Changes in microbial communities associated with the conditioning of filter material in recirculating aquaculture systems of the pufferfish Takifugu rubripes Shiro Itoi, Ayako Niki, Haruo Sugita ⁎ Department of Marine Science and Resources, Nihon University, Kameino, Fujisawa, Kanagawa 252-8510, Japan Received 18 November 2005; received in revised form 24 January 2006; accepted 10 February 2006 Abstract We investigated changes in microflora associated with the conditioning of filter material in a recirculating aquaculture system for the culture of the pufferfish Takifugu rubripes using a clone library method of partial 16S rRNA gene sequences. Total bacteria on the pebbles used as filter material increased from 8.4 × 10 9 cells g - 1 at peak ammonia concentrations (8 days) to 1.3 × 10 10 cells g - 1 at the end of the study (44 days). As filter material became conditioned, the occurrence of Clostridia, α-Proteobacteria and γ-Proteobacteria on the surface of the pebbles increased, whereas Flavobacteria, Sphingobacteria and Mollicutes decreased. The occurrence of ammonia-oxidizing bacteria related to Nitrosomonas decreased from 3.00% at day 8 to 0.95–1.04% at days 15–44. Phylogenetic analysis of the clones related to the genus Nitrosomonas using a Bayesian method revealed that two clones obtained in this study formed a cluster with N. aestuarii in the N. marina sublineage of the N. oligotropha lineage, whereas another two clones formed a cluster with Nitrosomonas sp. Nm143 of the Nitrosomonas sp. Nm143 lineage with high Bayesian posterior probabilities support. Two clones formed a separate cluster from those of the other Nitrosomonas lineages. Our results demonstrated the importance of effective utilization of nitrifying bacteria in aquaculture, since number of these bacteria did not vary for the duration of the experiment. © 2006 Elsevier B.V. All rights reserved. Keywords: Nitrifying bacteria; Nitrosomonas; Recirculating aquaculture systems; Filter material; Microflora 1. Introduction Aquaculture has considerable potential for meeting the increasing demand for aquatic products in many regions of the world. According to the latest Food and Agriculture Organization (FAO) statistics, the contribu- tion of aquaculture to global supplies of fish, crustaceans and mollusks has increased from 3.9% of total production by weight in 1970 to 29.9% in 2002 (FAO Fisheries Department, 2004). Aquaculture is undertaken using various systems including cage culture, pond culture, recirculating water systems, longlines and flow- through systems. Recirculating aquaculture systems have been widely applied to the culture of aquatic animals in various regions of the world, since the systems reuse water with mechanical and biological treatment between each use. This is primarily due to the benefits associated with their operation, including the optimal use of space, lower water requirements Aquaculture 256 (2006) 287 – 295 www.elsevier.com/locate/aqua-online Abbreviations: DAPI, 4′, 6-diamidino-2-phenylindole; PCR, polymerase chain reaction; rRNA, ribosomal RNA. ⁎ Corresponding author. Tel./fax: +81 466 84 3679. E-mail address: sugita@brs.nihon-u.ac.jp (H. Sugita). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.02.037