311 JOURNAL OF BIOSCIENCE AND BIOENGINEERING © 2007, The Society for Biotechnology, Japan Vol. 103, No. 4, 311–317. 2007 DOI: 10.1263/jbb.103.311 Acetylation of Loofa (Luffa cylindrica) Sponge as Immobilization Carrier for Bioprocesses Involving Cellulase Akihiro Hideno, 1 James C. Ogbonna, 2 * Hideki Aoyagi, 1 and Hideo Tanaka 1 Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8572, Japan 1 and Department of Microbiology, University of Nigeria, University Road, Nsukka, Enugu, Nigeria 2 Received 7 November 2006/Accepted 5 January 2007 The feasibility of using loofa sponge for immobilization of cellulase-producing microorganisms was investigated by acetylating loofa sponge. Acetylation was achieved by autoclaving process of loofa sponge immersed in acetic anhydride at various temperatures for various times. The degree of acetylation, as inferred by the weight percentage gain (WPG), was enhanced by increasing both temperature and the duration of acetylation. The acetylation of a piece of loofa sponge in an auto- clave at 120°C for 20 min resulted in a WPG of about 8%, which was sufficient to protect the loofa sponge against cellulose degradation. The acetylated loofa sponge prepared under this condition was not decomposed by commercial cellulase and its structure was maintained for more than 720 h during repeated-batch treatments with commercial cellulase. A flocculating yeast (Saccha- romyces cerevisiae IR-2) and a fungus (Trichoderma reesei QM9414) were successfully immobi- lized in the acetylated loofa sponge. In each case, the percentage of immobilized cells was as high as that obtained using nonacetylated loofa sponge. Acetylation had no adverse effects on cell growth and immobilization of T. reesei QM9414, as well as on cell growth and ethanol production by S. cerevisiae IR-2. T. reesei QM9414 immobilized on an acetylated loofa sponge was success- fully used for repeated-batch cellulase production from commercial cellulose powder. Although the acetylated loofa sponge showed a slight weight loss, it was not disintegrated by activated sludge. The results obtained in this study showed that acetylated loofa sponge is suitable as an im- mobilization carrier for bioprocesses involving cellulase. [Key words: loofa (Luffa cylindrica) sponge, acetylation, cell immobilization, cellulase, flocculating yeast, fungus] Cell immobilization techniques using biological materials are ecofriendly and have many advantages over suspended cell systems. Many studies have shown that the adsorption method for immobilization of microorganisms has many ad- vantages over the entrapment method using gel beads (1, 2). Many synthetic materials such as polyurethane foams, poly- ester foams, and synthetic cellulose sponges have been used as carriers for the adsorption method. However, these mate- rials are often very expensive and, because they are nonbio- degradable, their disposal causes a lot of environmental problems after their use. It is therefore desirable to use re- newable and biodegradable natural materials as carriers for cell immobilization. It has been reported that loofa (Luffa cylindrica) sponge is an excellent carrier for immobilization of microorganisms and plants and animal cells (3–6). However, loofa sponge is a lignocellulosic material composed mainly of cellulose, hemicellulose, and lignin (7). The chemical composition of this material makes it susceptible to degradation by cellu- lase. Thus, it is not suitable for immobilization of cells in bioprocesses involving production or utilization of cellu- lase, such as those in cellulase production, ethanol produc- tion from lignocellulosic biomass, and wastewater treatment using activated sludge. Recently, production of renewable energy from biomass materials has been emphasized as a means of solving cur- rent environmental problems such as global warming. In this regard, production of ethanol from lignocellulosic ma- terials has a great potential because such materials are the cheapest and the most abundant biomass materials on earth. Among the systems for ethanol production from lignocel- lulose, the simultaneous saccharification and fermentation (SSF) process seemed to be one of the promising options (8, 9). In this process, cellulase enzymes or cellulase-pro- ducing microorganisms are used for saccharification. The use of immobilized cells for this SSF process requires that the carrier used for immobilization of both the cellulase- producing strain and the ethanol-producing strain must be resistant to cellulase because both of them are in constant contact with the enzyme. The treatment of wastewater using immobilized cells has * Corresponding author. e-mail: jctogbonna@yahoo.com phone: +81-(0)29-853-7212 fax: +81-(0)29-853-4605