Enhanced saccharification of alkali-treated rice straw by cellulase from Trametes hirsuta and statistical optimization of hydrolysis conditions by RSM Marimuthu Jeya a,b , Ye-Wang Zhang a , In-Won Kim a , Jung-Kul Lee a,b, * a Department of Chemical Engineering, Konkuk University, Seoul 143-701, Republic of Korea b Institute of Biomedical Science and Technology, Konkuk University, Seoul 143-701, Republic of Korea article info Article history: Received 17 February 2009 Received in revised form 17 May 2009 Accepted 20 May 2009 Available online 18 June 2009 Keywords: Cellulase Rice straw RSM optimization Saccharification Trametes hirsuta abstract A white rot fungus, identified as Trametes hirsuta based on morphological and phylogenetic analysis, was found to contain efficient cellulose degrading enzymes. The strain showed maximum endoglucanase (EG), cellobiohydrolase (CBH) and ß-glucosidase (BGL) activities of 55, 0.28 and 5.0 U/mg-protein, respec- tively. Rice straw was found to be a potentially good substrate for growth of T. hirsuta for cellulase pro- duction. Statistical experimental design was used to optimize hydrolysis parameters such as pH, temperature, and concentrations of substrates and enzymes to achieve the highest saccharification yield. Enzyme concentration was identified as the limiting factor for saccharification of rice straw. A maximum saccharification rate of 88% was obtained at an enzyme concentration of 37.5 FPU/g-substrate after opti- mization of the hydrolysis parameters. The results of a confirmation experiment under the optimum con- ditions agreed well with model predictions. T. hirsuta may be a good choice for the production of reducing sugars from cellulosic biomass. Ó 2009 Published by Elsevier Ltd. 1. Introduction Lignocellulosic materials are cheap renewable resources avail- able in large quantities (Zhang and Cai, 2008). Cellulose, the major fraction of lignocellulosic biomass, can be hydrolyzed to glucose by cellulase enzymes. Cellulosic biomass is an abundant global renewable resource and includes a wide variety of materials, including various agricultural residues, fruit and vegetable wastes, woods, municipal solid wastes, wastes from the pulp and paper industry, as well as herbaceous energy crops. The degradation of cellulosic material is gaining increasing research attention due to its worldwide availability and the immense potential for its trans- formation into sugars, alternative fuels, and chemical feedstocks. In particular, there is intense interest in utilizing renewable cellulosic resources as starting materials for biofuel production (Cassman and Liska, 2007). There are three categories of cellulolytic enzymes that are required for the complete breakdown of cellulose to simple sug- ars. Endoglucanase (EG) (1,4-ß-D-glucan-4-glucano-hydrolases; EC3.2.1.74) cleaves glycosidic bonds randomly within the interior of cellulose polymer chain. Exoglucanases (EC 3.2.1.91 and EC 3.2.1.74) act progressively on the reducing or non-reducing ends of cellulose chains, releasing either cellobiose or glucose as major products. The ß-glucosidases (BGL) (EC 3.2.1.21) hydrolyze soluble cellodextrins and cellobiose to glucose (Eriksson et al., 1990; Saha et al., 1994). The steps involved in the production of fuels and chemicals from lignocellulosic biomass consist of feedstock preparation, pre- treatment, fractionation, enzymatic hydrolysis (saccharification), fermentation, product recovery, and waste treatment (Saha, 2004). Saccharification is the critical step for sugar production. The hydrolysis of cellulose can be affected by the porosity of ligno- cellulosic biomass, by cellulose crystallinity, and by lignin and hemicellulose content (Zhang and Cai, 2008). Pretreatment proce- dures are essential for removal of the hemicellulose and lignin, for reducing cellulose crystallinity and for increasing the porosity of the materials. Enzymatic saccharification of cellulosic biomass has been considered as an environmentally friendly method that replaces chemical sulfuric acid saccharification treatments. The conventional technique for the optimization of a multifac- torial system is to deal with one-factor at a time. However, this type of method is time-consuming and also does not reveal the alternative effects between components. In the study presented here, an attempt was made to employ response surface methodol- ogy (RSM) to identify the optimum conditions for reducing sugar production from rice straw by analyzing the relationships among a number of parameters that affect the overall process. Here we re- port on the enzymatic saccharification of alkali-treated rice straw and on the optimization of hydrolysis parameters for maximum sugar production. 0960-8524/$ - see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.biortech.2009.05.040 * Corresponding author. Address: Department of Chemical Engineering, Konkuk University, Seoul 143-701, Republic of Korea. Tel.: +82 2 450 3505; fax: +82 2 458 3504. E-mail address: jkrhee@konkuk.ac.kr (J.-K. Lee). Bioresource Technology 100 (2009) 5155–5161 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech