Optimization of microwave pretreatment and enzymatic hydrolysis of pith bagasse with Trichoderma cellulase

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1 Indian Journal of Biotechnology Vol 13, January 014, pp Optimization of microwave pretreatment and enzymatic hydrolysis of pith bagasse with Trichoderma cellulase M A Farid 1 *, A M Noor El-Deen 1 and H M Shata 1 Natural and Microbial Products Department, National Research Center, Dokki, Cairo, Egypt Microbial Chemistry Department, National Research Center, Dokki, Cairo, Egypt Received 15 November 01; revised 18 June 013; accepted August 013 Pith bagasse was subjected to microwave/chemical pretreatments at 900 W under atmospheric pressure for a short time (-6 min) through several cycles (30 sec/cycle). The pith bagasse lost about 48, 1 and 40% of its wt after treatment with microwave/naoh, microwave/h SO 4 and microwave/h O /acetic acid, respectively. Enzymatic hydrolysis of pretreated substrates with Trichoderma cellulase indicated that pith bagasse pretreated with microwave/h O /acetic acid and microwave/naoh had the highest hydrolysis rate and sugar yield. The mean value for the total reducing sugars (TRS) and glucose yields from both treatments are approx 4 to 5 times than microwave/h SO 4 pretreated sample. Optimization of treatment process using Box-Behnken design at 6 and 4 h showed that the linear and quadratic terms of the 3 variables tested [H O /acetic acid treatment time (X 1 ), solid:liquid ratio (X ) and microwave treatment time (X 3 )] had significant effects. Maximal concentrations of TRS and glucose ( and mg/ml, respectively) could be obtained after 4 h hydrolysis time when X 1, X and X 3 were set at 60 min, 1:0 (w/v) and 4 min, respectively. The yield of TRS reached about 99% (g/g total dry matter). Keywords: Box-Behnken design, hydrolysis, microwave treatment, optimization, pith bagasse, Trichoderma cellulase Introduction Lignocellulosic materials are the most economical and highly renewable natural resources in the world with an estimated annual production of more than 150 billion tons on the earth 1. These materials are highly resistant to enzymatic hydrolysis. There are three major components in lignocellulosic materials: hemicellulose, lignin, and cellulose. The susceptibility of lignocellulosic materials to enzymatic saccharification of cellulose is limited due to the presence of the complex structure of lignin and hemicellulose with the cellulose. Therefore, various pretreatment techniques of lignocellulosic materials to enhance saccharification by cellulase enzyme are developed,3. A number of physical, chemical, and biological treatments have been reported for the delignification of crop residues to make cellulose more accessible to the enzymatic complex 4-7. A promising pretreatment method involves the application of microwave radiation to biomass in an aqueous environment. The rationale for microwave pretreatment stems from two aspects. First, from a *Author for correspondence: Tel: ; Fax: nrcfarid@yahoo.com physical aspect, microwave radiation supplies internal heat to the biomass resulting from the vibrations of polar bonds in the biomass and surrounding aqueous medium. The radiation generates a continuously changing magnetic field causing the polar bonds to vibrate as they align with the magnetic field. This disruption and shock to the polar bonds accelerates chemical, biological, and physical processes 8. Second, from a chemical aspect, the thermal treatment of lignocellulosic materials in an aqueous medium is known to release acetic acid, hence providing an acidic environment for auto-hydrolysis 9. The earliest known study involving microwave pretreatment has examined the effect of microwave radiation on rice straw and bagasse immersed in water 10. Other studies on sugar cane bagasse and rice straw have involved extraction of hemicellulose and lignin, following application of microwave radiation 3. Published work in this area has combined microwave radiation with alkali reagents in the pretreatment of rice straw and wheat straw 11,1. Some studies have shown microwave irradiation could change the ultra structure of cellulose, degrade lignin and hemicellulose in rice straw and increase the enzymatic susceptibility of the substrate Bagasse is the main by-product of sugar industry. It contains % carbohydrates, mostly in the form

2 FARID et al: MICROWAVE PRETREATMENT AND ENZYMATIC HYDROLYSIS OF PITH BAGASSE 99 of cell wall polysaccharides. It consists of fiber bundles and other structural elements like vessels, parenchyma and epithelial cells. The latter ones can be summarized under the technical term pith. The pith of bagasse has always been considered as an undesired raw material for pulping. The major limitation of bagasse pith as feed is its low digestibility due to association of lignin with cellulose and hemicellulose 16. In the present study, pith bagasse was chosen as a model of lignocellulosic material for microwave/ chemical pre-treatment and enzymatic hydrolysis because it is one of the main agricultural residues and has always been considered as an undesired raw material of sugar industry. Materials and Methods All experiments were carried out thrice and data presented in the text and tables were expressed as the mean values±standard deviation. The standard deviation values were expressed as the error bar on the graphs. Pith bagasse was obtained from El Hawamdia Company for Sugar and Distillation, Cairo, Egypt. Enzyme Source Crude cellulase [1,4-(1,3.1,4)-B-D-glucan 4- glucanohydrolase; EC ] was prepared from the culture filtrate of Trichoderma reesei NRRL 6156 [obtained from Agricultural Research Service (ARS) Culture Collection (NRRL), Peoria, Illinois, USA] using solid state fermentation (SSF). The enzyme was extracted by distilled water and lyophilized 17. Its carboxymethyl cellulase (CMC-ase) activity in international units (IU) was 8.3 IU/mg solid, measured as the initial rate of reducing sugar formation during hydrolysis of 1% carboxymethyl cellulose (CMC) at ph 4.6 and 50 o C 18 using phosphate-citrate buffer 19. Filter paper-cellulase (FP-ase) activity was IU/mg solid as measured also by the standard procedure recommended by the Commission on Biotechnology of the International Union of Pure and Applied Chemistry (IUPAC) 0. Its cellobiase activity in cellobiase units (CBU) was 0.08 CBU/mg, measured as the initial rate of hydrolysis of mm cellobiose to glucose at ph 4.6 and 50 o C 1. Sulphuric Acid and Sodium Hydroxide Pretreatment Biomass samples of 1 g pith bagasse were immersed in 0 ml of 3% (v/v) dilute sulphuric acid (H SO 4 ) or 3% (w/v) dilute sodium hydroxide (NaOH) individually in conical flasks of 100 ml capacity for 4 h at room temperature. They were then heated in a water bath for 30 min at 100 o C according to Keshwani et al. The residues were collected, washed extensively with tap water until neutral ph, dried at 50 o C and weighed. H O /Acetic Acid Pretreatment (PA) Mixture of equal volume of acetic acid (96%) and hydrogen peroxide (30%) was prepared. 1 g of pith bagasse was suspended, unless otherwise stated, in 0 ml H O /acetic acid solution in a conical flask, left for 4 h at room temperature and then heated at 100 o C in a water bath for 30 min. The samples were filtered, washed with tap water until acid-free (ph ), then suspended in 5 ml ethanol, filtered, air dried at room temperature and weighed. Microwave Pretreatment The microwave based pretreatments were carried out in a general purpose microwave oven made by JAC Corporation (Model NGM-35, China). The apparatus provided microwave radiation level of 900 W and microwave frequency of 450 MHz. The microwave pretreatment was carried out as follows. 1 g of pith bagasse sample suspended in 0 ml of each chemical reagent mentioned earlier in a 50 ml round bottom flask was covered with a small conical flask (50 ml) upside down on the opened side of the round bottom flask to decrease water evaporation. The flask was then positioned at the center of the rotating circular glass plate in the microwave oven and the pretreatment time was ranged from to 6 min. To avoid water evaporation and increasing the acidity or alkalinity during microwave treatment for long time, the treatment process was carried out through several cycles (30 sec/cycle). Distilled water may be added between the cycles to keep the reagent volume at the starting point. After microwave pretreatment the residues were collected, washed with tap water until neutral ph, dried at 50 o C and weighed. Their weight loss was determined before their enzymatic hydrolysis was carried out. Enzymatic Hydrolysis The hydrolysis experiments were carried out, according to Mandels et al 18 with some modifications in 5 ml conical flasks, containing 1 ml phosphatecitrate buffer, ph , 5 mg pith substrate and 0.5 ml of the enzyme solution (50 mg/ml crude enzyme). Unless otherwise stated, each experiment was run for 48 h in a shaking water bath thermostat at

3 100 INDIAN J BIOTECHNOL, JANUARY o C and at 180 rpm. In all experiments, the phosphate-citrate buffer was supplemented with antibiotics oxytetracycline (40 µg/ml) and fluconazole (30 µg/ml) to prevent microbial contamination. Samples were taken from the reaction mixtures at different times were heated immediately to 100 o C for 3 min, cooled to room temperature and then centrifuged for 10 min at 5000 rpm. The supernatant of each sample was used for determination of total reducing sugars (TRS) and glucose yield. Yields for TRS and glucose are defined as follows: ( ) ( ) ( ) Released reducing sugar mg Yield TRS (%) = Total dry matter TDM mg ( ) ( ) ( ) Released reducing sugar mg Yield Glucose (%) = Total dry matter TDM mg Where 0.9 is the mass ratio of anhydroglucose to its free glucose. Analysis The concentration of TRS in the hydrolysate was measured according to Somogyi 3 and Nelson 4. The glucose content in the hydrolysate was determined by means of a glucose-oxidase method 5. Glucose kits were obtained from BioMereuxs Vitek Inc., USA. Box-Behnken Design Response surface methodology (RSM) was used to optimize microwae/h O /acetic acid pretreatment of pith bagasse for enhanced enzymatic hydrolysis process using Box-Behnken design 6. The behavior of the system was explained by the following quadratic equation: Y=β0+βiXi+βijXiXj+βiiX i (1) Where Y is the predicted response variable, β0, βi, βii, βij are constant regression coefficients of the model, and Xi, Xj (i=1, 3; j=1, 3; i j) represent the independent variables in the form of coded values. Statistical software package Design-Expert (Version 8.0., State-Ease, Minneapolis, MN, USA) was used to design and analyze the experiment. A 3 factorial design with 5 replicates at the centre point and with total number of 17 trial was employed. The accuracy and general ability of the above polynomial model could be evaluated by the coefficient of determination R. The coded values of the variables at various levels are given in Table 1. Results and Discussion Effect of Different Chemical Treatments and Microwave/ Chemical Treatment on Weight (wt) Loss of Pith Bagasse In Table, comparative results are reported related to three microwave/chemical pretreatment processes: (a) alkaline treatment (NaOH), (b) acid treatment (H SO 4 ) and (c) H O /acetic acid treatment. The results indicate that sodium hydroxide pretreatment of pith bagasse resulted in the highest level of wt loss per cent (4.45%) in comparison to H O /acetic acid (40.4%) and sulphuric acid (30%). The main effect of NaOH pretreatment on lignocellulosic biomass is delignification by breaking the ester bonds crosslinking lignin and xylan, thus increasing the porosity of biomass 4. On the other hand, pretreatment of pith bagasse with microwave-assisted chemical pretreatment at 900 W for 4 min increased the wt loss per cent when combined with alkali treatment (48%) compared to the alkali treatment alone. These results are in accordance with that reported by Zhu et al 11,1, while working on microwave-assisted alkali pretreatment of wheat straw (WS) and rice straw (RS). They mentioned that the wt loss of WS and RS was due to the solubilization of lignin and hemicellulose. They proved that microwave/alkali could enhance some reactions in the pretreatment, but the detailed mechanism is still unclear 7. The authors also indicated that microwave irradiation could enhance the solubilization of hemicellulose in the NaOH aqueous solution, which led to a close contact between lignin and the alkaline solution and thus enhanced the solubilization of lignin. The results in Table also show that microwave/acid treatment had Table 1 Coded values of variables used in Box-Behnken design Independent variables Level X 1 : H O /Acetic acid treatment time (min) X : Solid: liquid ratio (w: v) 1:15 1:0 1:5 X 3 : Microwave treatment time (min) 4 6 Table Weight loss of pith bagasse after each treatment Pretreatment method % wt loss 3% NaOH* 4.49 * 3% H SO H O /Acetic acid* Microwave/3% NaOH/4 min Microwave/3% H SO 4 /4 min 1.44 Microwave/H O /Acetic acid /4 min 40.9 *The treatment was carried out for 4h at room temperature and then heated in a water bath for 30 min at 100 o C

4 FARID et al: MICROWAVE PRETREATMENT AND ENZYMATIC HYDROLYSIS OF PITH BAGASSE 101 little effect on wt loss, especially with H SO 4. It seems that microwave/h SO 4 has weak delignification ability and only removed a small part of lignin, since some polysaccharides, especially hemicelluloses, are hydrolyzed during acid pretreatment. Zhao et al 8 have also mentioned that the poor delignification ability of H SO 4 is still a limit of mild acid pretreatment, because lignin is believed to be a major hindrance in enzymatic hydrolysis. Enzymatic Hydrolysis of Pretreated Pith Bagasse In order to evaluate the effect of microwave/chemical pretreatment of pith bagasse on its enzymatic hydrolysis, the above mentioned microwave pretreatment procedures (microwave/ NaOH, microwave/h SO 4 and microwave/h O / acetic acid) were employed with their controls and enzymatic hydrolysis was evaluated in terms of TRS and glucose yield. The results in Fig. 1 show that pith bagasse treated by combined microwave-chemical pretreatments gave almost higher TRS and glucose yield than the substrates treated with chemicals alone. Fig. 1 Time course of enzymatic hydrolysis of pith bagasse treated by chemical pretreatments and microwave/chemical pretreatment processes. The results of hydrolysis of pith bagasse pretreated by microwave/naoh and microwave/h O /acetic acid were almost similar and had the highest hydrolysis rate. The hydrolysis rate of sample treated with microwave/h O /acetic acid was slightly greater than sample treated with microwave/naoh. The highest concentration of TRS reached after about 4 h for both the treatments. It was also noticed that the hydrolysate from pith bagasse treated by both treatments of microwave irradiation had higher sugar content compared to microwave/h SO 4 pretreatment. The mean values for TRS and glucose yields from microwave/naoh or microwave/h O /acetic acid pretreatment samples were approximately 4 to 5 times in comparison to the values for microwave/h SO 4 pretreated sample. These results justify that microwave could enhance the enzymatic hydrolysis of pith bagasse by removing more hemicellulose and lignin and increasing its accessibility to hydrolytic enzymes. The pretreatment of lignocellulosic materials with H O in the presence of acidic or alkaline medium greatly enhanced the susceptibility of these materials to enzymatic hydrolysis. About 50% of the lignin and most hemicellulose were solubilized by treating the biomass with % H O at 30 o C within 8 h, giving 95% efficiency of glucose production from cellulose by enzymatic hydrolysis 8,9. On the other hand, the key factor that affects the removal of hemicelluloses and lignin from cellulose in rice straw and bagasse is the high temperature, i.e., o C 3. At this high temperature range, the acetic acid contained in the hemicelluloses chains can provide hydrogen ions to promote hydrolysis process and catalyze lignin hydrolysis 3. Therefore, in the present study, the combination of acetic acid that released during the microwave treatment with added H O /acetic acid might enhance the removal of more lignin and decrease the crystallinity of cellulose. The release of acetic acid in the reaction mixture might lead to obvious swelling of cellulose, increasing its internal surface area and possibly reducing its crystalline structure. These in turn resulted in the breaking of the structural chain between lignin and the major structures, so more lignin was dissolved in the acetic acid solution 10,14. On the other hand, microwave irradiation could enhance the pentosans and lignin degradation reaction in NaOH aqueous solution and partially disrupt the lignin structure and expose more accessible surface area of cellulose to cellulase 7,30. The probable mechanism

5 10 INDIAN J BIOTECHNOL, JANUARY 014 mentioned is that microwave irradiation enhances the saponification of intermolecular ester bonds cross linking xylan hemicelluloses and other components, for example, lignin, and other hemicelluloses. Optimization of Microwave/H O /Acetic Acid Pretreatment on Enzymatic Hydrolysis of Pith Bagasse In order to approach the optimum response region of pith bagasse pretreatment for enzymatic hydrolysis, the effectiveness of independent variables including H O /acetic acid treatment time (X 1 ), solid:liquid ratio (X ) and microwave treatment time (X 3 ) were investigated, each at three levels as indicated in Table 1 according to the Box-Behnken design 6. The TRS and glucose concentrations produced during the reaction time after 6 h and 4 h were determined as responses. The experimental design and the results obtained for TRS and glucose after 6 h of hydrolysis time are presented in Table 3. The results show a considerable variation in the yield of both TRS and glucose under different treatment conditions. Treatment runs 3, 6, 7, 11 and 13 showed the maximum level of TRS (0.66 mg/ml), while the minimum yield (9.73 mg/ml) was observed in run number 9. These results were analyzed by standard analysis of variance (ANOVA). The following quadratic regression equation no. is obtained in terms of TRS yield released during the enzymatic hydrolysis of pretreated pith bagasse after 6 h. Y 1 (TRS after 6 h)= x X 1.95X X 1 X +.64X 1 X X X X 1.87X 3.65X 3 () As shown in Table 4, the model F-value of 9.68 for TRS yield after 6 h of hydrolysis time implies that the model is significant as also evident from the Fisher's F-test with a very low probability value [(P model > F)=0.0034]. In this case, X 3, X 1 X 3, X 1, X, and X 3 are also significant model terms (less than 0.05). The goodness of fit of the model was also checked by determination coefficient (R ). The value of the determination coefficient (R TRS-6h=0.956) indicates that only 7.44% of the total variations are not explained by the model. The adjusted determination coefficient (Adj. R =0.899) was also high, which indicates a high significance of the model 31. The Adeq. Precision (9.697) of the model was greater than 4. Among glucose yield during the hydrolysis process after 6 h, maximum level of glucose (1.87 mg/ml) was obtained with the same runs mentioned before. The model F-value of implies that the model is significant as was evident from the Fisher's F-test with a very low probability value [(P model > F)=0.0019]. Values of "Prob > F" less than 0.05 indicate model terms X 1 X, X 1, X and X 3 are significant. At the same time a relatively lower value of the coefficient of variation (CV=9.1%) indicates improved precision Table 3 Design matrix presenting TRS and glucose concentrations after 6 h hydrolysis time of pretreated pith bagasse with microwave/ho/acetic acid Runs X 1 X X 3 Experimental % hydrolysis yield Predicted Experimental % hydrolysis yield Predicted TRS (g/g TDM) TRS glucose (g/g TDM) glucose (mg/ml) (mg/ml) (mg/ml) (mg/ml) X 1 : H O /acetic acid treatment time, X : solid:liquid ratio, X 3 : microwave treatment time 6 h

6 FARID et al: MICROWAVE PRETREATMENT AND ENZYMATIC HYDROLYSIS OF PITH BAGASSE 103 Table 4 Regression analysis for TRS and glucose yield for quadratic response surface model fitting (ANOVA) after 6 h hydrolysis time Source Some of square df Mean square TRS 6 h F-value Prob.> F-value Model * X X X * X 1 X X 1 X * X X X * X * X * Residual R = Glucose 6 h Model * X X X X 1 X * X 1 X X X X * X * X * Residual R = * Significant at 5% level (P<0.05) and reliability of the conducted experiments 31. The application of RSM for glucose production yielded the following regression equation (equation no. 3). Y (Glucose after 6h) = X X X X 1 X X 1 X X X X X.1365X 3 (3) The significance of each coefficient was also determined by P-values, which are listed in Table 4. These values imply that quadratic main effect of X 1, X and X 3 are significant compared to their respective first order effect. One cross product (X 1 X ) is also significant at 5% level. The results also show the analysis of variance (F-test) and the coefficient of determination (R Glucose-6h) is shown as 93.75%. This indicates that the accuracy and general ability of the polynomial model are good, and the analysis of response trends using the model is considered to be reasonable. This value indicates that only 6.5% of the total variations are not explained by the model. The value of the adjusted determination coefficient (Adj. R =0.857) was also high, which indicates the high significance of the model. In order to confirm the above mentioned results, other sets of experiments were carried out with the same variables for 4 h of hydrolysis time (Table 5). Maximum level of TRS (about mg/ml) and glucose (18.33 mg/ml) were obtained with the same Table 5 Design matrix presenting TRS and glucose concentration after 4 h hydrolysis time of pretreated pith bagasse with microwave/ho/acetic acid Runs X 1 X X 3 Experimental % hydrolysis yield Predicted Experimental % hydrolysis yield Predicted TRS (g/g TDM) TRS glucose (g/g TDM) glucose (mg/ml) (mg/ml) (mg/ml) (mg/ml) 4h X 1 : H O /acetic acid treatment time, X : solid:liquid ratio, X 3 : microwave treatment time

7 104 INDIAN J BIOTECHNOL, JANUARY 014 coded runs of 6 h. The results obtained were also submitted to ANOVA analysis. The application of RSM yielded the following regression equations: Y 3 (TRS after 4h) = X X 0.081X X 1 X +0.87X 1 X 3 0.7X X 3 +0.X 1.3X 1.69 X 3 (4) Y 4 (Glucose after 4h) = X X +0.6X X 1 X +.3X 1 X X X X X 3.71X 3 (5) The ANOVA of quadratic regression model for TRS yield demonstrates that equation no. 4 is a significant model as evident from the Fisher's F-test (Table 6) with a very low probability value [(P model > F)=0.0165]. Among model terms X and X 3 are also significant with probability of 99%. The interaction between X 1, X and X 3, however, has no significant influence on TRS yield when the hydrolysis process was carried out for 4 h. The goodness of fit of the model was checked by determination coefficient (R ). In this Source Table 6 Regression analysis for TRS and glucose yield for quadratic response surface model fitting (ANOVA) after 4 h hydrolysis time Some of square df Mean square TRS 4 h F-value Prob.> F-value Model * X X X X 1 X X 1 X X X X X * X * Residual R = Glucose 4 h Model * X X * X * X 1 X * X 1 X * X X X * X X * Residual R = * Significant at 5% level (P<0.05). case, the value of the determination coefficient (R TRS-4h=0.8785) indicates that only 1.15% of the total variation are not explained by the model. At the same time a relatively lower value of the coefficient of variation (CV=4.63%) indicates a good precision and reliability of the experiment. Statistical analysis of glucose yield after 4 h during the enzymatic hydrolysis of treated pith bagasse (Table 6) shows that, in range of the study, the 3 variables have a high significant effect on glucose yield. The Fisher's test with a very low probability value [(P model > F)=0.0001) demonstrates very high significance for the regression model. Value of "Prob > F" less than 0.05 indicates that model terms are significant. In this case, X, X 3, X 1 X, X 1 X 3, X 1 and X 3 are significant model terms. The goodness of fit of the model was checked by the determination coefficient (R ). The value of the determination coefficient (R Glucose-4h=0.9711) indicates that only.89% of the total variation are not explained by the model. The value of the adjusted determination coefficient (Adj. R =0.9338) was also very high, which indicates a high significance of the model. At the same time, a relatively lower value of the coefficient of variation (CV=4.3) indicates improved precision and reliability of the conducted experiments. Comparison of Observed and Predicted A regression model can be used to predict future observations on the response (TRS and glucose yields) during the hydrolysis process corresponding to particular values of repressor variables. Tables 3 and 5 show the observed TRS and glucose yields (the responses) and their predicted yields. The results prove that the predicted data of the response from the empirical model are in agreement with the observed ones in the range of the operating variables. Localization of Optimum Condition The 3D response surface plots described by the regression model were drawn to illustrate the effect of the independent variables and the interactive effects of each of them on the response variables. The shape of the corresponding contour plots indicates whether the mutual interactions between the independent variables are significant or not. An elliptical nature of the contour plots indicates that the interactions between the independent variables are significant. Since interactions between the three independents are observed from Table 4 (after 6 h) for X 1 X 3 (TRS) and

8 FARID et al: MICROWAVE PRETREATMENT AND ENZYMATIC HYDROLYSIS OF PITH BAGASSE 105 X 1 X (glucose), and from Table 6 (after 4 h) for X 1 X and X 1 X 3 (glucose), elliptical contour plots are found in Figs and 3. Figures of response surface plots for 4 h hydrolysis time are not shown. Evidently, TRS and glucose yields during the hydrolysis process (after 6 and 4 h) varied significantly upon changing the H O /acetic acid treatment time (X 1 ), solid:liquid ratio (X ) and Fig. Contour plots of glucose yield after 6 h hydrolysis time: a. X 1 (H O /acetic acid treatment time) vs X (solid:liquid ratio); b. X 1 (H O /acetic acid treatment time) vs X 3 (MW treatment time); c. X 3 (MW treatment time) vs X (solid:liquid ratio). Fig. 3 Contour plots of TRS yield after 6 h hydrolysis time: a. X 1 (H O /acetic acid treatment time) vs X 3 (MW treatment time); b. X (solid:liquid ratio) vs X 3 (MW treatment time); d. X 1 (H O /acetic acid treatment time) vs X (solid:liquid ratio).

9 106 INDIAN J BIOTECHNOL, JANUARY 014 microwave treatment time (X 3 ). The optimum values of each variable was identified based on the hump in the three dimensional plot, or from corresponding contour plot. An increase of H O /acetic acid pretreatment time from 30 to 60 min, solid:liquid ratio from 1:15 to 1:0 and microwave treatment time from to 4 min resulted in an increase of both TRS and glucose yield when hydrolysis time was carried out for 6 or 4 h. Under certain condition of X 1, X and X 3 (56-63 min, 1:19-1:0 (w/v) and min, respectively), and (57-63 min, 1:0-1: (w/v) and min, respectively), maximal contour of TRS and glucose could be detected from Figs and 3. Conclusions Pretreatment of pith bagasse in a loosely closed flask with high power microwave-irradiation for short time at different cycles of 30 sec could be used as a potential alternative method for the pretreatment of lignocellulosic materials and production of sugars by enzymatic hydrolysis. Results comparable to conventional chemical pretreatments were obtained at only 1/7 th of the heating treatment time. The microwave/h O /acetic acid pretreatment of pith bagasse was the most efficient pretreatment technique among the other microwave/chemical pretreatment processes and subsequently produced the highest sugar yield by cellulase hydrolysis. Thus these results could be applied to design large scale pretreatment of lignocellulosic biomass. Statistical optimization of pith bagasse treatment by microwave/h O /acetic acid and T. reesei cellulase using Box-Behnken design appeared to be a valuable tool for significant enhancement in the release of reducing sugars during the enzymatic hydrolysis. Acknowledgements The authors thank the financial assistance from Microbial Chemistry Department and Natural and Microbial Products Department, National Research Center, Dokki, Cairo, Egypt. 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