Microbial community structure in autotrophic nitrifying granules characterized by experimental and simulation analyses Shinya Matsumoto, 1 Mayu Katoku, 1 Goro Saeki, 1 Akihiko Terada, 2 Yoshiteru Aoi, 3 Satoshi Tsuneda, 1 * Cristian Picioreanu 4 and Mark C. M. van Loosdrecht 4 1 Department of Life Science and Medical Bioscience, Waseda University, Wakamatsu-cho 2–2, Shinjuku-ku, Tokyo 162-8480, Japan. 2 Institute of Environment and Resources, Technical University of Denmark, DK-2800 Lyngby, Denmark. 3 Waseda Institute for Advanced Study, Waseda University, Wakamatsu-cho 2-2, Shinjuku-ku, Tokyo 162-8480, Japan. 4 Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, the Netherlands. Summary This study evaluates the community structure in nitrifying granules (average diameter of 1600 mm) produced in an aerobic reactor fed with ammonia as the sole energy source by a multivalent approach combining molecular techniques, microelectrode measurements and mathematical modelling. Fluores- cence in situ hybridization revealed that ammonia- oxidizing bacteria dominated within the first 200 mm below the granule surface, nitrite-oxidizing bacteria a deeper layer between 200 and 300 mm, while het- erotrophic bacteria were present in the core of the nitrifying granule. Presence of these groups also became evident from a 16S rRNA clone library. Micro- profiles of NH 4 + , NO2 – , NO3 – and O2 concentrations measured with microelectrodes showed good agreement with the spatial organization of nitrifying bacteria. One- and two-dimensional numerical biofilm models were constructed to explain the observed granule development as a result of the multiple bacteria–substrate interactions. The interaction between nitrifying and heterotrophic bacteria was evaluated by assuming three types of heterotrophic bacterial growth on soluble microbial products from nitrifying bacteria. The models described well the bacterial distribution obtained by fluorescence in situ hybridization analysis, as well as the measured oxygen, nitrite, nitrate and ammonium concentration profiles. Results of this study are important because they show that a combination of simulation and experimental techniques can better explain the inter- action between nitrifying bacteria and heterotrophic bacteria in the granules than individual approaches alone. Introduction The low growth rate and low fraction of autotrophic bacteria in activated sludge processes make nitrification the rate-limiting step in most wastewater treatment pro- cesses. Thus, a simpler and effective method of immo- bilizing nitrifying bacteria within wastewater treatment processes is desired. Granulation without using any car- riers has been proposed for immobilizing nitrifying bacte- ria in inorganic wastewater treatment processes (De Beer et al., 1997; Tay et al., 2002; Tsuneda et al., 2003). Our group successfully produced nitrifying granules using an aerobic upflow fluidized bed (AUFB) reactor (Tsuneda et al., 2003). Furthermore, the time required for producing nitrifying granules could be greatly reduced by use of pre-aggregating seed sludge with hematite (Tsuneda et al., 2004). However, detailed information on granule formation and nitrogen conversion in primarily autotrophic systems is still lacking. To date, there are reports indicating ecophysiological interaction between nitrifying bacteria and heterotrophic bacteria in autotrophic nitrifying suspended cultures (Ritt- mann et al., 1994) and biofilms (Kindaichi et al., 2004; Okabe et al., 2005) grown without an external organic carbon source. In such systems, nitrifying bacteria may consume inorganic carbon not only to form cell mass but also to excrete organic carbon (soluble microbial prod- ucts, SMP), which will support heterotrophic bacterial growth (Rittmann et al., 2002). Although very much needed, a detailed analysis on microbial ecology of nitri- fying granules including nitrifying and heterotrophic bac- teria has never been reported. Better understanding on the formation of nitrifying granule’s structure (especially Received 3 April, 2009; accepted 11 August, 2009. *For correspon- dence. E-mail stsuneda@waseda.jp; Tel. (+81) (0)3 5369 7325; Fax (+81) (0)3 3341 2684. Environmental Microbiology (2010) 12(1), 192–206 doi:10.1111/j.1462-2920.2009.02060.x © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd