SHORT COMMUNICATION Cellulase-producing Bacillus strains isolated from the intestine of Amazon basin fish Samanta Balsini Peixoto, Florencia Cladera-Olivera, Daniel Joner Daroit & Adriano Brandelli Laborato¤ rio de Bioqu|¤ mica e Microbiologia Aplicada, Instituto de Cie Œ ncia eTecnologia de Alimentos, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil Correspondence: A Brandelli, Laborato¤ rio de Bioqu|¤mica e Microbiologia Aplicada, Instituto de Cie Œ ncia eTecnologia de Alimentos, Uni- versidade Federal do Rio Grande do Sul, Av. Bento Gonc° alves 9500,91501-970 Porto Alegre, RS, Brazil. E-mail: abrand@ufrgs.br The intensi¢cation of aquaculture practices has gen- erated a demand for alternative feed ingredients. In this sense, plant biomass is an inexpensive and highly available nutrient source mainly composed of cellulose, a recalcitrant polymer consisting of glucose units linked by b -1,4-glucosidic bonds (Lynd,Weimer, van Zyl & Pretorius 2002). As ¢sh usually do not produce cellulolytic en- zymes, the cellulase activity observed in ¢sh is mainly produced by the intestinal microbiota (Saha & Ray 1998). Various cellulolytic bacteria have been isolated from ¢sh intestines (Bairagi, Ghosh, Sen & Ray 2002; Saha, Roy, Sen & Ray 2006; Kar & Ghosh 2008; Mondal, Roy, Sen & Ray 2008), which may contribute to the digestion of plant materials. The proposed mechanism of enzymatic cellulolysis, based on fungal cellulase systems, is essentially com- posed of endo-b-1,4-glucanase, exo-b-1,4-glucanase (cellobiohydrolase) and b-glucosidase (cellobiase) activities. The endo- and exo-glucanases act syner- gistically to produce mainly cellobiose, which is cleaved by b -glucosidases (Singh & Hayashi 1995; Lynd et al . 2002). The South American warm water teleosts pacu ( Piaractus mesopotamicus Holmberg 1887) and piau- com-pinta ( Leporinus friderici Bloch 1794) possess herbivorous/omnivorous feeding habits (Albrecht, Ferreira & Caramaschi 2001; Abimorad, Carneiro & Urbinati 2007). Because of the ingestion of cellulosic materials, herbivorous/omnivorous ¢sh often har- bour cellulase-producing intestinal microorganisms. As the intestinal microbiota of Amazon basin ¢sh is poorly studied, and microbial diversity is a major re- source for novel products and processes in aquacul- ture and biotechnology, the present study evaluated the cellulolytic ability of bacterial strains isolated from the intestine of Amazonian ¢sh. Bacterial strains, P6 and P11, were isolated pre- viously from the midgut of P. mesopotamicus and L. friderici , respectively, on Luria^Bertani (LB) agar plates (10 g L À 1 lacto-peptone, 5 g L À 1 yeast extract, 10 g L À 1 NaCl and 15 g L À 1 agar) for 24 h at 30 1C. The strains were identi¢ed as Bacillus species based on 16S rDNA sequencing, clustering with Bacillus subtilis and Bacillus velesensis respectively (Giongo, Lucas, Casarin, Heeb & Brandelli 2007). Strains were maintained on LB agar plates at 4 1C. These bacteria were kindly provided by Dr Spartaco Astol¢-Filho (UFAM, Manaus, Brazil). Bacterial growth was inves- tigated on LB agar plates at di¡erent temperatures (15^55 1C) for 24h. In submerged cultivations, LB broths with di¡erent pH (5.0^10.0, adjusted with either 5 M NaOH or 5 M HCl) were inoculated, incu- bated at 30 or 37 1C and growth was determined by optical density at 600 nm. Alternatively, bacteria were cultivated in 250 mL Erlenmeyer £asks containing 50 mL of Bushnell^ Haas medium (0.2 g L À 1 MgSO 4 , 0.02 g L À 1 CaCl 2 , 1gL À 1 KH 2 PO 4 ,1gL À 1 K 2 HPO 4 ,1gL À 1 NH 4 NO 3 and 0.05 g L À 1 FeCl 3 , pH 7.0) plus10 g L À 1 of di¡erent carbon sources (Table 1), and incubated at 30 1C for 24 h with shaking (125 rpm). At de¢ned periods, ali- quots of culture were centrifuged (10 000 g for 10 min), the supernatant was ¢ltered (0.22 mm mem- brane) and used as a crude extracellular enzyme (Giongo et al . 2007). The pellet was washed with Aquaculture Research, 2011, 42 , 887^891 doi: 10.1111/j.1365-2109.2010.02727.x r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd 887