Introduction {#s1} ============ The field of biofuels, especially cellulosic ethanol, has been developed extensively in the last three decades ([@B10]). However, the use of agricultural residues in production of biofuels is not a new idea ([@B14]). In recent years, with the ever-increasing energy demand and environmental concerns, there has been a rising demand for renewable and efficient biofuels to replace the use of petroleum-based fuels. Among them, cellulosic ethanol is one of the most promising alternatives to the present fossil fuel-based fuel system due to the availability of the vast non-agricultural land ([@B34]). It is estimated that around the world 4 billion hectares of suitable agricultural lands are being utilized for crops used for feed, food, and energy purposes ([@B2]). In addition, due to the significant decline in the fossil fuel reserves, the ever-rising energy demand, environmental pollution, and the depletion of the existing sources of energy, there is a desperate need for alternative feedstock for biofuel production. A substantial amount of rice straw is generated as agricultural residues in Asia ([@B8]). One of the potential applications of rice straw is a biofuel as a second generation feedstock for biorefinery that may be used for producing biofuels through thermochemical, biochemical, and microbiological routes ([@B37]; [@B2]; [@B36]). Several studies have demonstrated that both rice straw and rice husks could be successfully converted into bioethanol by chemical, biological, and biotechnological routes ([@B16]; [@B21]; [@B13]). In addition to fuel, these residues also have the potential for producing a large amount of fiber that could be used as animal feed, soil conditioner, etc. ([@B21]). Since the biomass to biofuel conversion has been done commercially with corn stover using a multistage system of cellulolysis, lignocellulolysis, fermentation, pretreatment, and cellulose hydrolysis and saccharification, cellulosic ethanol from rice straw has also been studied to some extent for the past few decades ([@B18]). Based on *in vitro* saccharification assay, [@B24] demonstrated that rice straw biomass has higher sugar recovery compared to the same amount of wheat straw and corn stover. However, the saccharification efficiency obtained by these experiments is highly dependent on the composition of the biomass (cellulose, hemicellulose, lignin, etc.) as well as the method used for biomass pretreatment ([@B12]). Many reports have suggested that the cellulose to ethanol conversion rate could be significantly increased if hemicellulose is removed before hydrolysis because of its inhibitory effects on cellulose hydrolysis ([@B26]). Therefore, before any pretreatment, it is necessary to remove a substantial amount of hemicellulose in order to improve the cellulose to ethanol conversion efficiency. The current knowledge on the effects of different rice straw varieties on the saccharification efficiency are limited ([@B3]). However, more recent studies have indicated that hemicellulose removal from biomass has a great influence on the quality of hydrolysis liquor and the subsequent hydrolysis of cellulose ([@B38]; [@B26]). One of the approaches is the use of hemicellulase enzymes in the pretreatment and hydrolysis of cellulosic biomass. The most important property of hemicellulase enzymes is the ability to remove a significant amount of hemicellulose in the pretreatment step. Most studies to date have focused on cellulase enzymes. The main role of cellulase enzymes is cellulose hydrolysis into monomeric glucose in the saccharification step. However, some researchers have investigated the use of these enzymes in rice straw pretreatment for enhancing hemicellulose removal and improving saccharification efficiency ([@B30]). This study evaluated the effects of cellulase, xylanase and laccase pretreatment on the efficiency of steam explosion using rice straw as feedstock. The purpose of this study was to investigate the effect of the combination of enzymes used on the saccharification efficiency of the rice straw pretreated by the steam explosion process. Materials and Methods {#s2} ===================== Biomass Handling {#s2_1} ---------------- This experiment was carried out at the Food Processing Laboratory, University of California, Riverside, CA. In this study, rice straw was taken from the campus field. Two varieties of rice straw, one red colored (RS-red) and another brown colored (RS-brown), were randomly selected. Rice straw was threshed and air dried and then milled using a Wiley mill with a 1-mm sieve. The particle size was about 0.5--3.0 mm. Chemicals {#s2_2} --------- Glucose, xylose, and laccase used in this study were purchased from Sigma-Aldrich (St. Louis, MO). Hydrolyzed cellulase, xylanase, and laccase used in this study were purchased from Novozymes (Bagsvaerd, Denmark). All chemicals were stored in a refrigerator at 4°C. Steam Explosion Pretreatment {#s2_3} ---------------------------- The untreated and pretreated rice straw (0.25 g) was loaded into a 15-ml, 5-mm diameter quartz reactor with a 50-ml capacity. The water to solid ratio was 15:1 (w/w) and the reactor was placed in an oil bath for steam explosion pretreatment for 5 min at 190°C. The pressure in the reactor was adjusted to 2.5 atm. After pretreatment, the pretreated material was separated from the liquid under vacuum (1.0 Torr). The solids were dried for 12 h at 80°C and then used for composition analysis. Analysis {#s2_4} -------- Compositional analysis was conducted using the Association of Official Analytical Chemists (AOAC) standard methods of analysis ([@B34]). The ash content was determined after burning at 550°C for 4 h. The lignin and carbohydrate content were determined as acid-insoluble ash and acid-soluble lignin after ashing samples at 550°C for 4 h. Cellulose, hemicellulose, and lignin were determined using the Van Soest method ([@B34]). The total lignin content was estimated by multiplying the acid-insoluble lignin content by a factor of 0.85. The conversion efficiency of substrate sugars was determined by the following formula: C S = C T ( G L ) × 100 \% where *CS* is the conversion rate (%), *CL* is the cellulose, hemicellulose and lignin content in the untreated sample (%), and *GL* is the weight of glucan in the untreated sample. The chemical pretreatment was performed in a 25 ml volume at 4 mg/ml (protein). Glucan and xylan were determined by enzymatic hydrolysis using Whatman no. 1 filter paper ([@B34]). The chemical-treated sample was filtered from the liquid, washed thrice with deionized water, and then dried in an oven at 80°C for 24 h. The sample was transferred to a tube and dried in a 60°C oven until the weight of the sample is constant. The sugars produced from hydrolysis of each substrate were collected by evaporation and was then concentrated to 1 ml for quantitation. The final saccharification yield was expressed in terms of percentage. To determine glucose yield after acid hydrolysis, the treated solids were analyzed for the presence of glucose and xylose using High-Performance Liquid Chromatography (HPLC) analysis. After the enzymatic saccharification was completed, the sugar samples were diluted with deionized water and analyzed for glucose and xylose using an HPLC system (Agilent, model 1260 Infinity). The flow rate of the mobile phase was 0.6 ml/min with a 5-mm HPX-87H column and a Shodex RI-71 detector. The sample concentration was 100 mg/ml. Laccase Treatment {#s2_5} ----------------- Laccase pretreatment (1.0 ml, 1.52 mM) was done in 15-ml Pyrex bottle in a water bath at 65°C. After pretreatment, the pretreated material was separated from the liquid under vacuum (1.0 Torr). The saccharification of the pretreated material was determined by the same procedures described for composition analysis. Enzyme Treatment {#s2_6} ---------------- The pretreated solids (5 g) were mixed with the cellulase (3 ml) and incubated in a water bath shaker at a constant speed of 300 rpm at 50°C for 6 h. Enzyme hydrolysis was stopped by cooling the sample at 4°C and then centrifuged for 15 min at 10,000 rpm. The supernatant was diluted with 10-ml DI water and then analyzed for glucose and xylose using the aforementioned HPLC system. The weight of solids in the supernatant was determined by the difference between the dry weight and supernatant weight. The solids were dried in an oven at 80°C for 24 h. Statistical Analysis {#s2_7} -------------------- All experiments were conducted in triplicate. The data was subjected to analysis of variance (ANOVA), and the mean values with standard deviations were compared by one-way ANOVA using the Bonferroni multiple comparison test (SAS version 9.2). Results {#s3} ======= Composition Analysis of the Rice Straw Feedstock {#s3_1} ------------------------------------------------