Aggregation of ammonia-oxidizing bacteria in microbial biofilm on oyster shell surface Volodymyr Ivanov 1 , Olena Stabnikova 1, *, Prakitsin Sihanonth 2 and Piamsak Menasveta 3,4 1 School of Civil and Environmental Engineering, Nanyang Technological University, 20 Nanyang Avenue, Singapore, Singapore 2 Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand 3 Department of Marine Science, Faculty of Science, Chulalongkorn University, Bangkok, Thailand 4 Marine Biotechnology Research Unit at Chulalongkorn University, BIOTEC, Bangkok, Thailand *Author for correspondence: Tel.: +65-6790-4740, Fax: +65-6791-0676, E-mail: costab@ntu.edu.sg Received 27 June 2005; accepted 17 December 2005 Keywords: Ammonia-oxidizing bacteria, biofilm, confocal laser scanning microscopy, fluorescence in situ hybrid- ization, microbial aggregates, oligonucleotide probe, shrimp cultivation Summary Formation and activity of bacterial nitrifying biofilms play an important role in the closed seawater systems for shrimp cultivation. The structure of microbial biofilm on empty oyster shells, used as a biofilm carrier in biofil- tration of aquacultural water, was studied using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy. FISH was performed with specific oligonucleotide probes for Bacteria and ammonia- oxidizing Nitrosomonas spp. The bacterial cells were arranged within the biofilm as a layer of vertically elongated aggregates. Aggregates of ammonia-oxidizing bacteria were embedded within the matrix formed by other bacteria. Vertically elongated cell aggregates may be ecologically important in bacterial biofilms because they have a higher surface-to-volume ratio than that of laminated biofilms. Introduction Accumulation of toxic ammonia in aquacultural water is a major problem in intensive shrimp culture (Briggs & Funge-Smith 1994; Lin & Chen 2001; Jackson et al. 2003; Kir et al. 2004). Activity of nitrifying biofilms plays an important role in the functioning of closed seawater aquacultural systems by reducing the levels of ammonia in it (Thompson et al. 2002; Tal et al. 2003). There has been extensive research on the structure of microbial biofilms, which are used in aquatic biosystems for water treatment (Cooksey & Wigglesworth-Cooksey 1995; Gillan & De Ridder 1997; Fenchel 1998; Knoll et al. 2001). One of most informative approaches in microbial biofilm studies is application of specific flu- orochromes, fluorescent-labelled antibodies, lectins, and rRNA-targeted oligonucleotide probes, which are detected by confocal laser scanning microscopy (Norton et al. 1998; Neu 2000; Knoll et al. 2001; Strathmann et al. 2002; Venugopalan et al. 2005). It is known that oxidation of organic matter by het- erotrophic bacteria and oxidation of ammonia by nitrifying bacteria occur simultaneously in microbial biofilms. This may lead to competition for oxygen among these bacterial groups. Biofilms, which oxidize simultaneously organic matter and ammonia, consist of either microbial colonies embedded in slime attached to a carrier surface (Okabe et al. 1999) or microbial flocs anchored to the carrier (Kim et al. 2000). Representa- tives of genus Nitrosomonas are usually mainly ammo- nia-oxidizing microorganisms in marine recirculating aquaculture systems (Schramm et al. 1996; Tal et al. 2003). Ammonia-oxidizers from the genus Nitrosomonas form a dense layer of cell clusters in the upper layer of the biofilm (Schramm et al. 1996). However, it has been shown in other studies that ammonia-oxidizing bacteria are present only in the deeper layer of the plain or spherical biofilm (Bishop & Tong 1999; Tay et al. 2002; Ivanov et al. 2005). It is not sufficiently clear how the biofilm of marine recirculating aquaculture systems, containing hetero- trophic and ammonia-oxidizing bacteria, is arranged. Data on biofilm structure and formation can be used in the design of optimal marine recirculating aquaculture systems and in evaluation of different carriers for microbial biofilm. World Journal of Microbiology & Biotechnology (2006) 22: 807–812 Ó Springer 2006 DOI 10.1007/s11274-005-9107-z