Enhanced nutrients removal in membrane bioreactors (ENREM) combines enhanced biological phosphorus removal (EBPR), post-denitrification without additional carbon supply, and membrane filtration in a relatively compact wastewater treatment process [Gnirss et al. 2003]. Since 2006, a demonstration plant of 10 m³ is serving a peripheral area of Berlin to treat the wastewater of about 250 people, following an anaerobic – aerobic – anoxic process scheme [Gnirss et al. 2008]. Post-denitrification without additional carbon supply is quite uncommon because the lack of carbon as electron donor usually results in low endogenous denitrification rates (DNR) below 0.6 mgNO3-N/(goTS h), leading to larger reactor volumes and thus higher investment costs [Kujawa & Klapwijk 1999]. In contrast to that, the ENREM process showed enhanced denitrification rates of 1-2 mgNO3-N/(goTS h), raising the question which carbon source is used to obtain these rates [Adam 2004; Lesjean et al. 2005; Vocks et al. 2005]. To address this question, several batch experiments were conducted using acetate as reactor feed, which is completely consumed by the biomass within the anaerobic phase. These experiments ruled out soluble carbon sources such as extracellular polymeric substances (EPS), lysis/hydrolysis products, or adsorption of acetate [Vocks et al. 2005; Bracklow et al. 2007]. The analysis of polyhydroxyalkanoates (PHAs) and glycogen as intracellular carbon storage compounds typical for EBPR systems showed no clear trend for the anoxic phase. Furthermore, the results showed a carbon recovery rate for the anaerobic phase of only 50-70 %, accounting for PHAs, glycogen, carbon dioxide, soluble COD, and acetate. The experiments also showed that the DNR can be increased by adding higher acetate dosages at the beginning of the process [Nicke 2005; Baumer 2006; Stüber 2007]. These observations led to the assumption that an unknown intracellular carbon storage compound might be formed during the anaerobic phase which serves as carbon source for enhanced denitrification [Lesjean et al. 2008; Vocks 2008]. This study was conducted to prove the theory of an unknown intracellular carbon storage compound used for enhanced denitrification and to identify this compound. In-vivo nuclear magnetic resonance spectroscopy (NMR) has proven to be an adequate tool to analyse metabolic pathways of microorganisms and to identify also unknown compounds [Pereira et al. 1996; Maurer et al. 1997; Jeon & Park 2000; Lemos et al. 2003]. However, NMR requires the use of a single carbon source (monosubstrate) which can be labelled by 13C isotopes. Hence, this study included the adaption of the ENREM process to acetate as monosubstrate in lab scale. A 6 L sequencing batch membrane bioreactor (SBMBR) was inoculated with sludge from the ENREM demonstration plant and stepwise adapted to acetate as single carbon source. The reactor was operated successfully for a period of 190 days and showed phosphorus and nitrogen dynamics typical for the ENREM process. Furthermore, carbon mass balances showed the same recovery rates of 50-70 % like in previous studies, and fluorescence in-situ hybridisation (FISH) showed a high abundance of phosphorus accumulating organisms (PAOs), thus indicating a successful adaption of all ENREM process characteristics to monosubstrate. The continuous long-term operation with a readily biodegradable monosubstrate rules out the presence of slowly biodegradable COD (sbCOD) as carbon source for denitrification.