Effect of different cellulase dosages on cell viability and ethanol production by Kluyveromyces marxianus in SSF processes E. Tomás-Pejó * , M. García-Aparicio, M.J. Negro, J.M. Oliva, M. Ballesteros Renewable Energies Department-CIEMAT, Avda. Complutense, 22 28040-Madrid, Spain article info Article history: Received 9 April 2008 Received in revised form 3 July 2008 Accepted 12 July 2008 Available online 21 August 2008 Keywords: Bio-ethanol Cellulases Inhibition Cell growth Kluyveromyces marxianus abstract This study was aimed to study the effect of commercial cellulases (Celluclast 1.5 L FG) on Kluyveromyces marxianus CECT 10875 growth and ethanol production in SSF processes. Preliminary tests carried out in glucose (50 g/L) fermentation medium showed that high enzyme amounts (2.5–3.5 FPU/mL) could cause a negative effect on K. marxianus growth rate and viable cells number. However, the maximum ethanol production was not affected and about 86% of the theoretical (22 g/L) was reached in all cases indepen- dently of the enzyme dosage. In SSF experiments, cell viability was always affected by enzyme loading. Nervertheless, slight differences observed on cell viability during glucose fermentation processes with the detected concentrations of the additives did not justify the negative effect observed in SSF experiments. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Processes to obtain ethanol from lignocellulose based on enzy- matic hydrolysis are promising methods to produce bio-fuel with low cost. These processes consist basically of four stages: pretreat- ment, enzymatic hydrolysis, fermentation and distillation (Sun and Cheng, 2002). With pretreatment step the enzyme accessibility to cellulose is enhanced (Mosier et al., 2005), therefore, this process is directly related to the efficiency of cellulases to produce fer- mentable sugars. After pretreatment, enzymatic hydrolysis and fermentation can be carried out separately or simultaneously (Alfani et al., 2000). It has been shown that higher ethanol yields could be obtained in simultaneous saccharification and fermentation (SSF) processes compared with separate hydrolysis and fermentation (SHF) (Alfani et al., 2000; Stenberg et al., 2000; Tomás-Pejó et al., 2008). How- ever, one of the main drawbacks in an SSF process is the optimum temperature for both stages. Whereas, enzymatic hydrolysis has an optimum temperature around 50 °C, most fermenting microorgan- isms have an optimum temperature ranging between 30 and 37 °C (Alfani et al., 2000; Hari Krishna et al., 2001; Jorgensen et al., 2007). Therefore, ethanol yield could be improved in SSF processes by using thermotolerant yeasts such as Kluveromyces marxianus (Fonseca et al., 2007). It grows rapidly even at temperatures above 40 °C, it is very versatile and could be economically explored for a wide range of applications (Banat et al., 1998; Fonseca et al., 2007). In addition, there are several advantages which could be exploited with using thermotolerant yeast such as, energy savings through a reduction in cooling cost, higher saccharification yields, continuous ethanol removal and significant decreased risk of contamination (Banat et al., 1998; Limtong et al., 2007). An SSF process with K. marxianus is usually carried out at 42 °C, with an inoculum size in range 0.3–1.2 g/L, substrate concentration ranging from 5% to 10% (w/v) and 15 FPU/g cellulose of enzyme loading (Ballesteros et al., 2004). Taking into account the average glucose content in pretreated materials used as substrates for SSF processes (Ballesteros et al., 2004), substrate loadings about 15% (w/v) are needed to reach an ethanol concentration of 4% (v/v), which is considered as a benchmark for an economically viable dis- tillation (Wingren et al., 2003; Zacchi and Axelsson, 1989). So, higher matter content would imply, for the same cellulase concen- tration per gram of cellulose, higher volumes of enzyme solution in the SSF medium. The most commonly commercialized enzyme cocktails for lig- nocellulosic saccharification are mainly obtained from the filamen- tous fungus Trichoderma reesei (Howard et al., 2003). It produces cellulolytic enzymes by secreting an enzyme system capable to de- grade crystalline cellulose which consists of several cellobiohydro- lases, endoglucanases and ß-glucosidades (Domingues et al., 2000; Esterbauer et al., 1991). Previous studies in order to assess several available commercial cellulases have detected some additives in cellulase preparations from T. reesei (Golias et al., 2000; Nieves et al., 1998; Szczodrak and Targonski, 1987), which are suspected to be preservatives added to increase the product’s shelf-life (Golias et al., 2000). Furthermore, some researchers have pointed out that 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.07.012 * Corresponding author. Tel.: +34 91 346 6058; fax: +34 91 346 6037. E-mail address: mariaelia.tomas@ciemat.es (E. Tomás-Pejó). Bioresource Technology 100 (2009) 890–895 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech