Environmental Microbiology (2003) 5(4), 278–286 © 2003 Society for Applied Microbiology and Blackwell Publishing Ltd Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912Blackwell Publishing Ltd, 20035Original Article Nitrifying bacteria in an activated-sludge reactorJ. Tanaka et al. Received 1 October, 2002; accepted 29 November, 2002. *For correspondence. E-mail junichi.tanaka@mbio.jp; Tel. (+81) 193 26 6537; Fax (+81) 193 26 6584. † Present address (from April 2003): AT Research Laboratory, Kansai Paint Co., Ltd, 4-17-1 Higashi-Yawata, Hiratsuka, Kanagawa 254-8562, Japan. Activity and population structure of nitrifying bacteria in an activated-sludge reactor containing polymer beads Jun-ichi Tanaka, 1 * † Kazuaki Syutsubo, 1 Kazuya Watanabe, 1 Hitoshi Izumida 2 and Shigeaki Harayama 1 1 Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026–0001, Japan. 2 New Business Division, Kansai Paint Co. Ltd, 4-17-1 Higashi-Yawata, Hiratsuka, Kanagawa 254–8562, Japan. Summary Photo-crosslinked polymer beads were introduced into a laboratory activated-sludge unit containing municipal sewage sludge and the effect on nitrifying capacity was examined. The ammonia load started at a nitrogen-loading rate of 0.02 kg m -3 day -1 and was increased stepwise. It was found that the bead- containing unit could almost completely oxidize ammonia (over 95%) up to a nitrogen-loading rate of 0.216 kg m -3 day -1 , whereas the maximum loading rate of the control unit (without polymer beads) was 0.096 kg m -3 day -1 . The nitrifying potential of sus- pended and bead-associated organisms in the bead- containing unit was measured under different loading conditions. It was found that the bead-associated organisms exhibited high specific activities under high loading conditions and that the contribution of the bead-associated organisms to nitrification was greater than that of the suspended solids under these conditions. The bacterial population dynamics in the suspended solids and bead-associated organisms were analysed by denaturing gradient gel electro- phoresis (DGGE) of PCR-amplified 16S rRNA gene fragments and by fluorescence in situ hybridization with group-specific probes. Among the known nitrify- ing organisms, ammonia-oxidizing b-proteobacteria and Nitrospira-related organisms were detected by these approaches. A comparison of the activity dynamics and population dynamics, however, sug- gested a possibility that other organisms may also have been involved in the nitrification process under high loading conditions. Introduction Human activity discharges a large amount of nitrogen compounds (ammonia, nitrite and nitrate) into the environment and pollutes groundwater, rivers, lakes and inland seas. To solve this problem, nitrogen removal pro- cesses based on the combined action of two biological reactions, nitrification and denitrification, have been developed and used in biological wastewater treatment plants. Nitrifying bacteria play an important role in nitrifi- cation, whereby ammonia is oxidized by ammonia- oxidizing bacteria to nitrite, which is further oxidized to nitrate by nitrite-oxidizing bacteria (Prosser, 1989; Watson et al., 1989). The specific growth rate of nitrifying bacteria, however, is generally low (Watson et al., 1989) and these bacteria are readily washed out when the mean cell residence time [MCRT; also commonly termed the sludge residence time (SRT)] in a wastewater treatment system is short. Thus, immobilization techniques have been developed as solu- tion to this problem. Reactor systems such as those involving trickling filters (Fan et al., 2000), rotating biolog- ical contactors (Christine and Sabine, 1998) and fluidized beds allow biofilms of nitrifiers to develop on substrata, and the slow-growing organisms remain in the reactors by their attached growth. To have a better understanding of the community structure of such nitrifying biofilms and to optimize the nitrification process in wastewater treatment, molecular approaches have been taken to analyse these bacterial aggregates. Whole cell hybridization with rRNA- targeted fluorescent nucleic acid probes (fluorescence in situ hybridization; FISH), which can detect specific ammo- nia-oxidizing or nitrite-oxidizing bacteria, has been devel- oped (Mobarry et al., 1996; Juretschko et al., 1998), and the spatial distribution of nitrifiers has been elucidated in several biofilms. Furthermore, the community structures of nitrifying biofilms have been found to be related to the nitrifying activity as determined by microsensors (Schramm et al., 1996; 2000; Lee et al., 1999; Okabe et al., 1999; Gieseke et al., 2001). To reinforce the nitrification/denitrification activities, nitrifiers and/or denitrifiers have been cultivated and the resulting cells were embedded in appropriate matrices. For example, gel beads have been used to co-immobilize Nitrosomonas europaea (nitrifier) and either Pseudomo- nas denitrificans or Paracoccus denitrificans (denitrifier).