated the influence of the ratio So/Xo (ratio between initial substrate concentration and initial biomass concentration) on the observed sludge growth yield of an oxic-settling anaerobic (OSA) system. By measuring the substrate removed, the oxygen consumed and the carbon dioxide produced, we were able to estimate the substrate fraction used for maintenance purposes. The results indicate that at a high So/Xo ratio, a greater proportion of the substrate is devoted to maintenance thus significantly decreasing the observed growth yield. These findings are of particular importance in view of the cost associated with the disposal of excess sludge in aerobic wastewater treatment processes.