Received: 1 May, 2009. Accepted: 27 October, 2009. Original Research Paper Dynamic Biochemistry, Process Biotechnology and Molecular Biology ©2009 Global Science Books Effects of Substrate Composition on Growth and Fructo-Oligosaccharide Production by Gliocladium virens Rosemeire A. B. Pessoni 1 • Kelly Simões 2 • Marcia R. Braga 2 • Rita de Cássia L. Figueiredo-Ribeiro 2* 1 Faculdade de Ciências Biológicas e da Saúde, Universidade Metodista de São Paulo, CP 5002, 09735-460 São Bernardo do Campo, SP, Brazil 2 Seção Fisiologia e Bioquímica de Plantas, Instituto de Botânica, CP 3005, 01061-970 São Paulo, SP, Brazil Corresponding author: * ritarib@usp.br ABSTRACT Several species of filamentous fungi isolated from the rhizosphere of Asteraceae from the Brazilian cerrado have been shown to produce and metabolise fructose-containing sugars. Among them is Gliocladium virens, isolated from the rhizosphere of Vernonia herbacea, an inulin-accumulating species. In the present work, we investigated the ability of G. virens to produce fructo-oligosaccharides (FOS) when sucrose was used as the carbon source. We also studied the growth and FOS production in cultures of this fungus fed with different nitrogen sources. Significant increases in mycelium dry matter and production of FOS were observed when the sucrose concentration in the culture medium was increased to 3%. 1-Kestose, nystose, and 1-F-fructofuranosylnystose were the main FOS detected in fluids of G. virens cultured up to 18 d on 3% sucrose-containing media. The addition of complex sources of nitrogen, such as corn and yeast extracts, increased biomass production and reduced the content of extracellular proteins when G. virens was cultured in a sucrose-containing medium. Production of FOS was detected during the fungal growth cycle and was not affected by the nitrogen source. Although the production of oligo-fructans has gained tremendous commercial importance, only few microorganisms have the potential for industrial application. In this context, it is worth to find microbes from unexplored environments with the ability to synthesise these products. G. virens isolated from the rhizosphere of tropical plants has shown the ability to produce FOS, indicating that the Brazilian cerrado represents a profitable environment to search these microbes. _____________________________________________________________________________________________________________ Keywords: filamentous fungus, nitrogen sources, oligofructan production, sucrose INTRODUCTION Fructans are fructose-based oligo- and polysaccharides that are the main reserve carbohydrate in about 15% of flower- ing plants (Hendry and Wallace 1993) and in microbes, where they serve different functions (Heyer and Wenden- burg 2001). In plants, these carbohydrates are built up by the successive addition of fructose residues with different linkages to three trisaccharides, 1-kestose (1-F-fructosyl sucrose), 6-kestose (6-F-fructosyl sucrose) and neokestose (6-G-fructosyl sucrose), resulting in structurally different types of fructans, such as inulin, levan, mixed fructans and the so-called neo-series (Carvalho et al. 2007). In bacteria, fructans are generally of the levan type and produced by extracellular enzymes that use sucrose as a substrate, syn- thesising very high molecular mass polymers. Series of inulin-based, low or high molecular mass fructans are also synthesised by fungi, although less information on fungal fructans is available in the literature than for plants and bac- teria (Heyer and Wendenburg 2001; Velázquez-Hernández et al. 2009). Production of fructans has been reported for example in Aspergillus sydowi (Kawai et al. 1973), A. niger (Hirayama et al. 1989), A. foetidus (Rehm et al. 1998), A. aculeatus (Ghazi et al. 2007), Penicillium chrysogenum (Olah et al. 1993), Fusarium oxysporum (Patel et al. 1997) and Rhodotorula sp. (Hernalsteens and Maugeri 2008). In contrast to plants, which need at least two distinct enzymatic activities to catalyse priming and elongation of the fructan chain, microbes require only a single fructosyl- transferase activity for both reactions. Microbial -fructo- furanosidases are able to transfer a fructosyl residue to suc- rose or to a fructan acceptor molecule through different gly- cosidic bonds, depending on the enzyme source (Yun 1996; Velázquez-Hernández et al. 2009). Commercial production of FOS comes mainly from the enzymatic transformation of sucrose by -fructosyltransferases from microorganisms (Maiorano et al. 2008). Currently, Aspergillus fructosyl- transferase is the main industrial producer of inulin-type FOS, synthesising mixtures of oligosaccharides with 1-kes- tose, nystose, and 1-F-fructofuranosylnystose as the main components (Sangeetha et al. 2005; Linde et al. 2009). Interest in fructans and FOS has increased steadily since they are considered to be functional food ingredients acting as prebiotics and therefore being beneficial to human nutri- tion (Heyer and Wendenburg 2001). Studies have demons- trated that these sugars are low-calorie and non-cariogenic sweeteners that stimulate selectively the growth of bifido- bacteria. They have also been claimed to contribute to the prevention of colon cancer and to reduce cholesterol, phos- pholipids and triglyceride levels in serum (Ritsema and Smeekens 2003; Maiorano et al. 2008). A number of stu- dies have been carried out on methods of producing fruc- tose syrup or FOS from inulin and sucrose; many of these methods include the use of microbial -fructofuranosidases (Sangeetha et al. 2005). Therefore, the search for microor- ganisms capable of synthesising oligofructans is relevant for the industrial production of FOS (Linde et al. 2009). Although the production of FOS has been widely re- ported in microorganisms (Maiorano et al. 2008), most re- search has been carried out with Aspergillus and Penicil- lium spp. (Hayashi et al. 2000; Sangeetha et al. 2004). Little is known about the production of FOS by microbes from the rhizosphere of tropical plants. The savanna vegetation of the Brazilian cerrado in- cludes a number of native Asteraceae species that accumu- late inulin-type fructans in their thickened underground organs (Carvalho et al. 2007 and refs. therein). The rhizo- sphere of these species has been used as a source of micro- ®