Optimization of inulinase production by solid-state fermentation using sugarcane bagasse as substrate Marcio Mazutti, Jo˜ ao Paulo Bender, Helen Treichel, Marco Di Luccio ∗ Universidade Regional Integrada do Alto Uruguai e das Miss˜ oes Campus de Erechim, Departamento de Engenharia de Alimentos, Av. Sete de Setembro 1621, Erechim 99700-000, Brazil Abstract The production of enzymes by bioprocesses is a good alternative to add value to agroindustry residues. Sugarcane bagasse is an abundant by-product of sugar industry and was tested as support and carbon source for production of inulinase (2,1--d-fructanohydrolase, E.C. 3.2.1.7) from Kluyveromyces marxianus NRRL Y-7571 by solid-state fermentation. Corn steep liquor was used as nitrogen supplement. Factorial design and response surface analysis were carried out to evaluate the effects of temperature (30.4–41.6 ◦ C) and corn steep liquor (13–27.1%, w/v) on the production of inulinase. Optimum fermentation conditions were found to be: 36 ◦ C and 20 wt.% of corn steep liquor. Under optimized conditions, the extra-cellular enzyme concentration reached 391.9 U/g of dry fermented bagasse. Keywords: Inulinase; Optimization; Solid-state fermentation; Sugarcane; Bagasse 1. Introduction Solid-state fermentation (SSF) may be defined as a fermenta- tion process where the microorganisms grow in solid substrates with low water concentration [1–3]. Many studies about the application of SSF are focused in adding value to agroindustry residues, which have been extensively used as physical support or source of nutrients in SSF [2,4–7]. Brazil is known as one of the greatest producers of sugar from sugarcane in the world [8]. Sugarcane production in 2004 was 410 million t per month. The crop is mainly directed for production of ethyl alcohol, sugar and spirits [9]. Sugarcane bagasse is a by-product resulting from juice extraction. This waste basically consists of 50% of cellulose, 30% sugars and 2.4% of ashes [7]. The production of enzymes by SSF has gained much attention in biotechnology studies for production of lipases [5], inulinases [2], proteases [10], etc. The use of low cost residues, higher productivities, low energy requirements, lower wastewater pro- ∗ Corresponding author. Tel.: +55 54 520 9000; fax: +55 54 520 9090. E-mail address: diluccio@uricer.edu.br (M.D. Luccio). duction, extended stability of products and low production costs are some of the main advantages of SSF [3,11]. The selection of a suitable microorganism is an important aspect of SSF for production of enzymes [3]. The microorganism should be able to grow at low water activity, to be GRAS (“Generally Rec- ognized as Safe”) and be accepted by FDA (“Food and Drug Administration”) [12]. Inulinase production by Kluyveromyces marxianus NRRL Y- 7571 is then of great interest, since it attends the requirements of GRAS and just a few studies of the production of inulinase by SSF using this yeast are reported [2]. Inulinases are potentially useful enzymes for production of high fructose syrups (HFS) from inulin [13]. Fructose produc- tion by inulin hydrolysis is more advantageous than conventional process based on starch, which includes the action of -amylase, amyloglucosidase and glucose isomerase, yielding only 45% of fructose in the final product due to the thermodynamical equi- librium of the reaction. Inulinase based hydrolysis of inulin can yield products with 95% of fructose [14]. This work aimed to optimize inulinase production by SSF using sugarcane bagasse and corn steep liquor as substrates. The optimization was carried out by experimental design and surface analysis methodology.