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Yoon, L. W., Ngoh, G. C., and Chua, A. S. M. (2012). "Simultaneous production of cellulase and reducing sugar from alkali-pretreated sugarcane bagasse via solid state fermentation," BioRes. 7(4), 5319-5332.

Abstract

This study optimized alkali pretreatment of sugarcane bagasse (SCB) and investigated the potential of alkali-pretreated SCB in producing cellulase and reducing sugar by a white-rot fungus, P. sanguineus, via solid state fermentation (SSF). The fermentability of the reducing sugar produced during SSF was examined by co-culturing yeast, Saccharomyces cerevisiae, with P. sanguineus. Central composite design (CCD) was applied to optimize the pretreatment based on reducing sugar yield obtained from enzymatic hydrolysis of the pretreated SCB. The model developed from CCD fitted the data well, and the optimized conditions for alkali pretreatment were 128 °C, 0.62 M NaOH, and 30 min with a reducing sugar yield of 97.8%. The alkali-pretreated SCB after washing and drying was cultivated with P. sanguineus during SSF. It was found that cellulase and reducing sugar can be produced simultaneously from this SSF system. The maximum cellulase activities determined from filter paper assay (FPase), carboxylmethylcellulase (CMCase) assay and β-glucosidase assay were 0.02 IU/mL, 0.11 IU/mL, and 0.13 IU/mL on day 8, day 3, and day 6 of cultivation, respectively. The maximum reducing sugar concentration of 19.9 mg/g pretreated SCB was obtained on day 4 of SSF. The reducing sugar produced was converted into ethanol upon the addition of yeast into the SSF system. Evidently, the reducing sugar acquired can be further utilized to produce other valuable products in subsequent processes.


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Simultaneous production of cellulase and reducing sugar from alkali-pretreated sugarcane bagasse via solid state fermentation

Li Wan Yoon,Gek Cheng Ngoh,a,* and Adeline Seak May Chuaa

This study optimized alkali pretreatment of sugarcane bagasse (SCB) and investigated the potential of alkali-pretreated SCB in producing cellulase and reducing sugar by a white-rot fungus, P. sanguineus, via solid state fermentation (SSF). The fermentability of the reducing sugar produced during SSF was examined by co-culturing yeast, Saccharomyces cerevisiae, with P. sanguineus. Central composite design (CCD) was applied to optimize the pretreatment based on reducing sugar yield obtained from enzymatic hydrolysis of the pretreated SCB. The model developed from CCD fitted the data well, and the optimized conditions for alkali pretreatment were 128 °C, 0.62 M NaOH, and 30 min with a reducing sugar yield of 97.8%. The alkali-pretreated SCB after washing and drying was cultivated with P. sanguineus during SSF. It was found that cellulase and reducing sugar can be produced simultaneously from this SSF system. The maximum cellulase activities determined from filter paper assay (FPase), carboxylmethylcellulase (CMCase) assay and β-glucosidase assay were 0.02 IU/mL, 0.11 IU/mL, and 0.13 IU/mL on day 8, day 3, and day 6 of cultivation, respectively. The maximum reducing sugar concentration of 19.9 mg/g pretreated SCB was obtained on day 4 of SSF. The reducing sugar produced was converted into ethanol upon the addition of yeast into the SSF system. Evidently, the reducing sugar acquired can be further utilized to produce other valuable products in subsequent processes.

Keywords: Pretreatment; Sugarcane bagasse; Central composite design; Cellulase; Reducing sugar; Solid state fermentation

Contact information: a: Department of Chemical Engineering, Faculty of Engineering, University Malaya, 50603, Kuala Lumpur, Malaysia;*Corresponding author: ngoh@um.edu.my

INTRODUCTION

Bioproducts such as enzyme, fermentable sugar, organic acid, and biofuel have gained massive interest from various industries due to their vast and vital applications (Sun 2009; Yao et al.2007; Bansal et al. 2011; Mirahmadi et al. 2010). These bioproducts are favourable products from agricultural residues due to the lignocellulosic nature of the residues (Sun 2009). Oil palm fronds, sugarcane bagasse, and rice husks are a few examples of low-cost agricultural residues that are rich in cellulose, hemicellulose, and lignin (Sousa et al. 2009). Nevertheless, most agricultural residues have complex structures that may negatively affect the bioconversion of these biomasses into value-added products. In order to facilitate bioconversion processes such as enzymatic hydrolysis and fermentation, pretreatment is often necessary to alter the structure of the biomass prior to the conversion process (Mirahmadi et al. 2010).

Pretreatment techniques are generally classified as physical, chemical, biological, or physicochemical (Galbe and Zacchi 2007; Yang and Wyman 2008; Karunanithy and Muthukumarappan 2011). Alkali pretreatment is one of the effective techniques applied to alter the structure of biomass. Operating conditions such as temperature, duration, and alkali concentration are among the most commonly investigated parameters in the appli-cation of alkali pretreatment on various biomasses (Nlewem and Thrash 2010; McIntosh and Vancov 2010; Xu et al. 2010; Zhang et al. 2010). The alkaline solution acts as a delignification agent, and it plays a role in disrupting the bond between lignin and carbohydrates, thereby removing the lignin from the biomass (Galbe and Zacchi 2007). Due to the removal of lignin, the accessible surface area of cellulose and hemicellulose for enzyme binding and microbial attack increases (Singh et al. 2011). Furthermore, most of the cellulose in biomass can be retained after alkali pretreatment (Singh et al. 2011; Mirahmadi et al. 2010). This further contributes to the significant improvement in reducing sugar (RS) yield after enzymatic hydrolysis of alkali-pretreated biomass (Sukumaran et al. 2009; Zhao et al. 2009). Alkali-pretreated biomass, which has a lower lignin content and higher percentage of cellulose, is able to facilitate the production of various enzymes, especially cellulase, through fermentation (Damisa et al. 2008; Singh et al. 2011; Soni et al. 2010).

Solid state fermentation (SSF), being one of the most extensively studied fermentation methods, is widely applied in the production of industrial enzymes such as cellulase and xylanase, bioethanol, and other secondary metabolites (Tengerdy and Szakacs 2003). SSF offers a number of advantages as compared to submerged fermentation (SmF). SSF is particularly suitable for filamentous fungi because SSF provides a condition that is closer to their natural habitat compared to SmF (Raghavarao et al. 2003). Furthermore, the cost of dewatering can be greatly reduced, since SSF is carried out in the absence of free flowing water (Pandey et al. 2000). Most importantly, SSF offers benefits in terms of having a higher concentration of end products, higher fermentation productivity, and lower demand on sterilization of the equipment (Hölker et al. 2004). All of these results make SSF a technique worthy of investigation for the bioconversion of agricultural residues into value-added products with the aid of filamentous fungi.

The objective of this work was to perform an optimization study on the alkali pretreatment of sugarcane bagasse (SCB) and to investigate the potential of a white-rot fungus, Pycnoporus sanguineus, in the production of cellulase and reducing sugar from alkali-pretreated SCB via SSF. Ethanol production was used to examine the ferment-ability of the reducing sugar produced by adding yeast to the SSF system. SCB was selected due to its relatively high cellulose content of approximately 50% (Pandey et al. 2000). The potential of P. sanguineus in producing cellulase and reducing sugar has also been confirmed (Quiroz-Castaneda et al. 2009; Teoh and Mashitah 2010). Alkali pretreatment is suitable for application to SCB since this pretreatment method is effective on agricultural residue, which contains a relatively lower amount of lignin compared to woody materials (Galbe and Zacchi 2007). In the optimization study, the effect of pretreatment temperature, duration, and sodium hydroxide (NaOH) concentration on RS obtained from enzymatic hydrolysis of pretreated SCB was examined using Central Composite Design (CCD). It was expected that a higher RS yield would be obtained with a more effective pretreatment condition. The ability of P. sanguineus to produce cellulase and reducing sugar simultaneously was further investigated by cultivating the fungus on SCB pretreated under optimum conditions. Ethanol production as a result of co-culturing yeast and P. sanguineus was reported.

EXPERIMENTAL

Fungal Culture and Inoculum Preparation

White-rot fungus Pycnoporus sanguineus was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany). It was maintained on malt extract peptone agar at 4 °C and was sub-cultured every month. The fungus inoculum was prepared by washing the surface of the fungus, which was grown on an agar plate, with 5 mL of sterilized water. The mycelia suspension obtained was then transferred to an Erlenmeyer flask that contained 50 mL of 2% malt extract culture medium. The mycelia suspension was incubated at room temperature at 100 rpm for 5 days. The fungal pellets obtained were washed with sterile water by centrifugation and homogenized before suspending the pellets into a sterile Mandel’s medium prior to use in SSF. The Mandel’s medium consisted of 2 g/L KH2PO4, 1.4 g/L (NH4)2SO4, 0.3 g/L urea, 0.3 g/L CaCl2.2H2O, 0.3 g/L MgSO4.7H2O, 1 g/L peptone, 0.2% (v/v) Tween 80, 5 mg/L FeSO4.7H2O, 1.6 mg/L MnSO4.2H2O, 1.4 mg/L ZnSO4.7H2O, and 2 mg/L CoCl2.6H2O.

Alkali Pretreatment

SCB was collected from Purecane Manufacturing Sdn. Bhd., Malaysia. The SCB was thoroughly washed and sun dried. Alkali pretreatment of the SCB was carried out by adding NaOH of various concentrations to the test tube that contained the SCB. The mixture was then heated in an oil bath (Julabo, MC V.2, Germany) to the desired temperature. The operating conditions examined are shown in Table 2. After the reaction, the mixture was cooled and filtered. The pretreated solid was washed with deionized water and acetate buffer before drying at 60 °C prior to enzymatic saccharification.

Enzymatic Hydrolysis

Enzymatic hydrolysis was performed by adding a cellulase solution with a loading of 30 FPU/g substrate to the pretreated SCB at a ratio of 2% (w/v) (Zhao et al. 2009). The mixture was buffered with a 50 mM acetate buffer solution with a pH of 4.8. The reaction was carried out at 50 °C for 48 hours and halted by heating the mixture in boiling water for five minutes. The mixture was then centrifuged at 6000 rpm for 20 min. The concentration of RS in the supernatant was determined using the DNS method (Miller 1959; Ghose 1987). Glucose solutions were used as standards and the absorbance reading was taken at 540 nm. The RS yield obtained from the enzymatic hydrolysis was determined using Equation (1) (Li et al. 2009).

 (1)

Optimization of Alkali Pretreatment

Design Expert 6.0.6 software (STAT-EASE Inc., Minneapolis, USA) was used to determine the optimum conditions for alkali pretreatment of SCB. Central Composite Design (CCD) of Response Surface Methodology (RSM) was employed to optimize the pretreatment process. The effects of three independent variables; pretreatment temperature (°C), NaOH concentration (M), and duration (min) on alkali pretreatment were investigated (Table 1). The selection of the range of each independent variable was based on some preliminary studies. RS yield obtained after enzymatic hydrolysis was the response or the dependent variable. A total of 20 experimental runs were conducted to optimize the process. Analysis of variance (ANOVA) was used to evaluate the statistical significance of the model developed by the software.

Table 1. Levels of the Independent Variables in the Alkali Pretreatment

Solid State Fermentation (SSF)

SCB pretreated with alkali under the optimized operating conditions was subjected to SSF. The SSF was carried out in an Erlenmeyer flask with 2 g of pretreated-SCB inoculated with 1 mL of homogenized mycelia suspension. The moisture content of the SCB was adjusted to 75% with Mandel’s medium, and SSF was conducted at room temperature. The content of the flask was extracted within 3 to 8 days of fermentation with 10 mL of a citrate buffer (50 mM, pH 4.8). The flask was then agitated at 150 rpm for 1 hour, and subsequently the content was centrifuged at 4 °C and 3500 rpm for 20 min. The supernatant obtained from centrifugation was filtered, and thereafter the cellulase activities, total soluble protein content, and reducing sugar concentration were determined from the filtrate.

Ethanol Production

Ethanol production was examined by co-culturing yeast, Saccharomyces cerevisiae, with P. sanguineusS. cerevisiae used in this study was obtained from the Institute of Biological Science, Faculty of Science in University Malaya, Malaysia. After 4 days of SSF of P. sanguineus on the alkali-pretreated SCB, 19 mL of deionized water and 1 mL of the S. cerevisiaesuspension with a cell concentration approximately 108 cells/mL were added to the fermented SCB. Ethanol concentration was analyzed after 2 days of fermentation under static conditions.

Analytical Methods

The reducing sugar concentration was determined using the DNS method (Miller 1959). The total soluble protein content was determined using a TP0300 total protein kit, which is based on micro Lowry Peterson’s modification method (Sigma-Aldrich, USA).

The total cellulase (filter paper activity, FPase), carboxylmethyl cellulase (CMCase), and β-glucosidase activities were determined in accordance with the procedures reported (Ghose 1987). Whatman filter paper no.1, carboxylmethyl cellulose, and cellobiose were used as the substrates in the determination of FPase, CMCase, and β-glucosidase activities, respectively. One unit of enzyme activity was defined as the amount of enzyme required to liberate 1 µmolmL-1 min-1 of glucose (2 µmol mL-1 min-1 of glucose in the case of β-glucosidase) from the substrate under the assay conditions.

The ethanol concentration obtained from co-culturing S. cerevisiae and P. sanguineus was analyzed using high performance liquid chromatography (HPLC, Waters, USA) equipped with a Waters 410 refractive index detector. A Hi-Plex H column (7.7 mm x 300) (Agilent, USA) was adopted, and the analysis conditions were as follows: deionized water as the mobile phase at a flow rate of 0.6 mL min-1, injection volume of 10 µL, and a column temperature of 65 °C.

RESULTS AND DISCUSSION

Optimization of Alkali Pretreatment

The alkali pretreatment of SCB was optimized before subjecting the substrate to the subsequent SSF process. Alkali pretreatment plays a major role in partial lignin and hemicellulose removal from the biomass (Brodeur et al. 2011; Galbe and Zacchi 2007).Other products such as phenolic acid, furfural, and aldehydes, which are known as fermentation inhibitors, might be produced during alkali pretreatment (Brodeur et al. 2011). Thus, it is necessary to perform a washing step prior to enzymatic hydrolysis or solid state fermentation to remove lignin, soluble hemicellulose, and other inhibitors that were deposited on the surface of alkali-pretreated biomass. While washing the alkali-pretreated biomass, a neutral pH is attained, and this creates a condition that is favorable for enzymes and microbial attack in the subsequent bioconversion processes. However, substantial amount of wastewater is inevitably produced from the washing step, and this incurs extra cost in the wastewater treatment, which is not beneficial in the economical point of view. To improve the cost effectiveness of this process, the alkaline solution could be pressed out from the biomass after pretreatment (Williams et al. 2013). Alternatively, the washed water can also be reused after the desired pH adjustment for alkaline pretreatment process.

The experimental design matrix and RS yields obtained from enzymatic hydrolysis of the alkali-pretreated SCB are presented in Table 2. RS yield as high as 97.1% was obtained from the alkali-pretreated SCB. The RS yield obtained under the operating conditions investigated was fitted to a second order polynomial equation. The final equation in terms of their coded factors after elimination of the insignificant model terms is shown as Equation (2).

Y = 94.77 + 1.85 A + 22.75 B + 1.54 C – 2.17 A2– 23.27 B2 – 2.12 C2 – 1.43 AC + 2.52 BC (2)

The results of the analysis of variance (ANOVA) for the above reduced quadratic model are tabulated (Table 3). A, B, C, B2, and BC are the significant model terms, since their probability > F values are less than 0.05 in 95% confidence interval. Among these terms, B and B2 have the lowest probability > F values. This implies that changes in NaOH concentration have the greatest impact on the RS yield obtained from alkali-pretreated SCB. The model developed by the software was able to fit the experimental data well, as proven by a significant model term and an insignificant lack of fit term. This is further confirmed by the coefficient of determination, R-squared, value for the model (0.9952), which is close to 1.

Table 2. Experimental Design Matrix and RS Yield from Enzymatic Hydrolysis of Alkali-Pretreated SCB

Table 3. ANOVA Results for the Reduced Quadratic Model of Alkali Pretreatment

* DF = degree of freedom

Figures 1 and 2 show the three-dimensional response surface plots and the interaction plots of the effect of the parameters investigated on RS yield. Both response surfaces can be best described as elliptical systems, as suggested by Wu and Hamada (2000). By comparing both figures, the interaction between duration and NaOH concentration was shown to have a larger effect on the RS yield. This can be further proven by the lower F value of the AC term (F-value = 4.25) compared to the F-value of the BC term (13.35), as stated previously in Table 3.

At a lower temperature i.e. 100 °C, longer pretreatment duration was needed to improve the RS yield of the pretreated SCB [Fig. 1(b)]. This might be due to the fact that the amount of lignin removed during alkali pretreatment is proportional to the pretreatment duration. Higher lignin removal further facilitated the release of sugar from the pretreated SCB during enzymatic hydrolysis (McIntosh and Vancov 2010). On the other hand, pretreatment at 140 °C gave similar RS yields regardless of the duration employed. This might be the maximum amount of RS that can be derived from SCB, and hence prolonging pretreatment duration did not lead to any improve-ment in the RS yield. From Fig. 1(b), it seems that pretreatment temperature did not significantly influence the production of reducing sugar. Only little variation in the amount of RS was observed when the pretreatment temperature was increased from 100 °C to 140 °C.

Fig. 1. (a) Three-dimensional response surface plot of the effect of pretreatment temperature and duration on the RS yield at 0.5 M NaOH; (b) Interaction plot between pretreatment temperature and duration on the RS yield at 0.5 M NaOH

As shown in Fig. 2(b), RS yield was significantly affected by the concentration of NaOH used in the pretreatment process. RS yield improved drastically when a NaOH concentration higher than 0.1 M was employed. There was a slight decrease in the RS yield when a NaOH concentration greater than approximately 0.65 M was applied in pretreatment. The decrease might be attributed to the loss of cellulose and hemicellulose during pretreatment by alkaline solution with a greater strength (Nlewem and Thrash 2010). Apart from that, longer pretreatment duration led to higher RS yield and this phenomenon was more apparent when an alkali solution with a higher concentration was used.

Fig. 2. (a) Three-dimensional response surface plot of the effect of pretreatment duration and NaOH concentration on the RS yield at 120°C (b) Interaction plot between pretreatment duration and NaOH concentration on the RS yield at 120°C

The optimum conditions for alkali pretreatment were obtained using numerical optimization features of Design Expert software. The optimized pretreatment conditions attained were 128°C, 30 min, and 0.62 M with a predicted RS yield of 99.8%. The average experimental RS yield under these particular conditions was found to be 97.8%. The difference of 1.8% between the predicted and experimental values implies that the model is reliable in predicting the RS yield for the range of the examined pretreatment conditions.

Solid State Fermentation of Alkali-Pretreated SCB

Through SSF with the aid of a microorganism, lignocellulosic biomass can be further converted into other value-added products and consequently, the applicability of the biomass can be evaluated. SCB pretreated under optimized conditions was used as the substrate to cultivate P. sanguineus in SSF. The ability of P. sanguineus for simultaneous production of cellulase and reducing sugar from alkali-pretreated SCB was investigated. P. sanguineus was found to be one of the most suitable white-rot fungi to be cultivated on alkali-pretreated SCB in a prior screening test.

A complete cellulase system should consist of three cellulase components, namely endoglucanase, exoglucanase, and β-glucosidase (Cen and Xia 1999). Each component functions distinctively during the hydrolysis of lignocellulosic biomass. Endoglucanase hydrolyze cellulose chains to form new chain ends and these chain ends are further broken down into cellobiose and cello-oligosaccharides by exoglucanase (Dashtban et al. 2009; Kumar et al. 2008). Cellobiose is then further fractionated into glucose by the action of β-glucosidase. The cellulose content in the substrate can only be hydrolysed completely into its smallest basic unit, glucose, in the presence of all three cellulase components in significant amounts.

Figure 3 shows that all three cellulase components were detected in the cellulase system produced by P. sanguineus. Colonization of P. sanguineus on the pretreated SCB began after 2 days of SSF. Cellulase was detected on day 3 of fermentation, and these components of cellulase generally require different durations to reach their maximum activities. CMCase had the highest activity (0.11 IU/ mL) in the shortest fermentation duration (day 3) as compared to the two other cellulase components. On the other hand, FPase and β-glucosidase both had their highest activities recorded on day 8 and day 6 with titres of 0.02 IU/ mL and 0.13 IU/ mL, respectively.

From Fig. 3, it is obvious that β-glucosidase activity was the highest, followed by CMCase and FPase in the cellulase system produced by P. sanguineus. This finding is slightly different from the cellulase system of P. sanguineus as reported by Teoh and Mashitah (2010) and Quiroz-Castaneda et al. (2009). Teoh and Mashitah (2010) cultivated P. sanguineus on palm oil mill effluent and palm-pressed fiber under submerged fermentation and found that CMCase is higher than FPase and β-glucosidase during the course of fermentation. Quiroz-Castaneda et al. (2009) reported that the highest activity was recorded by CMCase, followed by β-glucosidase and FPase during SSF of P. sanguineus on wheat straw. This might indicate that substrates with different structural and chemical compositions are able to stimulate the production of different components in the cellulase system. On the other hand, the production of CMCase was always higher than FPase (Fig. 3), and this phenomenon is in accordance with other reported findings (Quiroz-Castaneda et al. 2009; Teoh and Mashitah 2010; Elisashvili et al. 2008; Kachlishvili et al. 2005; Khan et al. 2007).