Abstract
Simultaneous saccharification and fermentation (SSF) of alkaline pretreated cogongrass to ethanol was optimized using the commercial cellulase Accellerase 1500 and Ethanol Red dry yeast. Cogongrass was pretreated with 10% (wt) NaOH at room temperature for 24 hours, resulting in an increase in the cellulose percentage from 38.5% to 60.5%. Each SSF of alkali-pretreated cogongrass was carried out with 1 g/L of dry yeast loading at pH 5.0 under 150 rpm shaking. Response surface methodology (RSM) based on a three-level three-factor Box-Behnken design was employed to optimize the key variables within the following ranges: cellulase concentration per unit gram water-insoluble cellulose (WIS) (0.15-0.25 mL/g-WIS), substrate concentration (5-15 % WIS, w/w), and temperature (35-45°C) for the SSF process. The response surface model arrived at the optimum SSF conditions: cellulase concentration of 0.255 ml/g-WIS, temperature at 37.5°C, and substrate concentration of 7.28% WIS for obtaining 80.3 % ethanol yield in 72 h. The optimal conditions were verified experimentally with an average absolute relative deviation of 3.01 %. Also, the SSF was scaled up to a 5-L rotary drum reactor filled with 1 kg of substrate under the optimal conditions, and an ethanol yield of 76.2% was obtained.
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Simultaneous saccharification and fermentation of alkali-pretreated cogongrass for bioethanol production
Yu-Sheng Lin and Wen-Chien Lee*
Simultaneous saccharification and fermentation (SSF) of alkaline pretreated cogongrass to ethanol was optimized using the commercial cellulase Accellerase 1500 and Ethanol Red dry yeast. Cogongrass was pretreated with 10% (wt) NaOH at room temperature for 24 hours, resulting in an increase in the cellulose percentage from 38.5% to 60.5%. Each SSF of alkali-pretreated cogongrass was carried out with 1 g/L of dry yeast loading at pH 5.0 under 150 rpm shaking. Response surface methodology (RSM) based on a three-level three-factor Box-Behnken design was employed to optimize the key variables within the following ranges: cellulase concentration per unit gram water-insoluble cellulose (WIS) (0.15-0.25 mL/g-WIS), substrate concentration (5-15 % WIS, w/w), and temperature (35-45°C) for the SSF process. The response surface model arrived at the optimum SSF conditions: cellulase concentration of 0.255 ml/g-WIS, temperature at 37.5°C, and substrate concentration of 7.28% WIS for obtaining 80.3 % ethanol yield in 72 h. The optimal conditions were verified experimentally with an average absolute relative deviation of 3.01 %. Also, the SSF was scaled up to a 5-L rotary drum reactor filled with 1 kg of substrate under the optimal conditions, and an ethanol yield of 76.2% was obtained.
Keywords: Bioethanol; Simultaneous saccharification and fermentation; SSF; Cogongrass; Alkaline pretreatment
Contact information: Department of Chemical Engineering, Systems Biology and Tissue Engineering Research Center, National Chung Cheng University, Minhsiung, Chiayi 621, Taiwan; *Corresponding author: chmwcl@ccu.edu.tw
INTRODUCTION
Cogongrass [Imperata cylindrical (L.) Beauv. var. major. (Nees) C.E.Hubb] is an aggressive, perennial grass that is distributed worldwide in the tropical and subtropical regions. Although it has been used for forage and soil stabilization, cogongrass is considered to be one of the top 10 worst weeds in the world, reported by 73 countries as a pest in a total of 35 crops (Holm et al. 1977). Despite its being an invasive and exotic weed, it is regarded as a promising medicinal plant because secondary metabolites isolated from the rhizome of I. cylindrical can have medicinal uses (Matsunaga et al. 1994; Pinilla and Luu 1999; Yoon et al. 2006). Genomic and proteomic methods have been used to reveal the genetic and ecotypic variations among Imperata cylindrica ecotypes in Taiwan (Chou and Tsai 1999; Chang 2008). In Taiwan, cogongrass was used several decades ago as a material for making houses and raincoats for farmers. Because of the flat form and high density of its leaf, cogongrass has proved better and more durable than Miscanthus as material for the roofs of huts. As cogongrass can be grown on land considered unsuitable for row crop production and it can grow all year long in tropical countries, this grass can be developed as a bioresource for renewable energy. Roots of cogongrass have potential for medicinal application, and its stems and leaves can serve as the feedstock for biofuels. The present work describes the use of cogongrass for the production of bioethanol by simultaneous saccharification and fermentation (SSF).
After pretreatment, lignocellulosic feedstock such as cogongrass undergoes enzymatic hydrolysis of cellulose and yeast fermentation of the hydrolyzates to yield ethanol. The SSF process combines the enzymatic hydrolysis and ethanol fermentation in a single stage. SSF is usually preferred over separate hydrolysis and fermentation because higher ethanol yield can be achieved by minimizing product inhibition. Also, SSF has an advantage of low potential costs because lower amounts of enzyme are employed, reducing the capital investment (McMillan et al. 1999; Karimi et al. 2006; Olofsson et al. 2008). SSF studies, for example, have been carried out to produce ethanol from lignocellulosic wastes (sugar cane leaves and Antigonum leptopus leaves) using Trichoderma reesei cellulase and yeast cells (Krishna et al. 2001). Other lignocellulosic residues such as bermudagrass, reed, and rapeseed have also been used as the raw materials for the production of bioethanol by SSF (Li et al. 2009). SSF process variables that can influence the ethanol production efficiency include enzyme concentration, substrate concentration, temperature, and reaction time. SSF of alkaline hydrogen peroxide pretreated Antigonum leptopus (Linn) leaves to ethanol was optimized by using response surface methodology (RSM) (Krishna and Chowdary 2000). Response surface methodology (RSM) is a powerful tool for the optimization of complex processes, because it can offer several advantages. These include (1) an understanding of how the process variables affect the selected process response, (2) determination of any possible interrelationship among the test variables, and (3) characterization of the combined effect that all process variables may have on the process response (Domingos et al. 2003). RSM was used here for optimizing the SSF of alkaline pretreated cogongrass for bioethanol production. The obtained optimal SSF condition was verified by experiments carried out in a flask as well as in a rotary drum reactor.
EXPERIMENTAL
Alkaline Pretreatment of Cogongrass
Cogongrass stems and leaves were collected from the campus of National Chung Cheng University. Air-dried cogongrass was pre-cut into sticks of ca. 2 cm long. For the alkaline pretreatment, cogongrass sticks were incubated with 10% (wt) NaOH at a solid-to-liquid ratio of 1:20 (w/v) at room temperature for one day and then washed with tap-water until the pH became neutral. The pretreated cogongrass was stored in sealed plastic bags at 4 °C. To calculate its dried weight, the pretreated cogongrass was dried in a forced-air oven at 65 °C for 24 h. The composition of the cogongrass before and after pretreatment was determined according to a previously reported method (Sluiter et al. 2008). The scanning electron microscope (SEM) micrographs of cross-section of the cogongrass before and after pretreatment were taken with a Hitachi-S2400 SEM-EDX microscope (Japan). Samples were sputter-coated with gold prior to SEM observation.
Simultaneous Saccharification and Fermentation (SSF) of Pretreated Cogongrass
Pretreated cogongrass was simultaneously saccharified and fermented by using the commercial cellulase Accellerase 1500 (Genencor, USA) and Ethanol RedTM dry Saccharomyces cerevisiae yeast (Fermentis, France), respectively. Cogongrass that was pretreated by alkali under optimal conditions was transferred to a 250-mL flask containing 0.05 M citrate buffer at pH 5.0, to produce a final water-insoluble-solids (WIS) concentration of 10% (w/w) and then autoclaved at 121 °C for 30 min (Hayward et al. 1995). In addition, cellulase and dry yeast (1 g/L) were loaded into each substrate mixture and then incubated in water bath at 150 rpm for three days. Preliminary study indicated that the ethanol concentration could approach to a steady value after 3-d SSF. Fifteen runs of SSF were carried out with different combinations of independent variable values as described in the sub-section of Design of Experiments. Samples (1 mL) of SSF were centrifuged at 8050 g for 10 min to remove denatured enzyme and insoluble residues. The ethanol yield was calculated based on the conversion of glucan to ethanol.
Analytical Methods
Concentrations of ethanol and sugars in the SSF mixture were analyzed by HPLC using a Bio-Rad Aminex HPX-87H column (300 7.8 mm i.d.), operating at 65°C. The mobile phase was 5 mM H2SO4, and the flow-rate was 0.6 mL/min with an RI detector.
Design of Experiments
A three-level three-factor Box-Behnken design was adopted for the study. The important factors involved in ethanol production were substrate concentration, cellulase concentration, and temperature, which is in agreement with our previous study on the SSF of alkaline-pretreated rice straw, a similar raw material to cogongrass (Lin and Lee 2011). The amount of dry yeast loading was fixed at 1 g/L. The factors and their levels are given in Table 1, and the design of experiments employed is presented in Table 2. In Table 1 the level values of cellulase and substrate concentration were so chosen that at those conditions higher SSF efficiencies could be achieved. Temperature levels of 35, 40, and 45°C were chosen because that the yeast could ferment at higher temperature, and the elevated temperature favored SSF.
Table 1. Level of Variables Chosen for the Study
In Table 2, Yexperimental is the experimental ethanol yield, while Ypredicted is the calculated ethanol yield using the response surface model as described in the following section.
Table 2. Experimental Design Showing Coded Values of Variables, as well as the Experimental and Predicted Responses
Statistical Analysis
The experimental data (Table 2) were analyzed according to the response surface regression procedure to fit the following second-order polynomial equation, in which the level of significance (P value) of all coefficients was <0.05,
(1)
where Y is the ethanol yield (%, w/w), A0 is the intercept, A1–A3 are the linear coefficients, A4–A6 are the quadratic coefficients, A7–A9 are the cross-product coefficients, and Xi are the coded independent variables. The regression analyses, statistical significances, and response surfaces were carried out using STATISTICA software (version 8.0; StatSoft). Optimization of the reaction parameters for maximum ethanol yield was obtained through the software package.
SSF of Pretreated Cogongrass in a Rotary Drum Reactor
In a 5-L rotary drum reactor (the full volume was 7.7 liters), pretreated cogon-grass was mixed with 0.05 M citrate buffer at pH 5.0, making up a total of 1 or 2 kg mixture with a WIS concentration of 10% (w/w). The pH value of the mixture was adjusted to 5 using 1N HCl. SSF was run with 0.258 mL/g-WIS of enzyme and 1 g/L dry yeast. The reactor was operated at 37C in a temperature controlled box and rotated at a speed of 5 rpm for 1 min at the beginning of SSF. Samples (1 mL) of SSF were taken every 24 h with large-mouth pipette tips and were centrifuged at 8050 g for 10 min. After each sampling point the reactor was also rotated for 1 min.
RESULTS AND DISCUSSION
Pretreatment with NaOH
After harvesting, a constant weight of cogongrass was reached in two days in an oven at 65 °C. A water content of 59.9% (wt) was determined in the fresh cogongrass. When the harvested cogongrass was air-dried to a constant weight, 1 kg raw material could yield 415 g of dried material. After NaOH pretreatment, 1 kg dry material was converted to 492 g WIS. Results as shown in Table 3 indicated that NaOH pretreatment at room temperature led to an increase in cellulose content (% glucan) by the removal of some hemicellulose and lignin. The fact that alkali pretreatment can decrease the proportion of hemicellulose and lignin has been reported for a similar raw material, rice straw (Zhang and Cai 2008). Figure 1 shows the morphology of cogongrass before and after pretreatment with 10%(wt) NaOH. Significant morphological changes of the cogongrass from sticks (Fig. 1a) to fibrous clusters (Fig. 1b) were observed. It was confirmed by SEM that changes in microstructure occurred, since a large fraction of lignin and some xylan were removed by the alkaline pretreatment. In contrast to the untreated sample that exhibited a cover of material on the microfibrils (Fig. 1c), the microfibrils of pretreated sample became exposed after removal of the cover material by the pretreatment (Fig. 1d). A similar behavior was observed in SEM of corn stover pretreated with aqueous ammonia (Kim and Lee, 2005).
The use of 10% NaOH caused solubilization of xylan from cogongrass at room temperature. In order to decrease the consumption of alkali and water, the alkaline solution was repeatedly used. No significant decrease in the pretreatment efficiency was observed on the use of alkali solution after five recycles of pretreatment (Table 3), suggesting that the consumption of NaOH and water could be saved by at least 75%. Pretreatment of cogongrass rendered it much more accessible to the enzymes for cellulose and hemicellulose degradation due to the removal of lignin and its structural changes.
Table 3. Composition of Cogongrass Before and After 10% NaOH Pretreatment
