Polyphosphate-Accumulating and Denitrifying Bacteria Isolated from Anaerobic-Anoxic and Anaerobic-Aerobic Sequencing Batch Reactors Mohamed Merzouki, 1 Jean-Philippe Delgene `s, 1 Nicolas Bernet, 1 Rene ´ Moletta, 1 Mohamed Benlemlih 2 1 Institut National de la Recherche Agronomique, Laboratoire de Biotechnologie de l’Environnement, Avenue des e ´tangs, 11100 Narbonne, France 2 Laboratoire de Microbiologie de l’Environnement, Faculte ´ des Sciences Dhar Mehraz, B.P. 1796 Fe `s, Maroc Received: 24 February 1998 / Accepted: 21 July 1998 Abstract. In this study, phosphate-accumulating bacteria achieved complete phosphate removal in two different systems: an anaerobic-anoxic sequencing batch reactor and an anaerobic-aerobic sequencing batch reactor. This result shows that phosphate-accumulating bacteria in the A 2 SBR can use nitrate as terminal electron acceptor instead of oxygen. Phosphate-accumulating bacteria accumulated phosphate with a rates between 30 and 70 mg P/L/h in the A/O SBR and between 15 and 32 mg P/L/h in the A 2 SBR. Twenty denitrifying isolates were screened from A 2 SBR and nine from A/O SBR. Identification of these isolates by the Biolog system and the API 20 NE identification kit revealed that the most active denitrifiers in both SBRs reactors were species of Ochrobactrum, Pseudomonas, Corynebacterium, Agrobacterium, Aquaspirillum, Haemophilus, Xanthomonas, Aeromonas, and Shewanella. The most active phosphate accumulating and denitrifying bacteria were identified as Agrobacterium tumefaciens B, Aquaspirillum dispar, and Agrobacterium radiobacter. This study showed that the active phosphate accumulating- bacteria were also the most efficient denitrifying bacteria in both reactors. Phosphorus removal and denitrification by bacteria are important processes in biological nutrient removal from wastewaters. Biological phosphorus removal is based on the ability of some bacteria to accumulate excess ortho- phosphate as polyphosphate. Phosphate-accumulating bac- teria take up, in anaerobic conditions, short-chain fatty acids and transform them into poly--hydroxybutyrate (PHB), which they store as an endogenous source of carbon. The energy needed for this conversion comes from the hydrolysis of polyphosphate to phosphate, which is released into the extracellular medium. In aerobic conditions the bacteria accumulate phosphate as polyphosphate using the energy resulting from the oxida- tion of PHB via the Krebs cycle [8, 19, 21, 23]. The alteration of anaerobic and aerobic phases favors the growth of polyphosphate accumulators and their selec- tive enrichment [17, 30, 32, 33]. Isolation of denitrifiers and polyphosphate accumula- tors from laboratory-scale activated sludge constitutes a large field of research. However, recent studies have resulted in the isolation, from full-scale activated sludge plants, of polyphosphate-accumulating bacteria and deni- trifiers belonging to the genera Pseudomonas, Agrobacte- rium, Sphingomonas, Xanthomonas, Bacillus, and Alca- ligenes [17, 21, 31]. Species belonging to the genera Acinetobacter, Pseudomonas, Moraxella, Flavobacte- rium, Aeromonas, Bacillus, Xanthomonas, Sphingomo- nas, Alcaligenes, and Corynebacterium [17, 21, 30] and many other Gram-positive bacteria in activated sludge have been reported to accumulate polyphosphate [25]. On the whole species of Acinetobacter are considered to be responsible for most of the phosphate removal in activated sludge [1, 2, 10–15, 23]. However, some authors have shown that the biological phosphate removal observed is independent of the presence of Acinetobacter, which represents around 1% of the total flora in the activated sludge at the anaerobic and the aerobic phases [19]. Kuba and collaborators [22] studied biological phos- phorus removal in anaerobic-aerobic (A/O) and anaerobic- anoxic (A 2 ) SBR systems. They verified the ability of phosphorus-removing bacteria to use nitrate as sole electron acceptor, instead of oxygen, for biological phosphorus removal in an A 2 SBR. From a microbiologi- cal point of view, few studies have been done in the identification of phosphate-accumulating bacteria iso- Correspondence to: J.-P. Delgene `s CURRENT MICROBIOLOGY Vol. 38 (1999), pp. 9–17 An International Journal  Springer-Verlag New York Inc. 1999