214 JOURNAL OF BIOSCIENCE AND BIOENGINEERING © 2007, The Society for Biotechnology, Japan Vol. 104, No. 3, 214–217. 2007 DOI: 10.1263/jbb.104.214 Alginate-Loofa as Carrier Matrix for Ethanol Production Muenduen Phisalaphong, 1 * Rusdianto Budiraharjo, 1 Phoowit Bangrak, 1 Jirawan Mongkolkajit, 1 and Savitree Limtong 2 Department of Chemical Engineering, Chulalongkorn University, Bangkok 10330, Thailand 1 and Department of Microbiology, Kasetsart University, Bangkok 10900, Thailand 2 Received 7 May 2007/Accepted 22 June 2007 An alginate-loofa matrix was developed as a cell carrier for ethanol fermentation owing to its porous structure and strong fibrous nature. The matrix was effective for cell immobilization and had good mechanical strength and stability for long-term use. After a storage period of 4 months, yeast cells remained firmly immobilized and active. [Key words: loofa sponge, alginate, yeast, ethanol production] In response to energy crisis, ethanol has re-emerged as an alternative to, or extender for, petroleum-based liquid fuels. Ethanol production using an immobilized cell system offers many advantages such as higher productivity and protection of cells from inhibitions. Cell entrapment within alginate is one of the most widely studied because cell viability and ac- tivity are kept very high (1). However, the practical applica- tion of polymeric gel carriers including alginate beads has been limited by the problems of gel degradation, low physi- cal strength, and severe mass transfer limitation (1–8). Fur- thermore, large-scale production of these carriers often re- quires complex and sophisticated equipment leading to high cost of production (2). On the other hand, loofa sponges, lignocellulosic matrices from Luffa cylindrica, were found to be promising cell carriers for ethanol production by floc- culating cells (2–5). The sponges are light, strong, chemi- cally stable, and composed of interconnecting voids within an open network of fibers. Because of the random lattices of small cross sections of the sponges coupled with high po- rosity, the sponges are suitable for cell adhesion. Continu- ous fuel ethanol production had been realized using yeast cells immobilized in loofa sponges in a bubble column con- figuration (5). However, a low-shear environment and large aggregates of cells were required in order to prevent exces- sive cell sloughing from the carriers (3, 9). On the basis of the above, in the present work, we fo- cused on developing a new cell carrier by combining algi- nate gel and loofa sponge namely, the alginate-loofa matrix (ALM). Ethanol production by repeated batch fermentation using yeast cells immobilized within the ALM was then ex- amined and compared with that using suspended cells and cells immobilized in conventional calcium alginate beads. MATERIALS AND METHODS Yeast strains Saccharomyces cerevisiae M30 selected on the basis of its high efficiency in ethanol production from molasses at high temperature was used in this study. Culture media and cell preparation Starter cultures were prepared by transferring cells from stock PDA slants to 150 ml of sterilized medium followed by incubation at 33 °C, 150 rpm for 20 h. The medium for the starter culture contained 0.05% ammo- nium sulfate and 5% inverse sugar from palm sugar at pH 5.0. After that, the obtained cell suspension was concentrated by de- cantation and then transferred to the main culture. Cells immobilized on alginate-loofa matrix Sodium algi- nate (3% w/v) solution was formulated by dissolving Na-alginate powder in 0.9% (w/v) NaCl solution. It was autoclaved for 5 min at 121°C and kept overnight at 4°C to facilitate deaeration. Cell suspension of 5 ml was then added to 50 ml of 3% (w/v) alginate solution to form an alginate-cell mixture. To form ALM, 2 g of sterilized cubic sponges of loofa (8 × 8 × 2 mm) was dipped into the alginate-cell mixture. The gel carriers were transferred to 1.47% (w/v) CaCl 2 solution and left to harden in this solution with mild stirring for 15 min. The carriers were then rinsed 3 times with 0.9% (w/v) NaCl solution. Carriers were prepared under aseptic condi- tions and the average size of ALM was 9 × 9 × 3 mm 3 . Fermentations Repeated batch fermentations were carried out in duplicate using a medium contained 0.05% ammonium sul- fate and 21% (w/v) inverse sugar from cane molasses at pH 5.0. The prepared medium was sterilized at 121°C for 20 min. Experi- ments were initiated by transferring prepared cell suspension or immobilized cells into 250 ml of the medium in 500 ml Erlenm- eyer flasks. Fermentation flasks were then shaken in the incubator at 150 rpm, 33 °C for 48 h. The experiments were monitored by re- moving 2 ml samples every 6 h for cell, sugar and ethanol analyses. Analytical methods Free cell dry weight was determined from the absorbance at 660 nm with a UV-2450 UV-visible spec- trophotometer and converted to dry cell concentration on the basis of a corresponding standard curve. For immobilized cells, a known mass of cell carriers was dissolved in 0.05 M sodium citrate. After the sponge was removed, immobilized cell concentration was de- termined similarly for the free cells. The concentration of ethanol was determined by gas chromatography using a Shimadzu model GC 7A G (Shimadzu, Kyoto) equipped with a flame ionization de- tector. To measure reducing sugar concentration, the sample solu- tion was hydrolyzed in 33% HCl at 100 °C for 10 min and neutral- ized with NaOH solution. Reducing sugar content was then deter- mined by the dinitrosalicylic acid method (10). * Corresponding author. e-mail: muenduen.p@chula.ac.th phone: +66-2-218-6875 fax: +66-2-218-6877