COMBINED PHOSPHATE AND NITROGEN REMOVAL IN A SEQUENCING BATCH REACTOR USING THE AEROBIC DENITRIFIER, MICROVIRGULA AERODENITRIFICANS D. PATUREAU*, E. HELLOIN, E. RUSTRIAN M , T. BOUCHEZ, J. P. DELGENES M and R. MOLETTA M Institut National de la Recherche Agronomique, Laboratoire de Biotechnologie de l'Environnement, Avenue des Etangs, 11100, Narbonne, France (First received 20 September 1999; accepted in revised form 20 April 2000) AbstractÐA phosphate removal sludge was bioaugmented with the aerobic denitri®er, Microvirgula aerodenitri®cans in order to reduce the nitrate produced during the aerobic nitrifying-phosphate uptake phase. Fluorescent in situ hybridization (FISH) was used to follow the fate of the added strain. In order to maintain the pure strain in the complex ecosystem, diverse physiological and kinetic based strategies of bioaugmentation were tested under the sequencing batch reactor (SBR) type culture. The nature of the M. aerodenitri®cans inoculum (adapted to nitrate±aerobic conditions or to anoxic one) had no in¯uence on the SBR performances and did not enhance aerobic denitrifying performances. The optimum quantity of the added strain (10% of the total biomass) seemed to have much more positive in¯uence on the long term maintenance of the pure strain than on the SBR performances. A small but daily supply of M. aerodenitri®cans gave exactly the same result than a massive and 1-day supply, i.e. no enhancement of performances and no amelioration of the length of maintenance. A continuous supply of carbon during the ®rst hour of the aerobic phase combined to a 10% supply of M. aerodenitri®cans gave the best compromise in terms of phosphate removal, nitri®cation and aerobic denitri®cation performances. It was accompanied too by a decreased number of the ammonia and nitrite-oxidizing bacteria and a modi®cation of the nitrite-oxidizing ¯oc structure. FISH on M. aerodenitri®cans revealed that (i) before bioaugmentation, the strain was already present in the phosphate removal sludge and (ii) the added bacteria almost disappeared from the reactor after 16 HRT. In a last experiment, M. aerodenitri®cans embedded in alginate beads allowed enhancement of both aerobic denitrifying performances and length of strain maintenance. 7 2000 Elsevier Science Ltd. All rights reserved Key wordsÐphosphate removal, nitrogen removal, aerobic denitri®cation, Microvirgula aerodenitri®- cans, ¯uorescent in situ hybridization, bioaugmentation INTRODUCTION Removal of nitrogen and phosphate from waste- water is becoming a more and more acute problem for municipalities and industries as euent quality standards become more stringent. Indeed, the Euro- pean directive of 21 May 1991 has de®ned for sensi- tive areas (1) a maximum total nitrogen and phosphorus concentration in released wastewater of 15 and 2 mg/l for sites with 10,000±100,000 popu- lation equivalent (PE), of 10 and 1 mg/l for sites bigger than 100,000 PE, (2) a yield higher than 70 and 80%. However, the actual average yield for nitrogen and phosphorus treatment of urban waste- water surrounds 40%. This last ®gure underlines the importance of enhancement of existing pro- cesses. Conventional nitrogen removal plants are based on the combination of nitri®cation (aerobic oxi- dation of ammonia into nitrate) and denitri®cation (anoxic conversion of nitrate into nitrogen gas). In terms of reactor con®guration, this implies spatial or temporal separation of the two phases. Up to date, phosphate removal is realized either by chemical precipitation, or by phosphate incor- poration into biomass. Enhanced biological phos- phate removal (EBPR) systems can be considered as an eective alternative to chemical precipitation because of its low investment, low operational costs and better quality of sludge (Lan et al., 1983). An anaerobic/aerobic sequence is necessary to promote biological phosphate removal: a P release in the an- aerobic stage followed by an excess of P uptake in the aerobic stage, P accumulation as poly-phos- Wat. Res. Vol. 35, No. 1, pp. 189±197, 2001 7 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter 189 www.elsevier.com/locate/watres PII: S0043-1354(00)00244-X *Author to whom all correspondence should be addressed. Tel.: +33-468-425-169; fax: +33-468-425-160; e-mail: patureau@ensam.inra.fr