For decades, glycolysis has been generally accepted to supply the reducing power for the anaerobic conversion of volatile fatty acids (VFAs) to polyhydroxyalkanoates (PHAs) by polyphosphate accumulating organisms (PAOs). However, the importance of the tricarboxylic acid (TCA) cycle has also been raised since 1980s. The aim of this study is to demonstrate the involvement of the TCA cycle in the anaerobic metabolism of PAOs. To achieve this goal, the glycogen pool of an activated sludge highly enriched in Candidatus Accumulibacter Phosphatis (hereafter referred to as Accumulibacter), a putative PAO was reduced substantially through starving the sludge under intermittent anaerobic and aerobic conditions. After the starvation, acetate added was still taken up anaerobically and stored as PHA, with negligible glycogen degradation. The metabolic models proposed by Pereira, Hesselmann and Yagci, which predict the formation of reducing power through glycolysis and the full or partial TCA cycle, were used to estimate the carbon fluxes. The results demonstrate that Accumulibacter can use both glycogen and acetate to generate reducing power anaerobically. The anaerobic production of reducing power from acetate is likely through the full TCA cycle. The proportion of TCA cycle involvement depends on the availability of degradable glycogen.