War. Res. Vol. 26, No. 4, pp. 453-460. 1992 0043-1354/92$5.00+ 0.00 Printed tn Great gritain.All rights r--z,~rved Copyright ,~ 1992Pergamon Press pk: BIOLOGICAL PHOSPHATE REMOVAL BY ACTIVATED SLUDGE UNDER DEFINED CONDITIONS KLAAS J. APPELDOORN*, GERARD J. J. KORTSTEE and ALEXANDER J. B. ZEHNDERt Department of Microbiology, Wagcningen Agricultural University. Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands (First receired April 1991; accepted in rerised form September 1991) Abstract--A simple, one reactor vessel system, called a fill-and-drawsystem, was developed for the study of enhanced biological phosphate removal under defined conditions. Sludge was grown in a medium with acetate and glucose as sole energy and carbon sources. The sludge was exposed to cycleswith three distinct, consecutive periods: first an anaerobic period, then an aerobic period and finally a settlement period. In the period of settlement one third of the liquid was replaced with fresh medium. Sludge grown under this regime became considerably enriched with polyphosphate-accumulating bacteria. The polyphosphate content reached up to 110 mg P/g dry weight. The amount of polyphosphate in the cells during steady state depended on the acetate:glucose ratio, the nitrate and phosphate concentration in the medium. Highest phosphate accumulation was obtained with an acetate:glucose ratio of 9:1. lntracellular polyphosphate was formed during the aerobic period and was anaerobically hydrolysed and released as phosphate into the medium, in the absence of oxygen and in the presence of 2 g acetate-COD/I. 80-90% of phosphate was released by sludge containing 100 mg P/g dry weight. In the absence of acetate only 2-19% of the accumulated phosphate was excreted. Key words--activated sludge, biological phosphate removal, phosphate, polyphosphate, wastewater treatment INTRODUCTION Enhanced phosphate removal from wastewater can be achieved by introducing an anaerobic zone at the front end of the conventional activated sludge pro- cess. In treatment plants modified in this way, polyphosphate-containing bacteria are enriched in the sludge (Buchan, 1983; R6skc et al., 1989). In gencral, bacteria of the genus Acinetobacter are con- sidered to be responsible for the excess phosphate removal (Fuhs and Chert, 1975; Buchan, 1983; Deincma et aL, 1985). According to recent studies other bacterial species may also be of importance in this process, however. Nakamura et aL (1989) have isolated a number of Gram-positive bacteria from sludge, which are able to accumulate high amounts of phosphorus. Cloete and Stein (1988a, b) could not detect enough acinetobacters in their sludge to account for the observed phosphate removal. For the application of enhanced biological phos- phate removal several types of activated sludge plants have been designed, mainly based on empirical data and undefined sewages as influents (Eckenfelder, 1985). Studies with sludges and with pure cultures have not, until now, revealed any fully corresponding pictures about the fundamental principles of the enhanced biological phosphate removal. To increase *Present address: BKH Consulting Engineers, Smidswater 23. 2509 AE The Hague, The Netherlands. ?Author to whom all correspondence should be addressed. the reliability of the process on a large scale more knowledge about these principles is required. The current investigation was undertaken to develop a simple lab-scale system for studying the enhanced biological phosphate removal under well-defined conditions and to determine some properties of the resulting sludge types. Some preliminary results of this study have bccn prcscnted elsewhere (Appeldoorn and Dcincma, 1987). MATERIALSAND METHODS lnocula Renpho sludge originated from a pilot plant of the Department of Water Pollution Control of the Wageningen Agricultural University, The Netherlands, showing en- hanced biological phosphate removal (Rensink et al., 1989). The full-scale activated sludge plants Bunnik and Renkum were described previously by Janssen and Rensink (1987) and Mulder and Rensink (1987), respectively. Both plants were run as biological phosphate removal plants. Rhenen sludge originated from a carousel activated sludge plant located at Rhenen, The Netherlands, and was mainly fed with municipal wastewater. "Potato flour" sludge was sampled from a low loaded oxidation ditch located in the province of Zeeland, The Netherlands. This plant, divided into aerated and not aerated zones, treats (for 5.5 days a week) potato washing water and a starch-containing influent which had firat passed through an anaerobic treatment plant. The resulting influent for the oxidation ditch had a very high COD:N ratio (700:255). Sediments were taken from Lake Loosdrecht, The Netherlands. Media The basic media contained (in g/I demineralized water): 0.32, NH, CI, 0.6, MgSO,.7H20; 0.07, CaCI2.2H20; 0.1, 453