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Statistical optimization of dilute sulfuric acid pretreatment of corncob for xylose recovery and ethanol production
b i o m a s s a n d b i o e n e r g y 3 6 ( 2 0 1 2 ) 2 5 0 e2 5 7
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Statistical optimization of dilute sulfuric acid pretreatment of corncob for xylose recovery and ethanol production Bai-Yan Cai a, Jing-Ping Ge a,*, Hong-Zhi Ling a, Ke-Ke Cheng b,*, Wen-Xiang Ping a a b
Key Laboratory of Microbiology, College of Life Sciences, Heilongjiang University, Harbin 150080, PR China Institute of Nuclear and New Energy Technology, Tsinghua University, Beijing 100084, PR China
article info
abstract
Article history:
The interest on use of lignocellulose for producing fuels and chemicals is increasing as
Received 14 July 2010
these materials are low cost, renewable and widespread sources of sugars. Corncob is an
Received in revised form
attractive raw material for xylose and fuel ethanol production due to its high content of
22 September 2011
xylan and glucan. In this study, the central composite design of response surface method
Accepted 21 October 2011
was used to optimize dilute H2SO4 pretreatment of corncob, in respect to acid concentra-
Available online 25 November 2011
tion (0.16e1.84%), treatment time (0.16e1.84 h) and temperature (105e130 C) for xylose production. Enzymatic hydrolysis of the remaining solid was carried out further to evaluate
Keywords:
the acid pretreatment conditions for maximizing glucose production. The results showed
Corncob
that pretreatment conditions for the highest xylose production was 1% sulfuric acid for
Ethanol
1.5 h at 123 C, corresponding to 87.2% total xylan converted to xylose and that for the
Fermentation
highest glucose þ cellobiose recovery was 0.5% sulfuric acid for 30 min at 125 C, corre-
Hemicellulose
sponding to 78.1% total glucan converted to glucose þ cellobiose. In the subsequent
Pretreatment
simultaneous saccharification and fermentation (SSF) experiments using 14% glucan
Xylose
substrates pretreated under above two kinds of conditions, 47 g l1 ethanol with a 65.8% theoretical yield and 50.2 g l1 ethanol with a 70.4% theoretical yield were obtained, respectively. ª 2011 Elsevier Ltd. All rights reserved.
1.
Introduction
Lignocellulosic materials represent an abundant and inexpensive source of sugars which can be microbiologically converted to industrial products [1]. Diluted acids are commonly used for hydrolysis of the hemicellulosic fraction at moderate temperature. Research investigations on dilute acid hydrolysis of various raw materials, such as sugarcane bagasse, sorghum straw, eucalyptus wood and oil palm fiber, for xylose production have been carried out by several researchers [2e6]. From these research studies it was revealed that the acids attack the polysaccharides, especially hemicelluloses that are easier to be hydrolyzed than cellulose and
lignin. On the other hand, the condition is not severe enough to hydrolyze the crystalline structure of cellulose which remains as insoluble solid. Therefore, the cellulose and lignin fractions remain almost unaltered in the solid phase. Although xylose was the main sugar obtained from hemicellulose, other sugars such as glucose, arabinose, were also produced in low amount during the hydrolysis process. Xylose can be used as substrate for production of a wide variety of compounds by chemical and biochemical processes. One such compound is xylitol, which is extensively used in food, pharmaceutical and thin coating applications [7,8]. Many studies have been conducted utilizing the hemicellulose portion of agricultural residues like eucalyptus, rice straw, corncob,
* Corresponding authors. Tel./fax: þ86 10 89796086. E-mail addresses: [email protected] (J.-P. Ge), [email protected] (K.-K. Cheng). 0961-9534/$ e see front matter ª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biombioe.2011.10.023
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Table 1 e Variables and experimental design levels for response surface. Variables
Symbol
Temperature ( C) Time (h) Acid concentration (%)
X1 X2 X3
Range and levels 1
0
1
110 0.5 0.5
117.5 1 1
125 1.5 1.5
brewer’s spent grain, sugarcane bagasse, and corn stover for xylitol production [9e16]. Among the various agricultural crop residues, corncob is one of the most abundant agricultural materials in
Northeastern China. Corncob is an attractive raw material for xylose and fuel ethanol production due to its high content of xylan and glucan. The xylan fraction of corncob represents up to 30% of the total carbohydrates that can readily be hydrolyzed into xylose by diluted acids. After the xylan in corncob has been hydrolyzed for xylitol production, the corncob cellulosic residue is porous and can easily be hydrolyzed by cellulases into glucose, and further converted to ethanol, another high added-value chemical [17,18]. In this study, corncob was chosen as the raw lignocellulosic material for integrated xylose and ethanol production. The central composite design of response surface method was used to optimize dilute H2SO4 pretreatment of corncob, in respect to acid concentration (0.16e1.84%), treatment time (0.16e1.84 h)
Table 2 e Material balances for pretreatment conditions g/100 g raw material corncorb liquid fraction after pretreatment by dilute H2SO4. Run
X1 ( C)
X2 (h)
X3 (%)
Glucose (g)
Xylose (g)
Arabinose (g)
Acetic acid (g)
Furfural þ 5-HMF (g)
Xylose yield (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
110 125 110 125 110 125 110 125 105 130 117.5 117.5 117.5 117.5 117.5 117.5 117.5 117.5 117.5 117.5
0.50 0.50 1.50 1.50 0.50 0.50 1.50 1.50 1.00 1.00 0.16 1.84 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.50 0.50 0.50 0.50 1.50 1.50 1.50 1.50 1.00 1.00 1.00 1.00 0.16 1.84 1.00 1.00 1.00 1.00 1.00 1.00
0.56 1.65 1.34 2.51 1.40 2.42 1.79 3.31 1.15 2.86 1.19 2.63 1.00 3.06 2.17 2.13 2.07 2.00 2.06 2.17
15.57 27.05 24.09 28.55 26.64 27.57 27.07 28.45 23.81 27.83 22.75 29.44 17.30 29.36 28.55 28.06 28.54 28.18 27.67 28.81
2.12 2.62 2.40 3.17 2.93 3.08 2.82 3.60 2.64 3.21 2.52 3.30 2.24 3.47 2.98 2.97 2.97 2.89 2.85 3.00
2.41 3.39 3.27 3.74 3.32 3.53 3.48 3.73 3.25 3.57 3.14 3.45 2.58 3.73 3.36 3.27 3.39 3.37 3.31 3.41
0.02 0.02 0.1 0.23 0.09 0.13 0.11 0.65 0.03 0.36 0.00 0.25 0.05 0.45 0.05 0.04 0.04 0.06 0.06 0.06
45.7 79.3 70.6 83.7 78.1 80.9 79.4 83.4 69.8 81.6 66.7 86.3 50.7 86.1 83.7 82.3 83.7 82.6 81.1 84.5
Table 3 e ANOVA for response surface quadratic model for xylose yield. Source Model X1 X2 X3 X1 X2 X1 X3 X2 X3 X1^2 X2^2 X3^2 Residual Lack of Fit Pure Error Cor Total
Sum of squares
df
Mean square
F-value
297.8745 25.58029 61.16189 113.6149 0.007813 6.679513 28.23761 10.44147 8.19236 52.48139 3.085595 2.236512 0.849083 300.9601
9 1 1 1 1 1 1 1 1 1 10 5 5 19
33.09716 25.58029 61.16189 113.6149 0.007813 6.679513 28.23761 10.44147 8.19236 52.48139 0.30856 0.447302 0.169817
107.2635 82.90228 198.2175 368.2105 0.025319 21.6474 91.5143 33.83939 26.55034 170.0851
CV ¼ 2.14%; R2 ¼ 0.9897; Pred R2 ¼ 0.9386; Adj R2 ¼ 0.9805; Adeq Precision ¼ 35.981.
2.634031
Probe > F < < < <
0.0001 0.0001 0.0001 0.0001 0.8767 0.0009 < 0.0001 0.0002 0.0004 < 0.0001 0.1557
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according to corresponding Chinese National standards [19]. Xylan and glucan in material were analyzed using the modified Tappi T-222 om-88 method as described by Bura [20]. The pretreatment was carried out in 500 ml glass flasks. 20 g corncobs at a solid loading of 10% (w/w) was mixed with dilute sulfuric acid and pretreated in an autoclave with designed temperature and residence time. The liquid fraction was separated by filtration and the unhydrolyzed solid residue was washed with 40 ml warm water (60 C) for twice. The filtrate and wash liquid were pooled together and analyzed for sugar, acetic acid, furfural and 5-hydroxymethylfurfural (5-HMF) content. The solid phase was washed with water until neutrality and dried at 105 C for 6 h. The oven-dried samples were stored in valve bags for further analysis and enzymatic hydrolysis.
2.2.
Enzymatic hydrolysis
Before enzymatic hydrolysis, the glucan content in the treated samples was determined. Enzymatic hydrolysis experiments were carried out in 50 ml stoppered conical flasks with 20 ml medium. The cellulase used in the experiments was Cellulase ZC-1700, which was produced by CTA-TEX Chemical Co. LTD in China. The cellulase activity was determined by the method recommended by Ghose [21], and expressed in terms of filter paper units (FPU). One FPU was defined as the amount of enzyme capable of producing 1 mmol of reducing sugars in 1 min. The glucan concentration in enzymatic hydrolyzate was in the presence of 8% (w/v) medium. The samples were digested by cellulase loading of 30 FPU g1 glucan. The enzymatic digestibility tests were conducted as follows: 45 0.1 C, pH 4.8 (0.1 M sodium acetate buffer), 140 rpm in an air-bath shaker.
2.3. Fig. 1 e a) Contour plot of the combined effects of temperature and acid concentration on xylose yield. (b) Contour plot of the combined effects of acid concentration and time on xylose yield.
and temperature (105e130 C) for xylose production. Enzymatic hydrolysis of the remaining solid was carried out also to evaluate the acid pretreatment conditions for maximizing glucan conversion. The bioethanol yields from the solid phase were evaluated further by SSF.
2.
Materials and methods
2.1.
Dilute H2SO4 pretreatment
Saccharomyces cerevisiae CICC 31014 was grown on the preculture medium containing 30 g l1 glucose, 2 g l1 (NH4)2SO4, 5 g l1 KH2PO4, 0.5 g l1 MgSO4, 5 g l1 yeast extract. The pretreated solid sample, supplemented with 1 g l1 (NH4)2SO4, 2.5 g l1 KH2PO4, 0.2 g l1 MgSO4, 2 g l1 yeast extract and cellulase was used as the fermentation medium. The medium was autoclaved for 15 min at 121 C before adding enzymes and inoculum. The seed cells were prepared in a 250 ml flask containing 100 ml of preculture medium at 35 C in an air-bath shaker at 140 rpm for 14 h. 250 ml flasks containing 50 ml fermentation medium were used in SSF with a 5% (v/v) inoculum. The cellulase loading was 30 FPU g1 glucan. All SSF were performed at 35 C for 72 h on an orbital shaker agitated at 130 rpm. Samples were withdrawn periodically for ethanol determination.
2.4. Corncob from Heilongjiang province in Northeastern China was used as raw material. Particles in the size ranged from 2 mm to 4 mm (5e10 mesh) were used in the experiments. The compositions of corncob, such as benzene-ethanol extractives, cellulose, hemicellulose, and lignin, were determined
Microorganism and culture conditions for SSF
Analytical methods
The samples were analyzed by HPLC, equipped with UV and RI detectors. The concentrations of glucose, xylose, and arabinose were determined using refractive index detector and Aminex HPX-87H column at 65 C with 5 mM H2SO4 as mobile
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Table 4 e Sugar recovery during enzymatic digestion (g/100 g raw material corncorb) at each pretreatment condition. Run
Compositions of solid residue after acid pretreatment (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Glucan
Xylan
Lignin
Glucose (g)
Xylose (g)
Cellobiose (g)
64.1 66.4 65.2 68.1 65.5 68.7 66.0 70.1 64.9 69.6 65.0 68.1 64.7 68.6 67.1 67.1 67.0 67.1 67.1 67.1
12.4 8.6 10.9 6.0 9.8 5.0 9.1 2.6 11.2 3.3 11.0 5.4 11.7 5.1 7.3 7.4 7.4 7.5 7.4 7.4
23.5 24.9 23.9 26.0 24.7 26.3 24.9 27.3 23.9 27.1 24.1 26.5 23.6 26.3 25.6 25.4 25.6 25.4 25.5 25.5
31.48 33.31 33.35 30.70 30.28 30.24 32.38 26.54 32.60 28.59 33.19 29.82 33.10 30.59 30.60 32.15 31.26 32.42 31.77 31.51
6.77 4.77 6.15 2.98 5.05 2.46 4.92 1.11 6.23 1.53 6.18 2.61 6.62 2.54 3.72 3.94 3.81 4.03 3.87 3.86
2.92 3.83 3.11 4.10 3.63 4.05 3.87 4.08 3.09 4.46 3.38 4.61 2.84 4.21 4.09 4.06 4.26 4.06 4.08 4.09
phase at 0.8 ml min1. Furfural and 5-HMF was detected on UV chromatograms at 250 nm.
2.5.
Glucose þ cellobiose yield (%)
Sugar recovery in enzymatic hydrolysis
Experimental design
CCD was used to investigate the significance of temperature, acid concentration and retention time. The experiments were designed by using the Design-Expert 7.0.3 Trial (State Ease Inc., Minneapolis, MN. USA). The levels of variables were given in Table 1. A three-level, three-factor factorial central composite design and six replicates at the center points leading to 20 runs was employed for acid and enzymatic hydrolysis. The variables were coded according to the following equation:
xi ¼ ðXi X0 Þ=DXi
72.3 78.1 76.6 73.2 71.3 72.2 76.2 64.5 75.0 69.6 76.9 72.5 75.5 73.2 73.0 76.2 74.8 76.7 75.4 74.9
i ¼ 1; 2; .; k
(1)
Where xi and Xi are the dimensionless and the actual values of the independent variable i, X0 is the actual value of the independent variable at the center point, and DXi is the step change of Xi corresponding to a unit variation of the dimensionless value. The quadratic model for predicting the optimal point was expressed as Eq. (2): yi ¼ b0 þ
P
bi xi þ
P
bii x2i þ
P
bij xi xj i ¼ 1; 2; .; k; j ¼ 1; 2; .; k; isj (2)
Where yi is the predicted response, b0 is the interception coefficient, bi is the linear term, bii is the quadratic term, and bij is the interaction term.
Table 5 e ANOVA for response surface quadratic model for glucose D cellobiose yield. Source Model X1 X2 X3 X1 X2 X1 X3 X2 X3 X1^2 X2^2 X3^2 Residual Lack of Fit Pure Error Cor Total
Sum of squares
df
Mean square
F-value
Probe > F
38.61994 7.160523 3.187589 4.561361 9.850186 7.877632 0.845169 4.631377 0.383743 0.685611 1.584997 0.881545 0.703452 40.20494
9 1 1 1 1 1 1 1 1 1 10 5 5 19
4.291105 7.160523 3.187589 4.561361 9.850186 7.877632 0.845169 4.631377 0.383743 0.685611 0.1585 0.176309 0.14069
27.07327 45.17689 20.11101 28.77836 62.14641 49.70125 5.332307 29.2201 2.421098 4.325628
< 0.0001 < 0.0001 0.0012 0.0003 < 0.0001 < 0.0001 0.0436 0.0003 0.1508 0.0642
CV ¼ 1.12%; R2 ¼ 0.9606; Pred R2 ¼ 0.8083; Adj R2 ¼ 0.9251; Adeq Precision ¼ 22.471.
1.25317
0.4052
254
2.6.
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Statistical analysis
All experiments were carried out in triplicate and data were expressed as average values. Data obtained from CCD for optimization of the reaction conditions were used for determining the regression coefficients of the second-order multiple regression model. The analysis of variance (ANOVA) was evaluated using the Design-Expert 7.0.3 Trial. The quality of the fit of the polynomial model equation was evaluated by the coefficient of determination R2, its statistical and regression coefficient significance were checked with Ftest and t-test, respectively. The optimum values of the selected variables were obtained by analyzing the response surface contour plot and solving the regression equation.
3.
Results and discussion
3.1.
Raw material composition
The initial composition of the biomass was 42.21 0.27% cellulose, 34.47 0.35% hemicellulose, and 17.88 0.42% lignin. The hemicellulose part consisted of 85% xylose and small amounts of arabinose and glucose. The lignin content of corncob is in the range of some reported data for corncob (17e18%) [22,23]. There were also 2.99 0.06% benzeneeethanol extractives and 2.11 0.02% ash. Based on the original substrate composition of 43.1% glucan and 30.2% xylan and the appropriate change in mass due to hydrolysis, the maximum potential recovery of glucose and xylose was 47.8 g and 34.1 g, respectively, per 100 g of the dry weight of corncob. The sum of xylan and glucan accounted for about 73% of the dry material which makes corncob a very promising substrate for xylose and ethanol production.
3.2.
Effect of pretreatment on hydrolyzate composition
After dilute acid pretreatment, the slurry was separated into liquid phase (hydrolyzate) and solid phase. The composition in sugars and other compounds of the hydrolyzate, as a result of the pretreatment were measured and shown in Table 2. The variable considered in the analysis of the liquid phase was xylose yield. The results of the second-order response surface models for the xylose yield in the form of analysis of variance (ANOVA) were given in Table 3. Using the designed experimental data (Table 2), the second-order polynomial model for the yield of xylose in terms of coded factors was shown as the following equation: Y ¼ 28:31 þ 1:37X1 þ 2:12X2 þ 2:88X3 0:031X1 X2 0:91X1 X3 1:88X1 X3 0:85X21 0:75X22 1:91X23
(3)
Where Y is the predicted xylose yield (g/100 g raw material corncorb), X1 is temperature, X2 is time and X3 is acid concentration. As shown in Table 3, the “Model F-value” of 107.26 implied the model was significant and there was only a 0.01% chance that a “Model F-Value” this large could occur due to noise. The “Lack of Fit F-value” of 2.63 implied the lack of fit was not
significant relative to the pure error and there was a 15.57% chance that a “Lack of Fit F-value” this large could occur due to noise. The goodness of fit of the model was examined by determination coefficient (R2 ¼ 0.9897), which implied that the sample variation of more than 99% was attributed to the variables and only 1.03% of the total variance could not be explained by the model. The predicted determination coefficient (Pred R2 ¼ 0.9368) was in reasonable agreement with the adjusted determination coefficient (Adj R2 ¼ 0.9805), which was also satisfactory to confirm the significance of the model. A lower value of coefficient of variation (CV ¼ 2.14%) showed the experiments conducted were precise and reliable. The signal to noise ratio was measuered by “Adeq Precision”. A ratio greater than 4 is desirable. The ratio of 35.981 indicated an adequate signal, which implied this model could be used to navigate the design space. Table 3 also showed the significance of each coefficient, which was determined by t-test and P-value. The larger the magnitude of t-test and smaller the P-value, the more significant of the corresponding coefficient is. Values of “Prob > F” less than 0.05 indicate model terms are significant whereas values greater than 0.1 indicate the model terms are not significant. In this case, X1, X2, X3, X1X3, X2X3, X21, X22and X23 were significant model terms whereas X1X2 were not significant. This suggested temperature, time and acid concentration had significant effects on the xylose yield. Further temperature and acid concentration, time and acid concentration were interacted (Fig. 1a,b). This means xylose yield doesn’t always increase with the increase of acid concentration and temperature because the interaction of temperature and acid concentration negatively affected the xylose yield. For example, the xylose yield under 125 C, 1.5 h, 1.5% sulfuric acid is lower than that under 125 C, 1.5 h, 0.5% sulfuric acid. More furfural and 5-HMF (0.65 g/100 g corncorb) were produced which suggest that there exist some decomposition reaction which leads to dehydration of sugar to furfural or 5HMF. To overcome this problem, it is necessary to select satisfactory reaction conditions to obtain the high xylose yield and to keep the degradation products at low levels. The response surface showed that the highest xylose yield appeared at 123 C, 1.5 h, 1% sulfuric acid. Verification of the calculated optimum conditions for xylose yield was done by performing the experiment at optimized conditions. Under these conditions, xylose yield of 87.2% was obtained which is in agreement with the predicted value 88.1% suggesting that Eq. (3) is valid for xylose yield.
3.3.
Enzymatic hydrolysis of solid phase
After the hemicellulose in corncob has been hydrolyzed for xylose production, the remaining solid (mainly cellulose and lignin) is porous and can easily be hydrolyzed by cellulases into glucose. In order to evaluate the effects of the pretreatment process on consequent enzymatic hydrolysis, the solids after acid pretreatment were hydrolyzed by cellulase of 30 FPU g1 glucan for 72 h. The composition of sugars in enzymatic hydrolyzate, also as a result of the pretreatment were measured and shown in Table 4. Table 4 shows the glucose, cellobiose and xylose concentrations during the enzymatic hydrolysis. The glucose yield
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ranged from 26.5 g/100 g corncob to 33.4 g/100 g corncob. A cellobiose accumulation was detected during the enzymatic hydrolysis process. Considering during SSF process, the cellobiose is easily converted to glucose. The sum of glucose and cellobiose was calculated together as a response for statistical analysis. Y ¼ 36:12 0:72X1 0:48X2 0:58X3 1:11X1 X2 0:99X1 X3 0:33X1 X3 0:57X21 0:16X22 0:22X23
(4)
Where Y is the predicted glucose þ cellobiose yield (g/100 g raw material corncorb), X1 is temperature, X2 is time and X3 is acid concentration. The model used to evaluate the glucose þ cellobiose yield in enzymatic hydrolyzate fitted with the experimental data well (Table 5, P < 0.0001). Fig. 2aec presented the effect of two variables on the glucose þ cellobiose yield, while aother variable was held at constant level. An elliptical profile of the contour plots indicates remarkable interaction between the independent variables. The response surface showed that the highest glucose þ cellobiose yield appeared at 125 C, 0.5 h, 0.5% H2SO4, which is in the design. From Tables 2 and 3, we can observed that not only a significant portion of the xylan not liberated in acid pretreatment was released by enzyme digest, but also conditions for the highest xylose yield in acid hydrolysis stage did not give the best glucose yields in enzymatic hydrolysis stage. Pretreatment conditions required for best sugar yields depended on which sugars and which products were targeted. Similar observations were reported during dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis of the remaining solids [24].
3.4.
SSF
SSF processes combine enzymatic hydrolysis of cellulose with simultaneous fermentation of glucose obtained to ethanol. Thus, the presence of yeast together with cellulase reduces the accumulation of cellobiose, thereby increasing saccharification rate and ethanol yield. Two kinds of pretreatment conditions were selected before SSF. One is for the highest xylose recovery, and the other is for the highest glucose þ cellobiose recovery. To increase the ethanol concentration, SSF at high dry matter loading (10% and 14% glucan (w/v)) are conducted to obtain high ethanol content. The SSF experiments were performed under nonaseptic conditions with uncontrolled pH starting at 5 at the beginning of the fermentation and dropping steadily to 4.6 at the end. No cellobiose accumulation was observed. The highest 50.2 g l1 ethanol was reached in the SSF with 14% glucan concentration (Table 6). This concentration corresponded to 70.4% overall ethanol yield based on the glucose content in the raw material. It should to be pointed out that compared to 10% glucan substrate loading, the ethanol yield with 14% glucan substrate loading was reduced, Fig. 2 e (a) Contour plot of the combined effects of temperature and pretreatment time on glucose D cellobiose yield. (b) Contour plot of the combined effects of acid concentration and temperature on
glucose D cellobiose yield. (c) Contour plot of the combined effects of pretreatment time and acid concentration on glucose D cellobiose yield.
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Table 6 e Ethanol concentration and yield (expressed as percentage of theoretical yield) with different substrate loading. Substrate loading (%, w/v)
Glucose (g l1)
Xylose (g l1)
Arabinose (g l1)
Ethanol (g l1)
Percentage of theoretical yield (%)
Pretreated at 123 C, 1.5 h, 1% sulfuric acid, the solid residue after pretreatment contains 5.2% xylan and 69.3% glucan 10% glucan 0.7 7.7 1.2 38.3 67.6 14% glucan 0 10.6 1.9 47 65.8 Pretreated at 125 C, 0.5 h, 0.5% sulfuric acid, the solid residue after pretreatment contains 8.6% xylan and 66.4% glucan 10% glucan 0 10.9 1.4 40.3 71.2 14% glucan 0 13.9 2.1 50.2 70.4
though the ethanol concentration was higher. The reason for that may be insufficient mass transfer caused by stirring hindrance and lignin inhibition on cellulase.
4.
Conclusions
This study has demonstrated that xylan in the corncob can be removed efficiently by dilute H2SO4 pretreatment. The optimal combination of pretreatment conditions was found to be 1% sulfuric acid and treatment time of 1.5 h at 123 C, corresponding to 87.2% total xylan converted to xylose. Conditions for the highest xylose yield in acid hydrolysis stage did not give the best glucose yields in enzymatic hydrolysis stage. The highest glucose þ cellobiose recovery was 0.5% sulfuric acid for 30 min at 125 C, corresponding to 78.1% total glucan converted to glucose þ cellobiose. Pretreatment conditions required for best sugar yields depended on which sugars and which products were targeted. In the subsequent simultaneous saccharification and fermentation (SSF) experiments using 14% glucan substrates pretreated under above two kinds of conditions, 47 g l1 ethanol with a 65.8% theoretical yield and 50.2 g l1 ethanol with a 70.4% theoretical yield were obtained. This study showed some xylose release during enzymatic hydrolysis or SSF process. If the xylose and arabinose presented in the broth at the end of the SSF could be fermented to ethanol, another 8.0 g l1 ethanol could theoretically be produced (0.51 g ethanol/g pentose).
Acknowledgments This study was supported by National Basic Research Program of China (2011CB707406), National Natural Science Foundation of China (No. 31170466), High-level talents (innovation team) Projects of Heilongjiang University (Hdtd2010-17), Science Fund of Young Scholars (Heilongjiang University) and Research Fund of Key Laboratory of Microbiology (College of Life Sciences, Heilongjiang University, China).
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0.2 0.3 0.1 0.4
[2] Rodriguez CA, Ramirez JA, Garrote G, Vazquez M. Hydrolysis of sugarcane bagasse using nitric acid: a kinetic assessment. J Food Eng 2004;61:143e52. [3] Lavarack BP, Griffin GJ, Rodman D. The acid hydrolysis of sugarcane bagasse hemicellulose to produce xylose, arabinose, glucose and other products. Biomass Bioenerg 2002;23:367e80. [4] Herrera A, Te´llez-Luis SJ, Ramı´rez JA, Va´zquez M. Production of xylose from sorghum straw Using Hydrochloric Acid. J Cereal Sci 2003;37:267e74. [5] Rahman SHA, Choudhury JP, Ahmad AL. Production of xylose from oil palm empty fruit bunch fiber using sulfuric acid. Biochem Eng J 2006;30:97e103. [6] Garrote G, Dominguez H, Parajo JC. Generation of xylose solutions from eucalyptus wood by autohydrolysiseposthydrolysis processes: posthydrolysis kinetics. Bioresour Technol; 2001:155e64. [7] Eva P, Ba¨rbel H. Fermentation of lignocellulosic hydrolysates. I. Inhibition and detoxification. Bioresour Technol 2000;74:17e24. [8] Washuttle J, Riederer P, Banchen E. Qualitative and quantitative study of sugarealcohols in several foods. J Food Sci 1973;38:1262e3. [9] Emodi A. Xylitol: its properties and food applications. Food Technol 1978;32:28e32. [10] Carvalho W, Santos JC, Canilha L, Silva SS, Perego P, Converti A. Xylitol production from sugarcane bagasse hydrolysate: metabolic behaviour of Candida guilliermondii cells entrapped in Ca-alginate. Biochem Eng J 2005;25:25e31. [11] Liaw WC, Chen CS, Chang WS, Chen KP. Xylitol production from rice straw hemicellulose hydrolyzate by polyacrylic hydrogel thin films with immobilized Candida subtropicalis WF79. J Biosci Bioeng 2008;105:97e105. [12] Mancilha IM, Karim MN. Evaluation of ion exchange resins for removal of inhibitory compounds from corn stover hydrolyzate for xylitol fermentation. Biotechnol Prog 2003; 19:1837e41. [13] Solange IM, Giuliano D, Ineˆs CR. Influence of the toxic compounds present in brewer’s spent grain hemicellulosic hydrolysate on xylose-to-xylitol bioconversion by Candida guilliermondii. Process Biochem 2005;40:3801e6. [14] Villarreal MLM, Prata AMR, Felipe MGA, Almeida JBES. Detoxification procedures of eucalyptus hemicellulose hydrolysate for xylitol production by Candida guilliermondii. Enzyme Microb Technol 2006;40:17e24. [15] Canilha L, Almeida SJB, Solenzal AIN. Eucalyptus hydrolysate detoxification with activated charcoal adsorption or ionexchanger resins for xylitol production. Process Biochem 2004;39:1909e12. [16] Cheng KK, Zhang JA, Ling HZ, Ping WX, Huang W, Ge JP, et al. Optimization of pH and acetic acid concentration for bioconversion of hemicellulose from corncobs to xylitol by Candida tropicalis. Biochem Eng J 2009;43:203e7. [17] Cheng KK, Zhang JA, Erik C, Li JP. Integrated production of xylitol and ethanol using corncob. Appl Microbiol Biotechnol 2010;87:411e7.
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[18] Chen M, Xia LM, Xue PJ. Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate. Int Biodeter Biodegr 2007;59:85e9. [19] Shi SL. Analysis and detection of pulping and papermaking. 1st ed. Beijing: Chinese Light Industry Press; 2003. [20] Bura R, Mansfield SD, Saddler JN, Bothast RJ. SO2-catalyzed steam explosion of corn fiber for ethanol production. Appl Biochem Biotechnol 2002;98e100:59e72. [21] Ghose TK. Measurement of cellulase activities. Pure Appl Chem 1987;59:257e68.
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[22] Garrote G, Dominguez H, Parajo JC. Autohydrolysis of corncob: study of non-isothermal operation for xylooligosaccharide production. J Food Eng 2001;52:211e8. [23] Zhang MJ, Wang F, Su RX, Qi W, He ZM. Ethanol production from high dry matter corncob using fed-batch simultaneous saccharification and fermentation after combined pretreatment. Bioresour Technol 2010;101:4959e64. [24] Lloyd TA, Wyman CE. Combined sugar yields for dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis of the remaining solids. Bioresour Technol 2005;96:1967e77.
Available online at www.sciencedirect.com
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Statistical optimization of dilute sulfuric acid pretreatment of corncob for xylose recovery and ethanol production Bai-Yan Cai a, Jing-Ping Ge a,*, Hong-Zhi Ling a, Ke-Ke Cheng b,*, Wen-Xiang Ping a a b
Key Laboratory of Microbiology, College of Life Sciences, Heilongjiang University, Harbin 150080, PR China Institute of Nuclear and New Energy Technology, Tsinghua University, Beijing 100084, PR China
article info
abstract
Article history:
The interest on use of lignocellulose for producing fuels and chemicals is increasing as
Received 14 July 2010
these materials are low cost, renewable and widespread sources of sugars. Corncob is an
Received in revised form
attractive raw material for xylose and fuel ethanol production due to its high content of
22 September 2011
xylan and glucan. In this study, the central composite design of response surface method
Accepted 21 October 2011
was used to optimize dilute H2SO4 pretreatment of corncob, in respect to acid concentra-
Available online 25 November 2011
tion (0.16e1.84%), treatment time (0.16e1.84 h) and temperature (105e130 C) for xylose production. Enzymatic hydrolysis of the remaining solid was carried out further to evaluate
Keywords:
the acid pretreatment conditions for maximizing glucose production. The results showed
Corncob
that pretreatment conditions for the highest xylose production was 1% sulfuric acid for
Ethanol
1.5 h at 123 C, corresponding to 87.2% total xylan converted to xylose and that for the
Fermentation
highest glucose þ cellobiose recovery was 0.5% sulfuric acid for 30 min at 125 C, corre-
Hemicellulose
sponding to 78.1% total glucan converted to glucose þ cellobiose. In the subsequent
Pretreatment
simultaneous saccharification and fermentation (SSF) experiments using 14% glucan
Xylose
substrates pretreated under above two kinds of conditions, 47 g l1 ethanol with a 65.8% theoretical yield and 50.2 g l1 ethanol with a 70.4% theoretical yield were obtained, respectively. ª 2011 Elsevier Ltd. All rights reserved.
1.
Introduction
Lignocellulosic materials represent an abundant and inexpensive source of sugars which can be microbiologically converted to industrial products [1]. Diluted acids are commonly used for hydrolysis of the hemicellulosic fraction at moderate temperature. Research investigations on dilute acid hydrolysis of various raw materials, such as sugarcane bagasse, sorghum straw, eucalyptus wood and oil palm fiber, for xylose production have been carried out by several researchers [2e6]. From these research studies it was revealed that the acids attack the polysaccharides, especially hemicelluloses that are easier to be hydrolyzed than cellulose and
lignin. On the other hand, the condition is not severe enough to hydrolyze the crystalline structure of cellulose which remains as insoluble solid. Therefore, the cellulose and lignin fractions remain almost unaltered in the solid phase. Although xylose was the main sugar obtained from hemicellulose, other sugars such as glucose, arabinose, were also produced in low amount during the hydrolysis process. Xylose can be used as substrate for production of a wide variety of compounds by chemical and biochemical processes. One such compound is xylitol, which is extensively used in food, pharmaceutical and thin coating applications [7,8]. Many studies have been conducted utilizing the hemicellulose portion of agricultural residues like eucalyptus, rice straw, corncob,
* Corresponding authors. Tel./fax: þ86 10 89796086. E-mail addresses: [email protected] (J.-P. Ge), [email protected] (K.-K. Cheng). 0961-9534/$ e see front matter ª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biombioe.2011.10.023
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Table 1 e Variables and experimental design levels for response surface. Variables
Symbol
Temperature ( C) Time (h) Acid concentration (%)
X1 X2 X3
Range and levels 1
0
1
110 0.5 0.5
117.5 1 1
125 1.5 1.5
brewer’s spent grain, sugarcane bagasse, and corn stover for xylitol production [9e16]. Among the various agricultural crop residues, corncob is one of the most abundant agricultural materials in
Northeastern China. Corncob is an attractive raw material for xylose and fuel ethanol production due to its high content of xylan and glucan. The xylan fraction of corncob represents up to 30% of the total carbohydrates that can readily be hydrolyzed into xylose by diluted acids. After the xylan in corncob has been hydrolyzed for xylitol production, the corncob cellulosic residue is porous and can easily be hydrolyzed by cellulases into glucose, and further converted to ethanol, another high added-value chemical [17,18]. In this study, corncob was chosen as the raw lignocellulosic material for integrated xylose and ethanol production. The central composite design of response surface method was used to optimize dilute H2SO4 pretreatment of corncob, in respect to acid concentration (0.16e1.84%), treatment time (0.16e1.84 h)
Table 2 e Material balances for pretreatment conditions g/100 g raw material corncorb liquid fraction after pretreatment by dilute H2SO4. Run
X1 ( C)
X2 (h)
X3 (%)
Glucose (g)
Xylose (g)
Arabinose (g)
Acetic acid (g)
Furfural þ 5-HMF (g)
Xylose yield (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
110 125 110 125 110 125 110 125 105 130 117.5 117.5 117.5 117.5 117.5 117.5 117.5 117.5 117.5 117.5
0.50 0.50 1.50 1.50 0.50 0.50 1.50 1.50 1.00 1.00 0.16 1.84 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
0.50 0.50 0.50 0.50 1.50 1.50 1.50 1.50 1.00 1.00 1.00 1.00 0.16 1.84 1.00 1.00 1.00 1.00 1.00 1.00
0.56 1.65 1.34 2.51 1.40 2.42 1.79 3.31 1.15 2.86 1.19 2.63 1.00 3.06 2.17 2.13 2.07 2.00 2.06 2.17
15.57 27.05 24.09 28.55 26.64 27.57 27.07 28.45 23.81 27.83 22.75 29.44 17.30 29.36 28.55 28.06 28.54 28.18 27.67 28.81
2.12 2.62 2.40 3.17 2.93 3.08 2.82 3.60 2.64 3.21 2.52 3.30 2.24 3.47 2.98 2.97 2.97 2.89 2.85 3.00
2.41 3.39 3.27 3.74 3.32 3.53 3.48 3.73 3.25 3.57 3.14 3.45 2.58 3.73 3.36 3.27 3.39 3.37 3.31 3.41
0.02 0.02 0.1 0.23 0.09 0.13 0.11 0.65 0.03 0.36 0.00 0.25 0.05 0.45 0.05 0.04 0.04 0.06 0.06 0.06
45.7 79.3 70.6 83.7 78.1 80.9 79.4 83.4 69.8 81.6 66.7 86.3 50.7 86.1 83.7 82.3 83.7 82.6 81.1 84.5
Table 3 e ANOVA for response surface quadratic model for xylose yield. Source Model X1 X2 X3 X1 X2 X1 X3 X2 X3 X1^2 X2^2 X3^2 Residual Lack of Fit Pure Error Cor Total
Sum of squares
df
Mean square
F-value
297.8745 25.58029 61.16189 113.6149 0.007813 6.679513 28.23761 10.44147 8.19236 52.48139 3.085595 2.236512 0.849083 300.9601
9 1 1 1 1 1 1 1 1 1 10 5 5 19
33.09716 25.58029 61.16189 113.6149 0.007813 6.679513 28.23761 10.44147 8.19236 52.48139 0.30856 0.447302 0.169817
107.2635 82.90228 198.2175 368.2105 0.025319 21.6474 91.5143 33.83939 26.55034 170.0851
CV ¼ 2.14%; R2 ¼ 0.9897; Pred R2 ¼ 0.9386; Adj R2 ¼ 0.9805; Adeq Precision ¼ 35.981.
2.634031
Probe > F < < < <
0.0001 0.0001 0.0001 0.0001 0.8767 0.0009 < 0.0001 0.0002 0.0004 < 0.0001 0.1557
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according to corresponding Chinese National standards [19]. Xylan and glucan in material were analyzed using the modified Tappi T-222 om-88 method as described by Bura [20]. The pretreatment was carried out in 500 ml glass flasks. 20 g corncobs at a solid loading of 10% (w/w) was mixed with dilute sulfuric acid and pretreated in an autoclave with designed temperature and residence time. The liquid fraction was separated by filtration and the unhydrolyzed solid residue was washed with 40 ml warm water (60 C) for twice. The filtrate and wash liquid were pooled together and analyzed for sugar, acetic acid, furfural and 5-hydroxymethylfurfural (5-HMF) content. The solid phase was washed with water until neutrality and dried at 105 C for 6 h. The oven-dried samples were stored in valve bags for further analysis and enzymatic hydrolysis.
2.2.
Enzymatic hydrolysis
Before enzymatic hydrolysis, the glucan content in the treated samples was determined. Enzymatic hydrolysis experiments were carried out in 50 ml stoppered conical flasks with 20 ml medium. The cellulase used in the experiments was Cellulase ZC-1700, which was produced by CTA-TEX Chemical Co. LTD in China. The cellulase activity was determined by the method recommended by Ghose [21], and expressed in terms of filter paper units (FPU). One FPU was defined as the amount of enzyme capable of producing 1 mmol of reducing sugars in 1 min. The glucan concentration in enzymatic hydrolyzate was in the presence of 8% (w/v) medium. The samples were digested by cellulase loading of 30 FPU g1 glucan. The enzymatic digestibility tests were conducted as follows: 45 0.1 C, pH 4.8 (0.1 M sodium acetate buffer), 140 rpm in an air-bath shaker.
2.3. Fig. 1 e a) Contour plot of the combined effects of temperature and acid concentration on xylose yield. (b) Contour plot of the combined effects of acid concentration and time on xylose yield.
and temperature (105e130 C) for xylose production. Enzymatic hydrolysis of the remaining solid was carried out also to evaluate the acid pretreatment conditions for maximizing glucan conversion. The bioethanol yields from the solid phase were evaluated further by SSF.
2.
Materials and methods
2.1.
Dilute H2SO4 pretreatment
Saccharomyces cerevisiae CICC 31014 was grown on the preculture medium containing 30 g l1 glucose, 2 g l1 (NH4)2SO4, 5 g l1 KH2PO4, 0.5 g l1 MgSO4, 5 g l1 yeast extract. The pretreated solid sample, supplemented with 1 g l1 (NH4)2SO4, 2.5 g l1 KH2PO4, 0.2 g l1 MgSO4, 2 g l1 yeast extract and cellulase was used as the fermentation medium. The medium was autoclaved for 15 min at 121 C before adding enzymes and inoculum. The seed cells were prepared in a 250 ml flask containing 100 ml of preculture medium at 35 C in an air-bath shaker at 140 rpm for 14 h. 250 ml flasks containing 50 ml fermentation medium were used in SSF with a 5% (v/v) inoculum. The cellulase loading was 30 FPU g1 glucan. All SSF were performed at 35 C for 72 h on an orbital shaker agitated at 130 rpm. Samples were withdrawn periodically for ethanol determination.
2.4. Corncob from Heilongjiang province in Northeastern China was used as raw material. Particles in the size ranged from 2 mm to 4 mm (5e10 mesh) were used in the experiments. The compositions of corncob, such as benzene-ethanol extractives, cellulose, hemicellulose, and lignin, were determined
Microorganism and culture conditions for SSF
Analytical methods
The samples were analyzed by HPLC, equipped with UV and RI detectors. The concentrations of glucose, xylose, and arabinose were determined using refractive index detector and Aminex HPX-87H column at 65 C with 5 mM H2SO4 as mobile
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Table 4 e Sugar recovery during enzymatic digestion (g/100 g raw material corncorb) at each pretreatment condition. Run
Compositions of solid residue after acid pretreatment (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Glucan
Xylan
Lignin
Glucose (g)
Xylose (g)
Cellobiose (g)
64.1 66.4 65.2 68.1 65.5 68.7 66.0 70.1 64.9 69.6 65.0 68.1 64.7 68.6 67.1 67.1 67.0 67.1 67.1 67.1
12.4 8.6 10.9 6.0 9.8 5.0 9.1 2.6 11.2 3.3 11.0 5.4 11.7 5.1 7.3 7.4 7.4 7.5 7.4 7.4
23.5 24.9 23.9 26.0 24.7 26.3 24.9 27.3 23.9 27.1 24.1 26.5 23.6 26.3 25.6 25.4 25.6 25.4 25.5 25.5
31.48 33.31 33.35 30.70 30.28 30.24 32.38 26.54 32.60 28.59 33.19 29.82 33.10 30.59 30.60 32.15 31.26 32.42 31.77 31.51
6.77 4.77 6.15 2.98 5.05 2.46 4.92 1.11 6.23 1.53 6.18 2.61 6.62 2.54 3.72 3.94 3.81 4.03 3.87 3.86
2.92 3.83 3.11 4.10 3.63 4.05 3.87 4.08 3.09 4.46 3.38 4.61 2.84 4.21 4.09 4.06 4.26 4.06 4.08 4.09
phase at 0.8 ml min1. Furfural and 5-HMF was detected on UV chromatograms at 250 nm.
2.5.
Glucose þ cellobiose yield (%)
Sugar recovery in enzymatic hydrolysis
Experimental design
CCD was used to investigate the significance of temperature, acid concentration and retention time. The experiments were designed by using the Design-Expert 7.0.3 Trial (State Ease Inc., Minneapolis, MN. USA). The levels of variables were given in Table 1. A three-level, three-factor factorial central composite design and six replicates at the center points leading to 20 runs was employed for acid and enzymatic hydrolysis. The variables were coded according to the following equation:
xi ¼ ðXi X0 Þ=DXi
72.3 78.1 76.6 73.2 71.3 72.2 76.2 64.5 75.0 69.6 76.9 72.5 75.5 73.2 73.0 76.2 74.8 76.7 75.4 74.9
i ¼ 1; 2; .; k
(1)
Where xi and Xi are the dimensionless and the actual values of the independent variable i, X0 is the actual value of the independent variable at the center point, and DXi is the step change of Xi corresponding to a unit variation of the dimensionless value. The quadratic model for predicting the optimal point was expressed as Eq. (2): yi ¼ b0 þ
P
bi xi þ
P
bii x2i þ
P
bij xi xj i ¼ 1; 2; .; k; j ¼ 1; 2; .; k; isj (2)
Where yi is the predicted response, b0 is the interception coefficient, bi is the linear term, bii is the quadratic term, and bij is the interaction term.
Table 5 e ANOVA for response surface quadratic model for glucose D cellobiose yield. Source Model X1 X2 X3 X1 X2 X1 X3 X2 X3 X1^2 X2^2 X3^2 Residual Lack of Fit Pure Error Cor Total
Sum of squares
df
Mean square
F-value
Probe > F
38.61994 7.160523 3.187589 4.561361 9.850186 7.877632 0.845169 4.631377 0.383743 0.685611 1.584997 0.881545 0.703452 40.20494
9 1 1 1 1 1 1 1 1 1 10 5 5 19
4.291105 7.160523 3.187589 4.561361 9.850186 7.877632 0.845169 4.631377 0.383743 0.685611 0.1585 0.176309 0.14069
27.07327 45.17689 20.11101 28.77836 62.14641 49.70125 5.332307 29.2201 2.421098 4.325628
< 0.0001 < 0.0001 0.0012 0.0003 < 0.0001 < 0.0001 0.0436 0.0003 0.1508 0.0642
CV ¼ 1.12%; R2 ¼ 0.9606; Pred R2 ¼ 0.8083; Adj R2 ¼ 0.9251; Adeq Precision ¼ 22.471.
1.25317
0.4052
254
2.6.
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Statistical analysis
All experiments were carried out in triplicate and data were expressed as average values. Data obtained from CCD for optimization of the reaction conditions were used for determining the regression coefficients of the second-order multiple regression model. The analysis of variance (ANOVA) was evaluated using the Design-Expert 7.0.3 Trial. The quality of the fit of the polynomial model equation was evaluated by the coefficient of determination R2, its statistical and regression coefficient significance were checked with Ftest and t-test, respectively. The optimum values of the selected variables were obtained by analyzing the response surface contour plot and solving the regression equation.
3.
Results and discussion
3.1.
Raw material composition
The initial composition of the biomass was 42.21 0.27% cellulose, 34.47 0.35% hemicellulose, and 17.88 0.42% lignin. The hemicellulose part consisted of 85% xylose and small amounts of arabinose and glucose. The lignin content of corncob is in the range of some reported data for corncob (17e18%) [22,23]. There were also 2.99 0.06% benzeneeethanol extractives and 2.11 0.02% ash. Based on the original substrate composition of 43.1% glucan and 30.2% xylan and the appropriate change in mass due to hydrolysis, the maximum potential recovery of glucose and xylose was 47.8 g and 34.1 g, respectively, per 100 g of the dry weight of corncob. The sum of xylan and glucan accounted for about 73% of the dry material which makes corncob a very promising substrate for xylose and ethanol production.
3.2.
Effect of pretreatment on hydrolyzate composition
After dilute acid pretreatment, the slurry was separated into liquid phase (hydrolyzate) and solid phase. The composition in sugars and other compounds of the hydrolyzate, as a result of the pretreatment were measured and shown in Table 2. The variable considered in the analysis of the liquid phase was xylose yield. The results of the second-order response surface models for the xylose yield in the form of analysis of variance (ANOVA) were given in Table 3. Using the designed experimental data (Table 2), the second-order polynomial model for the yield of xylose in terms of coded factors was shown as the following equation: Y ¼ 28:31 þ 1:37X1 þ 2:12X2 þ 2:88X3 0:031X1 X2 0:91X1 X3 1:88X1 X3 0:85X21 0:75X22 1:91X23
(3)
Where Y is the predicted xylose yield (g/100 g raw material corncorb), X1 is temperature, X2 is time and X3 is acid concentration. As shown in Table 3, the “Model F-value” of 107.26 implied the model was significant and there was only a 0.01% chance that a “Model F-Value” this large could occur due to noise. The “Lack of Fit F-value” of 2.63 implied the lack of fit was not
significant relative to the pure error and there was a 15.57% chance that a “Lack of Fit F-value” this large could occur due to noise. The goodness of fit of the model was examined by determination coefficient (R2 ¼ 0.9897), which implied that the sample variation of more than 99% was attributed to the variables and only 1.03% of the total variance could not be explained by the model. The predicted determination coefficient (Pred R2 ¼ 0.9368) was in reasonable agreement with the adjusted determination coefficient (Adj R2 ¼ 0.9805), which was also satisfactory to confirm the significance of the model. A lower value of coefficient of variation (CV ¼ 2.14%) showed the experiments conducted were precise and reliable. The signal to noise ratio was measuered by “Adeq Precision”. A ratio greater than 4 is desirable. The ratio of 35.981 indicated an adequate signal, which implied this model could be used to navigate the design space. Table 3 also showed the significance of each coefficient, which was determined by t-test and P-value. The larger the magnitude of t-test and smaller the P-value, the more significant of the corresponding coefficient is. Values of “Prob > F” less than 0.05 indicate model terms are significant whereas values greater than 0.1 indicate the model terms are not significant. In this case, X1, X2, X3, X1X3, X2X3, X21, X22and X23 were significant model terms whereas X1X2 were not significant. This suggested temperature, time and acid concentration had significant effects on the xylose yield. Further temperature and acid concentration, time and acid concentration were interacted (Fig. 1a,b). This means xylose yield doesn’t always increase with the increase of acid concentration and temperature because the interaction of temperature and acid concentration negatively affected the xylose yield. For example, the xylose yield under 125 C, 1.5 h, 1.5% sulfuric acid is lower than that under 125 C, 1.5 h, 0.5% sulfuric acid. More furfural and 5-HMF (0.65 g/100 g corncorb) were produced which suggest that there exist some decomposition reaction which leads to dehydration of sugar to furfural or 5HMF. To overcome this problem, it is necessary to select satisfactory reaction conditions to obtain the high xylose yield and to keep the degradation products at low levels. The response surface showed that the highest xylose yield appeared at 123 C, 1.5 h, 1% sulfuric acid. Verification of the calculated optimum conditions for xylose yield was done by performing the experiment at optimized conditions. Under these conditions, xylose yield of 87.2% was obtained which is in agreement with the predicted value 88.1% suggesting that Eq. (3) is valid for xylose yield.
3.3.
Enzymatic hydrolysis of solid phase
After the hemicellulose in corncob has been hydrolyzed for xylose production, the remaining solid (mainly cellulose and lignin) is porous and can easily be hydrolyzed by cellulases into glucose. In order to evaluate the effects of the pretreatment process on consequent enzymatic hydrolysis, the solids after acid pretreatment were hydrolyzed by cellulase of 30 FPU g1 glucan for 72 h. The composition of sugars in enzymatic hydrolyzate, also as a result of the pretreatment were measured and shown in Table 4. Table 4 shows the glucose, cellobiose and xylose concentrations during the enzymatic hydrolysis. The glucose yield
b i o m a s s a n d b i o e n e r g y 3 6 ( 2 0 1 2 ) 2 5 0 e2 5 7
255
ranged from 26.5 g/100 g corncob to 33.4 g/100 g corncob. A cellobiose accumulation was detected during the enzymatic hydrolysis process. Considering during SSF process, the cellobiose is easily converted to glucose. The sum of glucose and cellobiose was calculated together as a response for statistical analysis. Y ¼ 36:12 0:72X1 0:48X2 0:58X3 1:11X1 X2 0:99X1 X3 0:33X1 X3 0:57X21 0:16X22 0:22X23
(4)
Where Y is the predicted glucose þ cellobiose yield (g/100 g raw material corncorb), X1 is temperature, X2 is time and X3 is acid concentration. The model used to evaluate the glucose þ cellobiose yield in enzymatic hydrolyzate fitted with the experimental data well (Table 5, P < 0.0001). Fig. 2aec presented the effect of two variables on the glucose þ cellobiose yield, while aother variable was held at constant level. An elliptical profile of the contour plots indicates remarkable interaction between the independent variables. The response surface showed that the highest glucose þ cellobiose yield appeared at 125 C, 0.5 h, 0.5% H2SO4, which is in the design. From Tables 2 and 3, we can observed that not only a significant portion of the xylan not liberated in acid pretreatment was released by enzyme digest, but also conditions for the highest xylose yield in acid hydrolysis stage did not give the best glucose yields in enzymatic hydrolysis stage. Pretreatment conditions required for best sugar yields depended on which sugars and which products were targeted. Similar observations were reported during dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis of the remaining solids [24].
3.4.
SSF
SSF processes combine enzymatic hydrolysis of cellulose with simultaneous fermentation of glucose obtained to ethanol. Thus, the presence of yeast together with cellulase reduces the accumulation of cellobiose, thereby increasing saccharification rate and ethanol yield. Two kinds of pretreatment conditions were selected before SSF. One is for the highest xylose recovery, and the other is for the highest glucose þ cellobiose recovery. To increase the ethanol concentration, SSF at high dry matter loading (10% and 14% glucan (w/v)) are conducted to obtain high ethanol content. The SSF experiments were performed under nonaseptic conditions with uncontrolled pH starting at 5 at the beginning of the fermentation and dropping steadily to 4.6 at the end. No cellobiose accumulation was observed. The highest 50.2 g l1 ethanol was reached in the SSF with 14% glucan concentration (Table 6). This concentration corresponded to 70.4% overall ethanol yield based on the glucose content in the raw material. It should to be pointed out that compared to 10% glucan substrate loading, the ethanol yield with 14% glucan substrate loading was reduced, Fig. 2 e (a) Contour plot of the combined effects of temperature and pretreatment time on glucose D cellobiose yield. (b) Contour plot of the combined effects of acid concentration and temperature on
glucose D cellobiose yield. (c) Contour plot of the combined effects of pretreatment time and acid concentration on glucose D cellobiose yield.
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Table 6 e Ethanol concentration and yield (expressed as percentage of theoretical yield) with different substrate loading. Substrate loading (%, w/v)
Glucose (g l1)
Xylose (g l1)
Arabinose (g l1)
Ethanol (g l1)
Percentage of theoretical yield (%)
Pretreated at 123 C, 1.5 h, 1% sulfuric acid, the solid residue after pretreatment contains 5.2% xylan and 69.3% glucan 10% glucan 0.7 7.7 1.2 38.3 67.6 14% glucan 0 10.6 1.9 47 65.8 Pretreated at 125 C, 0.5 h, 0.5% sulfuric acid, the solid residue after pretreatment contains 8.6% xylan and 66.4% glucan 10% glucan 0 10.9 1.4 40.3 71.2 14% glucan 0 13.9 2.1 50.2 70.4
though the ethanol concentration was higher. The reason for that may be insufficient mass transfer caused by stirring hindrance and lignin inhibition on cellulase.
4.
Conclusions
This study has demonstrated that xylan in the corncob can be removed efficiently by dilute H2SO4 pretreatment. The optimal combination of pretreatment conditions was found to be 1% sulfuric acid and treatment time of 1.5 h at 123 C, corresponding to 87.2% total xylan converted to xylose. Conditions for the highest xylose yield in acid hydrolysis stage did not give the best glucose yields in enzymatic hydrolysis stage. The highest glucose þ cellobiose recovery was 0.5% sulfuric acid for 30 min at 125 C, corresponding to 78.1% total glucan converted to glucose þ cellobiose. Pretreatment conditions required for best sugar yields depended on which sugars and which products were targeted. In the subsequent simultaneous saccharification and fermentation (SSF) experiments using 14% glucan substrates pretreated under above two kinds of conditions, 47 g l1 ethanol with a 65.8% theoretical yield and 50.2 g l1 ethanol with a 70.4% theoretical yield were obtained. This study showed some xylose release during enzymatic hydrolysis or SSF process. If the xylose and arabinose presented in the broth at the end of the SSF could be fermented to ethanol, another 8.0 g l1 ethanol could theoretically be produced (0.51 g ethanol/g pentose).
Acknowledgments This study was supported by National Basic Research Program of China (2011CB707406), National Natural Science Foundation of China (No. 31170466), High-level talents (innovation team) Projects of Heilongjiang University (Hdtd2010-17), Science Fund of Young Scholars (Heilongjiang University) and Research Fund of Key Laboratory of Microbiology (College of Life Sciences, Heilongjiang University, China).
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