DEVELOPMENT AND VALIDATION OF A FLUX-BASED STOICHIOMETRIC MODEL FOR ENHANCED BIOLOGICAL PHOSPHORUS REMOVAL METABOLISM J. PRAMANIK 1 , P. L. TRELSTAD 1 , A. J. SCHULER 2* M , D. JENKINS 2* M and J. D. KEASLING 1 * 1 Department of Chemical Engineering, University of California at Berkeley, Berkeley, CA 94720-1462, U.S.A. and 2 Department of Civil and Environmental Engineering, University of California at Berkeley, Berkeley, CA 94720-1462, U.S.A. (First received October 1997; accepted in revised form May 1998) AbstractÐEnhanced biological phosphorus removal (EBPR) is a wastewater treatment process invol- ving metabolic cycling through several biopolymers (polyphosphate, polyhydroxyalkanoates, and glyco- gen). This metabolic cycling is induced in microorganisms by treatment systems that alternate between initial carbon-rich, anaerobic environments followed by carbon-poor, aerobic environments. While the appearance and disappearance of these biopolymers has been documented, the intracellular pressures that lead to their synthesis and degradation are not well understood. To understand how carbon, energy, and redox potential are channeled through the metabolic pathways in each treatment process stage, a metabolic ¯ux model that contained a complete set of the pathways involved in biomass syn- thesis and energy production in bacteria was developed. The model accounts for the energy require- ments of macromolecule synthesis and of metabolite transport across the cell membrane. The equations for the 163 reversible and 166 irreversible reactions were solved using linear optimization. Data from a laboratory scale sequencing batch reactor performing EBPR were used as model inputs. Given polyhy- droxyalkanoate synthesis and glycogen degradation rates in the anaerobic phase, the model predicted reasonable anaerobic acetate uptake and polyphosphate consumption rates. In the aerobic phase, the polyphosphate and glycogen synthesis rates were used to predict the polyhydroxyalkanoate consump- tion rate. In addition, the model predicted the ratio of acetate uptake to phosphate release observed ex- perimentally, as well as an inverse relationship between polyhydroxyalkanoate and polyphosphate consumption. The model provides information on the pathways by which the energy-rich molecules ATP, NADH, and NADPH are produced and consumed during the EBPR processes. In doing so, it supports the hypothesis that biopolymer metabolism provides a means for organisms to balance intra- cellular energy supplies. Moreover, the model suggests pathways at which metabolic regulation should occur and provides a comprehensive account of EBPR metabolism. # 1998 Elsevier Science Ltd. All rights reserved Key wordsÐenhanced biological phosphorus removal, metabolic models, polyhydroxyalkanoates, glyco- gen, polyphosphate INTRODUCTION Enhanced biological phosphorus removal (EBPR) is an activated sludge process in which initial anaero- bic followed by aerobic cycling of the activated sludge results in the production of a higher than normal biomass phosphorus (P) content (greater than 2% P/volatile suspended solids (VSS)). Although it is widely used in practice, EBPR con- tinues to be the least understood of all activated sludge process modi®cations. Because of this, de- signs are highly empirical, process upsets are unpre- dictable, and prototype installations are usually equipped with backup systems (i.e., chemical pre- cipitation for P removal). Knowledge of the funda- mental mechanisms of EBPR will lead to more eective, robust, reliable and economical prototype processes. The EBPR process involves metabolic cycling through several biopolymers: polyphosphate (polyP), polyhydroxyalkanoates (PHAs), and glyco- gen. Cycling is induced through treatment systems that alternate between an initial carbon-rich, an- aerobic zone followed by a carbon-poor, aerobic reactor zone (Fig. 1). In the anaerobic zone, short- chain fatty acids (volatile fatty acids, VFAs) such as acetate are consumed and polymerized into PHAs, orthophosphate (P i ) generated from polyP depolymerization is secreted into the medium, and glycogen is degraded. In the subsequent aerobic stage, PHAs are degraded, glycogen is synthesized, and P i is taken up and polymerized into polyP. A simpli®ed depiction of these central EBPR reactions is shown in Fig. 2. Wat. Res. Vol. 33, No. 2, pp. 462±476, 1999 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/98 $19.00 + 0.00 PII: S0043-1354(98)00225-5 *Author to whom all correspondence should be addressed. [Tel: +510-642-4862; Fax: +510-642-4778, E-mail: keasling@socrates.berkeley.edu]. 462