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Comparison of the effects of five pretreatment methods on enhancing the enzymatic digestibility and ethanol production from sweet sorghum bagasse
Bioresource Technology 111 (2012) 215–221
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Comparison of the effects of five pretreatment methods on enhancing the enzymatic digestibility and ethanol production from sweet sorghum bagasse Weixing Cao, Chen Sun, Ronghou Liu ⇑, Renzhan Yin, Xiaowu Wu Biomass Energy Engineering Research Centre, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 21 November 2011 Received in revised form 29 January 2012 Accepted 7 February 2012 Available online 16 February 2012 Keywords: Sweet sorghum bagasse Enzymatic hydrolysis Pretreatment Bio-ethanol
a b s t r a c t To improve the enzymatic digestibility of sweet sorghum bagasse and bioethanol production, five pretreatment methods have been investigated and compared, including (1) dilute NaOH solution autoclaving pretreatment, (2) high concentration NaOH solution immersing pretreatment, (3) dilute NaOH solution autoclaving and H2O2 immersing pretreatment, (4) alkaline peroxide pretreatment and (5) autoclaving pretreatment. Among them, the best result was obtained when sweet sorghum bagasse was dilute NaOH solution autoclaving and H2O2 immersing pretreatment. The highest cellulose hydrolysis yield, total sugar yield and ethanol concentration were 74.29%, 90.94 g sugar/100 g dry matter and 6.12 g/L, respectively, which were 5.88, 9.54 and 19.13 times higher than the control. Moreover, the FTIR and SEM analysis illustrated significant molecule and surface structure changes of the sweet sorghum bagasse after pretreatments. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Sweet sorghum is a high photosynthetic efficiency energy crop, and it is adapted to growing in nutrient-poor soils, making it one of the most promising energy-crop for bio-ethanol production. Both its grain and stalk are ideal raw material for bioethanol production due to its high biomass and sugar-yielding (Billa et al., 1997). The starches in the grain can be hydrolyzed to fermentable sugars by amylase, while the cellulose and hemicellulose in the bagasse require pretreatment prior to enzymatic hydrolysis and further conversion to ethanol (Lin and Tanaka, 2006). Bio-ethanol production from sweet sorghum bagasse is more attractive in terms of energy balances and emissions, and it may be the case that the bagasse will become the vital supplementary material for ethanol production. The main components of sweet sorghum bagasse are cellulose, hemicellulose and lignin (Sipos et al., 2009), of which the intricate structure severely restricts the enzymatic hydrolysis. In order to improve the accessibility of the enzyme to cellulose, the studies on lignocellulose pretreatment are needed (Taherzadeh and Karimi, 2008). The pretreatment methods mainly include physical methods such as mechanical or thermal (Mais et al., 2002; Silva et al., 2010), chemical methods (Saha et al., 2005; Yamashita et al., 2010; Saha and Cotta, 2006; Beukes and Pletschke, 2011), biological methods and combination of these methods. The mechanism of physical pretreatment is to increase the accessible surface area and decrease the crystallinity degree of lignocellulose by chipping, milling, grinding ⇑ Corresponding author. Tel./fax: +86 21 34205744. E-mail address: [email protected] (R. Liu). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2012.02.034
or irradiation (Fan et al., 1980; Taherzadeh and Karimi, 2008; Mais et al., 2002; Silva et al., 2010). It is also effective for improving the enzymatic hydrolysis using thermal pretreatment such as steam explosion and liquid hot water pretreatment which can remove most of the hemicellulose (Shen et al., 2011; Laser et al., 2002). The typical conditions of liquid hot water pretreatment are high temperature and pressure (160–260 °C, 0.69–4.83 MPa) at short time (e.g. a few seconds to several minutes) or ambient temperature and normal pressure at relatively long time (several hours to several days) (Dien et al., 2006; Sreenath et al., 1999). Generally, the shortcoming of these physical pretreatment methods is high energy requirement. Alkaline (e.g. NaOH) and alkaline peroxide pretreatments which belong to chemical methods are effective processes for pretreating lignocellulose materials. NaOH is widely used for lignocellulose pretreatment. It can remove partial lignin and hemicellulose in the biomass by fracturing the ester bonds thereby increasing the porosity of the biomass (Xu et al., 2010; Zhang et al., 2011). Alkaline peroxide is used for lignocellulose pretreatment in recent years. It can improve the enzymatic hydrolysis by delignification. Mishima et al. (2006) also showed that the alkaline peroxide pretreatment method was one of the most effective methods for improving the enzymatic hydrolysis. In addition, no measurable furfural and hydroxymethylfurfural (HMF) which are harmful for yeast were detected in the process. That indicates it is easier for yeast to ferment the hydrolyzate compared to dilute-acid pretreatment using alkaline peroxide pretreatment method. Although several pretreatment methods have shown the effectiveness in many researches (Taherzadeh and Karimi, 2008), the combination of two
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or more pretreatment methods may be more effective than separate single one. Moreover, selection of pretreatment process for a certain material depends on the biomass material type. Besides, the enzymatic hydrolysis still needs to be improved when sweet sorghum bagasse is concerned. This study aims to compare the effects of five pretreatment methods on improving enzymatic hydrolysis of sweet sorghum bagasse and the ethanol production from the hydrolyzate. In addition, the lignocellulose compositions and structure changes of the sweet sorghum bagasse were also investigated. 2. Methods 2.1. Sweet sorghum Sweet sorghum (Chongming No. 1) was harvested in Qibao campus, Shanghai Jiao Tong University, China. The stalks were squeezed by a three-roller mill to obtain the liquid phase and bagasse separately. The bagasse was dried in the air and then ground to pass through sieve with 40 meshes. After that it was washed using boiling water three times to substantially remove the major soluble sugars (sucrose, glucose, fructose, etc.) present in the stalk liquid phase. The ratio of bagasse to water was 1:1 on a weight basis. Finally it was dried at 60 °C and stored in plastic bag at room temperature to avoid possible interference in the evaluation of the enzymatic hydrolysis of the cellulose (Wu et al., 2011). 2.2. Pretreatment process Five pretreatment methods were used in this study, labeled as A, B, C, D and E. Untreated bagasse was used as the control in the following experiments. Each pretreatment was conducted duplicated and the results were averaged. 2.2.1. Dilute NaOH solution autoclaving pretreatment (A) Ten grams of dry sweet sorghum bagasse was slurried with 100 mL 2% (w/v) sodium hydroxide solution for 5 min in a 500 mL flask with a silicone stopple and then autoclaved at 121 °C for 60 min. The residues were centrifuged and washed with distilled water until neutral pH was achieved and dried at 60 °C. The yield of product was 5.978 ± 0.212 g. 2.2.2. High concentration NaOH solution immersing pretreatment (B) Ten grams dry sweet sorghum bagasse was slurried with 100 mL 20% (w/v) sodium hydroxide solution for 5 min, and then stood for 2 h in a 500 mL flask with a silicone stopple. The residues were centrifuged and washed with distilled water until neutral pH was achieved and dried at 60 °C. The yield of product was 1.642 ± 0.141 g. 2.2.3. Dilute NaOH solution autoclaving and H2O2 immersing pretreatment (C) Ten grams dry sweet sorghum bagasse was slurried with 100 mL 2% (w/v) sodium hydroxide solution for 5 min in a 500 mL flask with a silicone stopple, and then autoclaved at 121 °C for 60 min. The 5% (w/v) hydrogen peroxide was mixed into the pretreated slurry after cooling down to room temperature. The mixture was kept airtight and in dark place for 24 h. The residues were centrifuged and washed with distilled water until neutral pH was achieved and dried at 60 °C. The yield of product was 5.432 ± 0.038 g. 2.2.4. Alkaline peroxide pretreatment (D) Ten grams dry sweet sorghum bagasse was slurried with 100 mL 2% (w/v) sodium hydroxide solution for 5 min, and then
stood for 2 h in a 500 mL flask with a silicone stopple. The 5% (w/v) hydrogen peroxide was mixed into the pretreated slurry. The mixture was kept airtight and in dark place for 24 h. The residues were centrifuged and washed with distilled water until neutral pH was achieved and dried at 60 °C. The yield of product was 6.856 ± 0.014 g. 2.2.5. Autoclaving pretreatment (E) Ten grams dry sweet sorghum bagasse was slurried with 100 mL distilled water for 5 min in a 500 mL flask with a silicone stopple, and then autoclaved at 121 °C for 60 min. After cooling down to room temperature, the residues were centrifuged and washed with distilled water until neutral pH was achieved and dried at 60 °C. The yield of product was 8.824 ± 0.156 g. 2.3. Enzymatic assay and hydrolysis A commercial cellulase (Celluclast 1.5L, Sigma Aldrich) supplemented with b-glucosidase (Novzymes 188, Denmark) was used for enzymatic hydrolysis of the sweet sorghum bagasse. The activities of Celluclast 1.5L and b-glucosidase were 45.8 FPU/mL and 302.1 U/mL, respectively. One unit of cellulase activity is defined as the amount of the enzyme that releases 1 mg of glucose per minute in the reaction mixture at 50 °C and pH 4.8. One unit of b-glucosidase activity is defined as the amount of the enzyme that releases 1 lmol of p-nitrophenol per minute in the reaction mixture at 50 °C and pH 4.8 (Saha and Cotta, 2006). The mixture was hydrolyzed in sodium citrate buffer (50 mM, pH 4.8) with the substrate loading of 2%. The enzyme loadings of Celluclast 1.5L and b-glucosidase were 20 FPU/g dry biomass and 40 IU/g dry biomass, respectively. The hydrolysis was conducted in a shaker at 50 °C. Samples (1 mL) were withdrawn at 12, 24, 48, 72 and 96 h and centrifuged at 10,000 rpm for 10 min, and the supernatants were preserved at 20 °C until they were used for sugar analysis. 2.4. Microorganism and batch fermentation Active dry yeast was bought from Angel Yeast Company of Hubei Province in China for ethanol fermentation. The medium was as follows: glucose 50 g/L, yeast 5 g/L, peptone 5 g/L, MgSO47H2O 1 g/ L, K2HPO4 1 g/L. 6 M HCl or NaOH solution was used for pH control. The medium was autoclaved at 121 °C for 20 min. The hydrolyzate was sterilized by autoclaving at 121 °C for 20 min before it was inoculated with the yeast medium at the volume ratio of 1:10 of the fermentation broth aseptically. The fermentation tests were conducted at 30 ± 0.5 °C in a shaker at 150 rpm. 2.5. Analytical methods The contents of cellulose, hemicellulose and lignin were determined according to the reference (Van Soest, 1963). The sugars in hydrolyzate including glucose, xylose, arabinose, galactose and mannose were measured with a HPLC system (Shimadzu LC-10A, Japan) with an Aminex HPX-87P column (7.8 mm I.D. 30 cm, Bio-Rad, USA) (Cara et al., 2007). Deionized water was used as the mobile phase with a flow rate of 1 mL/min and column temperature of 80 °C. The activities of the cellulase and b-glucosidase were measured according to the references (Xu et al., 2010). Ethanol concentration was analyzed by gas chromatography (Agilent 7890A GC system, USA) with a flame ionization detector and isopropanol was used as an internal standard (Liu and Shen, 2008). A scanning electron microscope (SEM, Siron 200, FEI Company, USA) was used to detect the microscopic structure of the sweet sorghum bagasse samples. The Fourier Transform Infrared Raman Spectroscopy (FTIR, EQUINOX 55, BRUKER Company, Germany)
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was recorded between 400 and 4000 cm1. Discs were prepared with 2 mg dried sample and 200 mg KBr (Zhang et al., 2011). The cellulose hydrolysis yield, total sugar yield and dry matter loss are defined as following three equations (Wu et al., 2011).
R1 ¼ C 1 =ðm W 1:11Þ 100ð1ÞR2 ¼ C 2 =ðm 100Þð2ÞR3 ¼ ðm0 mt Þ=m0 100ð3Þ where R1 is cellulose hydrolysis yield, %. C1 is the glucose mass in the hydrolyzate, g. m is the dry sweet sorghum bagasse mass, g. W is the cellulose content in dry sweet sorghum bagasse, %. 1.11 in Eq. (1) is the theoretical conversion from cellulose to glucose. R2 is the total sugar yield, g sugar/100 g dry matter. C2 is the total sugar mass in the hydrolyzate, g. R3 is the dry matter loss, %. m0 is the dry sweet sorghum bagasse mass before pretreatment, g. mt is the dry sweet sorghum bagasse after pretreatment, g. 2.6. Statistical analysis Data were analyzed for statistical significance by a one-way analysis of variance (ANOVA). A 5% probability level (p = 0.05) was used to accept or reject the null hypothesis. Duncan’s multiple range tests at the level of 5% were used to analyze the significances of different pretreatment methods. In this study, means in the tables followed by the same letter with a same column means not significantly different using Duncan’s multiple range tests at the level of 5% (Duncan, 1955). 3. Results and discussion 3.1. The effects of different pretreatment methods on the composition of sweet sorghum bagasse The aim of pretreatments was to change raw material properties, remove or dissolve lignin and hemicellulose and reduce the crystallinity of cellulose (Kumar et al., 2009). To be specific, with perfect pretreatment, the lignin will be mostly degraded while the cellulose and hemicellulose will be retained. The best pretreatment method and condition usually depend mainly on the type of lignocelluloses (Taherzadeh and Karimi, 2008). The dry matter loss has also been applied to evaluate the pretreatment effect in this study. The less dry matter loses, the more potential fermentation substrate is retained (Zhu et al., 2005). Table 1 shows the main composition, acid detergent lignin (ADL) removal and dry matter loss of sweet sorghum bagasse. The results of Table 1 showed that the orders of cellulose content, hemicellulose content, ADL content, ADL removal and dry matter loss were C > A > D > B > E > control, control > E > D > A > B > C, control > E > B > D > A > C, C > A > D > B > E and B > C > A > D > E, respectively. As it could be seen from Table 1, the cellulose content of the bagasse pretreated by method C was the highest one, while its ADL content and hemicellulose content were the lowest one. Dilute NaOH pretreatment of lignocellulose materials has been
found to cause swelling, leading to an increase in internal surface area of the sweet sorghum bagasse and disruption of the lignin structure. Millet reported (1976) that the NaOH pretreatment could decrease lignin content from 24% to 55%. But no effect was observed for softwoods with lignin content greater than 25% at the normal ambient conditions. Elevated temperatures can enhance lignin removal (Taherzadeh and Karimi, 2008). Silverstein et al. (2007) also reported that 2% NaOH in 90 min at 121 °C was the best pretreatment condition, resulting in 65% of delignification. In this study, both methods A and C were conducted at 121 °C, and the pretreatment time was 60 min, which resulted in more than 80% of ADL removal. Thus lignin removal increases enzyme effectiveness by eliminating nonproductive adsorption sites and by increasing access to cellulose and hemicellulose (Kumar et al., 2009). On the other hand, sweet sorghum bagasse pretreated by method A has higher cellulose content and lower ADL lignin than the others except method C. However, the hemicellulose content with method A was significantly higher than method C (p < 0.05). Meantime, the dry matter losses of bagasse pretreated by methods A and C were 40.75% and 46.10%, respectively. Both of which belonged to the medium level among all the pretreatment methods. The pretreatment methods A and C led to a comparatively low dry matter loss but significantly reduced the ADL lignin content and increased the cellulose content, which meant 2% sodium hydroxide pretreatment under autoclaving condition had shown sound effectiveness. The dilute NaOH pretreatment was beneficial for the follow-up enzymatic hydrolysis of the bagasse, which was supported by the results of the references (Wu et al., 2011; Curreli et al., 1997). By comparing methods A and C, it can be deduced that applying hydrogen peroxide during alkaline pretreatment will improve the effectiveness of hemicellulose and ADL lignin removal and cellulose retaining. But the cost of applying hydrogen peroxide is the increase of dry matter loss. The ANOVA showed that there was no significant differences (p > 0.05) in terms of the hemicellulose content between method B and C. Therefore, applying considerable amount of sodium hydrogen will do the same work in degrading and retaining hemicellulose as using small quantity of sodium hydrogen under autoclaving condition plus hydrogen peroxide immersing. However, the ADL lignin content of bagasse pretreated by method B was only superior to the control, which would definitely affect the enzyme hydrolysis process. In addition, the dry matter loss of method B strikingly came up to 83.7%, which would be disadvantageous for potential productivity of the follow-up fermentation. Also, this means that the concentration of 20% NaOH was too high for pretreatment due to the great loss in dry matter. Method D could be considered as the moderate method because of the middle level of its cellulose, hemicellulose, ADL lignin content and the dry matter loss. Compared with method C, method D pretreated raw material in rather low temperature and pressure. So it can be concluded that high temperature and pressure during oxydic alkaline pretreatment will be beneficial for retaining cellulose, removing hemicellulose and ADL lignin, but not good for retaining dry matter. On the other
Table 1 The main composition, ADL removal and dry matter loss of sweet sorghum bagasse. No. Control A B C D E A B
Cellulose (%) f,B
49.78 ± 0.86 78.44 ± 0.35b 69.90 ± 0.09d 82.08 ± 0.48a 72.45 ± 0.15c 54.40 ± 0.27e
Hemicellulose (%) a
27.72 ± 0.08 15.09 ± 0.91d 13.94 ± 0.35d 9.45 ± 0.57e 17.52 ± 0.91c 22.44 ± 0.77b
ADLA (%)
ADL removal (%)
Dry matter loss (%)
10.83 ± 0.18a 1.68 ± 0.16d 7.50 ± 0.29b 0.97 ± 0.12e 2.29 ± 0.01c 10.71 ± 0.06a
Null 84.52 ± 1.24b 30.82 ± 1.48d 91.02 ± 0.98a 78.84 ± 0.23c 1.16 ± 1.11e
Null 40.75 ± 2.19c 83.70 ± 1.56a 46.10 ± 0.57b 32.25 ± 0.21d 13.00 ± 1.70e
Acid detergent lignin. Means in the tables followed by the same letter with a same column means not significantly different using Duncan’s multiple range tests at the level of 5%.
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hand, when methods A and D are compared, it can be deduced that method A is more effective than method D in retaining cellulose and removing hemicellulose and ADL lignin than hydrogen peroxide. The lowest dry matter loss appeared in method E, but ANOVA showed that compared with control, there was no significant difference on ADL lignin removal (p > 0.05). Liquid hot-water (LHW) pretreatment is an environmental friendly pretreatment method with no addition of chemicals. Dien et al. (2006) reported that 75% of the xylan was dissolved at 160 °C, for 20 min, and higher temperatures, e.g. 220 °C, which can dissolve hemicellulose completely and remove lignin partially with 2 min (Dien et al., 2006; Sreenath et al., 1999). Laser et al. (2002) also showed that most hemicellulose was removed at 170–230 °C for 1–46 min. The pretreatment temperature of method E in this study was 121 °C, which is not sufficient to dissolve the hemicellulose and remove the lignin. Therefore, cellulose and hemicellulose might still be bundled by a great amount of lignin, which would harm the follow-up enzyme hydrolysis and ethanol production (Hendriks and Zeeman, 2009). So when compared with method E and control, it is clear that mere high temperature and high pressure will do limited work on effective pretreatment without alkali being involved. In brief, from the aspect of contents of cellulose, hemicellulose, ADL lignin, and the dry matter loss, methods A and C would be advisable as the pretreatment methods before enzymatic hydrolysis and ethanol fermentation. On the other hand, considering that not all of the yeast for ethanol production can utilize pentose such as xylose. And in such condition, hemicellulose would turn out to be another physical barrier which surrounds the cellulose fibers and can protect the cellulose from enzymatic attack (Wu et al., 2011; Curreli et al., 1997). Therefore, method C would be the best one for the follow-up enzymatic hydrolysis and ethanol formation.
3.2. The effects of different pretreatment methods on sugar concentration
16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
3.2.2. The effects of different pretreatment methods on the xylose concentration Fig. 2 shows the effects of different pretreatment methods on xylose concentration during the enzymatic hydrolysis. As it could be seen from Fig. 2, the variation tendency of xylose concentration in the hydrolyzate was similar to that of glucose concentration, but the xylose concentration was much lower than the glucose concentration in all the pretreatment samples. This was because hemicellulose content in both pretreated and unpretreated bagasse was lower than the cellulose content. Generally speaking, the majority of hydrolyzate of hemicellulose in ligno-cellulosic waste is xylose, and there are minor amounts of arabinose in the hydrolyzate of the bagasse (Taherzadeh and Karimi, 2008). In this sense, the hydrolysis of hemicellulose is related to the xylose concentration in the hydrolyzate. The order of final xylose concentration in hydrolyzate was A > D > C > B > E > control. According to Table 1, there was more hemicellulose while comparatively less lignin remained in
6
Control A B C D E
0
12
24
36
48
60
72
84
96
Hydrolysis time (h) Fig. 1. The effects of different pretreatment methods on glucose concentration during the enzymatic hydrolysis.
Concentration of xylose (mg/mL)
Concentration of glucose (mg/mL)
3.2.1. The effects of different pretreatment methods on glucose concentration Fig. 1 shows the effects of different pretreatment methods on glucose concentration during the enzymatic hydrolysis. As it could be seen from Fig. 1, the glucose concentration in the hydrolyzate increased as the hydrolysis time prolonged. The ANOVA analysis showed that the glucose concentration in the hydrolyzate for all pretreatment methods and control reached a plateau within 24 h (p > 0.05), which indicated that all the glucose production potential had been achieved in this short period (Saha and Cotta, 2006). The order of glucose concentration in the hydrolyzate after 96 h with
different pretreatment methods was C > B > A > D > E > control. Specifically, there was no significant difference of the glucose concentration between method E and the control after hydrolysis for 24 h, although the time for method E to achieve constant concentration of glucose was much shorter than the control. With regard to method C, the glucose concentration was the highest one among all samples after 12 h, which indicated that the pretreatment method C was the most effective one on converting the cellulose in sweet sorghum bagasse to glucose in these five pretreatment methods. The final glucose concentration in method C was up to 14.16 mg/mL, which was 9.8 times as much as the control. The glucose concentration in the hydrolyzate of bagasse pretreated with method A was higher than method B during the first 24 h, but the trend was reverse in the following 24 h. It demonstrated that the pretreatment with low concentration of sodium hydrogen plus autoclaving was much better than mere high concentration of alkali pretreatment for increasing the glucose concentration in hydrolyzate during 48 h. Although the glucose concentration in the hydrolysis pretreated by method D was almost constant after 12 h, the glucose concentration in method D was 26.5% lower than method C. This proved once again that it is much better for the cellulose hydrolysis to use relatively high temperature and pressure than the normal temperature and pressure during the pretreatment process. Both method C and D used alkaline hydrogen peroxide, which is good for improving the cellulose conversion by the removal of hemicellulose and lignin and increasing the accessibility of cellulose (Sreenath et al., 1999). In short, pretreatment method C yielded the highest glucose concentration in the hydrolyzate.
5 4
Control A B C D E
3 2 1 0 0
12
24
36 48 60 72 Hydrolysis time (h)
84
96
Fig. 2. The effects of different pretreatment methods on xylose concentration during the enzymatic hydrolysis.
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methods A and D than the others, except for method C. And both the hemicellulose and ADL lignin contents of method A were less than method D. But according to Fig. 2, firstly, method A could release xylose much faster than method D, and secondly, the xylose concentration in the hydrolyzate with pretreatment method A was the highest one. So lignin amount left after pretreatment is key factor for hemicellulose degrading during the enzymatic hydrolysis in the whole process. The same conclusion could also be deduced from the results of method B and C. Besides, the xylose concentration in method E was close to the control, and both method E and the control had less xylose in the hydrolyzate. However, both of which had more hemicellulose. This indicated that less hemicellulose in the bagasse of method E and control was hydrolyzed, because much of the cellulose and hemicellulose is unreachable for enzyme under the wrap of remained lignin (Beukes and Pletschke, 2011). Accordingly, xylose concentration with method A was the highest one in all pretreatment methods due to the reasons that method A yields a relatively high content of hemicellulose and low content of ADL lignin. 3.3. The effects of different pretreatment methods on enzymatic hydrolysis and ethanol fermentation of sweet sorghum bagasse Table 2 shows the effects of different pretreatment methods on cellulose hydrolysis yield, total sugar yield and ethanol concentration of sweet sorghum bagasse. The cellulose hydrolysis yield implies the efficiency of converting cellulose to glucose. The order of cellulose hydrolysis yield of sweet sorghum bagasse with different pretreatment methods for 96 h hydrolysis was C > B > A > D > control > E. This order was almost the same as that of glucose concentration in the hydrolyzate after 96 h. It indicated that alkaline pretreatment was good for improving the cellulose conversion. The cellulose hydrolysis yield of method C was 74.29%, which was 5.88 times higher than that of the control. It was higher than the result (70%) obtained from the steam pretreatment of Zhang’s research (Zhang et al., 2011). That may be due to the reasons that the content of hemicellulose and lignin was lower in the pretreated bagasse, and more cellulose was converted to glucose. Different pretreatment conditions can result in different pretreatment effects. In low alkaline concentration processes, with the concentration of 0.5– 4% NaOH at high temperature and pressure, the structure of lignocellulose can be destroyed in the pretreatment process while NaOH pretreatment at high temperature, the majority of the lignin and hemicellulose can be removed from the solid phase (Mirahmadi et al., 2010). On the other hand, the high concentration NaOH pretreatment (6–20%) is usually used at ambient conditions. Lignin was not significantly removed from the biomass (Mirahmadi et al., 2010). The ANOVA showed there was no significant difference of the cellulose hydrolysis yield between method E and the control. Also, it showed that mere autoclaving with distilled water cannot significantly improve the cellulose hydrolysis yield. Method E in this study was not similar to LHW pretreatment, because the pretreatment temperature (121 °C) of method E in this study was lower than the LHW pretreatment (160–260 °C). Therefore, it was not enough for method E to dissolve the hemicellulose and remove the lignin
for improving the cellulose hydrolysis yield (Sreenath et al., 1999). Totally, the pretreatment method C was the most effective one on increasing the cellulose hydrolysis yield in five pretreatment methods. During the enzymatic hydrolysis of sweet sorghum bagasse, the cellulose was converted to glucose, and the hemicellulose was converted to xylose, arabinose, mannose, galactose and other sugars. Table 3 shows the sugar concentration in the hydrolyzate after enzymatic hydrolysis for 96 h. As it could be seen from Table 3, there were minor amounts of arabinose in the hydrolyzate of the bagasse pretreated by methods A, B, C and D, while no arabinose was detected in the hydrolyzate of bagasse pretreated by method E and the control. The order of total sugar concentration in the hydrolyzate is C > A > D > B > E > control. According to Table 2, the order of total sugar yield of different pretreatment method was C > A > D > B > E > control. This order was exactly the same as that of cellulose contents after pretreatments. The total sugar yield with the method C was 9.54 times higher than the control. It is indicated that the total sugar yield is relevant to the residual cellulose content after pretreatment. Besides, total sugar yields of method A, D, and B were all more than 69.61 g sugar/100 g dry matter. Thus pretreatment method A, B and D can effectively improve the total sugar yield of sweet sorghum bagasse compared to others. The total sugar yield of method E was only 10.12 g sugar/100 g dry matter and there was no significant difference between method E and the control. Because of the addition of 2% NaOH solution, most of the hemicellulose and lignin in the bagasse with pretreatment method C, A and D were removed and the cellulose can be more easily converted to glucose by cellulase (Taherzadeh and Karimi, 2008). As a result, the total sugar yield was increased. The total sugar yield of bagasse pretreated by method B (69.61 g sugar/100 g dry matter) showed that relatively high concentration of NaOH solution (20%) had positive effect on the saccharification of sweet sorghum bagasse. But it had relatively high dry matter loss according to Table 1 (83.70%) which was not an ideal pretreatment method compared with the others. The objectives of sweet sorghum bagasse pretreatment were to improve the cellulose hydrolysis yield and total sugar yield, and to produce more glucose for the follow-up ethanol fermentation. As can be seen from Table 2, the order of the ethanol concentration in fermentation broth is C > B > A > D > E > control. This order was the same as those of glucose concentration in the hydrolyzate and cellulose hydrolysis yield. The ethanol concentration with the method C was 19.13 times higher than the control. So it can be inferred that the amount of cellulose content remained in the pretreatment residue may be a kind of criterion for telling the cellulose hydrolysis yield as well as the amount of ethanol concentration in the broth after fermentation. Due to high cellulose hydrolysis yield and high total sugar yield, the ethanol concentration of method C inevitably was the highest one in all methods. 3.4. SEM photos analysis Scanning electron microscope photos of unpretreated and pretreated sweet sorghum bagasse is shown in Supplemental Fig. 1.
Table 2 The effects of different pretreatment methods on cellulose hydrolysis yield, total sugar yield and ethanol concentration of sweet sorghum bagasse.
A
No.
Cellulose hydrolysis yield (%)
Total sugar yield (g sugar/100 g dry matter)
Ethanol concentration (g/L)
Control A B C D E
12.64 ± 0.19e,A 69.94 ± 0.93c 72.89 ± 0.12b 74.29 ± 0.81a 62.46 ± 0.56d 11.78 ± 0.19e
9.53 ± 0.02e 86.83 ± 0.68b 69.61 ± 0.90d 90.94 ± 0.39a 76.06 ± 0.17c 10.12 ± 0.03e
0.32 ± 0.01e 5.55 ± 0.23b 5.73 ± 0.06b 6.12 ± 0.06a 4.93 ± 0.16c 0.89 ± 0.04d
Means in the tables followed by the same letter with a same column means not significantly different using Duncan’s multiple range tests at the level of 5%.
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Table 3 The sugar concentration in the hydrolyzate after enzymatic hydrolysis for 96 h. No.
Control A B C D E A
Sugar concentration (mg/mL) Glucose
Xylose
Arabinose
Mannose
Galactose
Total sugar
1.45 ± 0.00 12.55 ± 0.41 12.45 ± 0.20 14.16 ± 0.00 10.41 ± 0.03 1.46 ± 0.03
0.53 ± 0.00 4.98 ± 0.15 2.56 ± 0.11 4.57 ± 0.00 4.98 ± 0.06 0.62 ± 0.03
NDA 0.35 ± 0.00 0.30 ± 0.00 0.28 ± 0.01 0.36 ± 0.00 ND
ND ND ND ND ND ND
ND ND ND ND ND ND
1.97 ± 0.00 17.88 ± 0.56 15.30 ± 0.09 19.01 ± 0.01 15.75 ± 0.10 2.08 ± 0.00
ND means not detected.
The result of Supplemental Fig. 1 indicated that there was an apparent net structure in sweet sorghum bagasse pretreated by method E and the control. This complex net structure in the bagasse will restrict the cellulose being attacked by cellulase (Fan et al., 1980), while it cannot be found in other samples. Autoclaving pretreatment of method E changed the structure a little, but the net structure could not be changed constitutionally. The alkali autoclaving pretreatment such as methods A and C could degrade the lignin and hemicellulose in the bagasse. The cellulose hydrolysis yield is closely related to the removal of hemicellulose and lignin (Yu et al., 2010). The structure of bagasse pretreated by method B using 20% NaOH solution was destroyed thoroughly because relatively high concentration of NaOH could dissolve the hemicellulose. The SEM photo of the bagasse pretreated by method D seemed different from those of method A, B and C, and it showed a smooth surface of the bagasse. This suggested that the pretreatment temperature or time might be not enough to destroy the bagasse thoroughly. Also, bigger pores could be found in bagasse pretreated by method C and smaller pores could found in bagasse pretreated by method D. Both of which were applied hydrogen peroxide. The analysis of the SEM photos indicated that there were distinct changes in the bagasse pretreated by method A, B, C and D. This was also corresponding to the analysis of the cellulose hydrolysis yield and total sugar yield.
lulose hydrolysis yield, total sugar yield and ethanol concentration were 74.29%, 90.94 g sugar/100 g dry matter and 6.12 g/L, respectively, which were 5.88, 9.54 and 19.13 times higher than the control. Some significant structure and chemical bonds changes after pretreatment were found. Cellulose content and total sugar yield were related to each other, so were glucose concentration, cellulose hydrolysis yield and ethanol concentration. Acknowledgements The authors are grateful for the support provided by the Ministry of Agriculture of the People’s Republic of China under the 948 Project entitled ‘‘Introduce and industrialization of advanced agriculture biomass energy technologies’’ (Grant No. 2008 G2) and Liaoning Natural Science Foundation (Grant No. 20092031). In addition, Yue Zhang from University of Southampton of UK is greatly acknowledged for her valuable suggestion and correction of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2012.02.034. References
3.5. FTIR analysis Supplemental Fig. 2 shows the FTIR spectra of untreated and pretreated sweet sorghum bagasse. As can be seen from Supplemental Fig. 2, there were no new peaks in the spectra of unpretreated and pretreated sweet sorghum bagasse, but there were some differences in parts of peak transmittance. The absorption at 3400 cm1 represents the stretching of hydroxyl and phenol in the bagasse. After pretreatment, the absorption reduced, which indicated that less hydroxyl and phenol existed compared to the control. The absorption at 2910 cm1 represents the stretching of –CH3 and –CH2. The transmittances of the bagasse pretreated by method A, B and C were less than others, which were indicators of fracture of carbon chains. The absorption at 1630 cm1 represents the bending mode stretching of the absorbed water and stretching of C@O in lignin (Zhang et al., 2011). The strong absorption at 1057 cm1 represents the stretching of C–O in cellulose and hemicellulose (Oh et al., 2005; Cao and Tan, 2004). The transmittances of the bagasse pretreated by method C, B and A were less than others, which are indicators of the structural destroy of the bagasse after pretreatment. 4. Conclusions In this study, dilute NaOH solution autoclaving and H2O2 immersing pretreatment was considered as the most suitable method for sweet sorghum bagasse pretreatment. The highest cel-
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Comparison of the effects of five pretreatment methods on enhancing the enzymatic digestibility and ethanol production from sweet sorghum bagasse Weixing Cao, Chen Sun, Ronghou Liu ⇑, Renzhan Yin, Xiaowu Wu Biomass Energy Engineering Research Centre, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 21 November 2011 Received in revised form 29 January 2012 Accepted 7 February 2012 Available online 16 February 2012 Keywords: Sweet sorghum bagasse Enzymatic hydrolysis Pretreatment Bio-ethanol
a b s t r a c t To improve the enzymatic digestibility of sweet sorghum bagasse and bioethanol production, five pretreatment methods have been investigated and compared, including (1) dilute NaOH solution autoclaving pretreatment, (2) high concentration NaOH solution immersing pretreatment, (3) dilute NaOH solution autoclaving and H2O2 immersing pretreatment, (4) alkaline peroxide pretreatment and (5) autoclaving pretreatment. Among them, the best result was obtained when sweet sorghum bagasse was dilute NaOH solution autoclaving and H2O2 immersing pretreatment. The highest cellulose hydrolysis yield, total sugar yield and ethanol concentration were 74.29%, 90.94 g sugar/100 g dry matter and 6.12 g/L, respectively, which were 5.88, 9.54 and 19.13 times higher than the control. Moreover, the FTIR and SEM analysis illustrated significant molecule and surface structure changes of the sweet sorghum bagasse after pretreatments. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Sweet sorghum is a high photosynthetic efficiency energy crop, and it is adapted to growing in nutrient-poor soils, making it one of the most promising energy-crop for bio-ethanol production. Both its grain and stalk are ideal raw material for bioethanol production due to its high biomass and sugar-yielding (Billa et al., 1997). The starches in the grain can be hydrolyzed to fermentable sugars by amylase, while the cellulose and hemicellulose in the bagasse require pretreatment prior to enzymatic hydrolysis and further conversion to ethanol (Lin and Tanaka, 2006). Bio-ethanol production from sweet sorghum bagasse is more attractive in terms of energy balances and emissions, and it may be the case that the bagasse will become the vital supplementary material for ethanol production. The main components of sweet sorghum bagasse are cellulose, hemicellulose and lignin (Sipos et al., 2009), of which the intricate structure severely restricts the enzymatic hydrolysis. In order to improve the accessibility of the enzyme to cellulose, the studies on lignocellulose pretreatment are needed (Taherzadeh and Karimi, 2008). The pretreatment methods mainly include physical methods such as mechanical or thermal (Mais et al., 2002; Silva et al., 2010), chemical methods (Saha et al., 2005; Yamashita et al., 2010; Saha and Cotta, 2006; Beukes and Pletschke, 2011), biological methods and combination of these methods. The mechanism of physical pretreatment is to increase the accessible surface area and decrease the crystallinity degree of lignocellulose by chipping, milling, grinding ⇑ Corresponding author. Tel./fax: +86 21 34205744. E-mail address: [email protected] (R. Liu). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2012.02.034
or irradiation (Fan et al., 1980; Taherzadeh and Karimi, 2008; Mais et al., 2002; Silva et al., 2010). It is also effective for improving the enzymatic hydrolysis using thermal pretreatment such as steam explosion and liquid hot water pretreatment which can remove most of the hemicellulose (Shen et al., 2011; Laser et al., 2002). The typical conditions of liquid hot water pretreatment are high temperature and pressure (160–260 °C, 0.69–4.83 MPa) at short time (e.g. a few seconds to several minutes) or ambient temperature and normal pressure at relatively long time (several hours to several days) (Dien et al., 2006; Sreenath et al., 1999). Generally, the shortcoming of these physical pretreatment methods is high energy requirement. Alkaline (e.g. NaOH) and alkaline peroxide pretreatments which belong to chemical methods are effective processes for pretreating lignocellulose materials. NaOH is widely used for lignocellulose pretreatment. It can remove partial lignin and hemicellulose in the biomass by fracturing the ester bonds thereby increasing the porosity of the biomass (Xu et al., 2010; Zhang et al., 2011). Alkaline peroxide is used for lignocellulose pretreatment in recent years. It can improve the enzymatic hydrolysis by delignification. Mishima et al. (2006) also showed that the alkaline peroxide pretreatment method was one of the most effective methods for improving the enzymatic hydrolysis. In addition, no measurable furfural and hydroxymethylfurfural (HMF) which are harmful for yeast were detected in the process. That indicates it is easier for yeast to ferment the hydrolyzate compared to dilute-acid pretreatment using alkaline peroxide pretreatment method. Although several pretreatment methods have shown the effectiveness in many researches (Taherzadeh and Karimi, 2008), the combination of two
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or more pretreatment methods may be more effective than separate single one. Moreover, selection of pretreatment process for a certain material depends on the biomass material type. Besides, the enzymatic hydrolysis still needs to be improved when sweet sorghum bagasse is concerned. This study aims to compare the effects of five pretreatment methods on improving enzymatic hydrolysis of sweet sorghum bagasse and the ethanol production from the hydrolyzate. In addition, the lignocellulose compositions and structure changes of the sweet sorghum bagasse were also investigated. 2. Methods 2.1. Sweet sorghum Sweet sorghum (Chongming No. 1) was harvested in Qibao campus, Shanghai Jiao Tong University, China. The stalks were squeezed by a three-roller mill to obtain the liquid phase and bagasse separately. The bagasse was dried in the air and then ground to pass through sieve with 40 meshes. After that it was washed using boiling water three times to substantially remove the major soluble sugars (sucrose, glucose, fructose, etc.) present in the stalk liquid phase. The ratio of bagasse to water was 1:1 on a weight basis. Finally it was dried at 60 °C and stored in plastic bag at room temperature to avoid possible interference in the evaluation of the enzymatic hydrolysis of the cellulose (Wu et al., 2011). 2.2. Pretreatment process Five pretreatment methods were used in this study, labeled as A, B, C, D and E. Untreated bagasse was used as the control in the following experiments. Each pretreatment was conducted duplicated and the results were averaged. 2.2.1. Dilute NaOH solution autoclaving pretreatment (A) Ten grams of dry sweet sorghum bagasse was slurried with 100 mL 2% (w/v) sodium hydroxide solution for 5 min in a 500 mL flask with a silicone stopple and then autoclaved at 121 °C for 60 min. The residues were centrifuged and washed with distilled water until neutral pH was achieved and dried at 60 °C. The yield of product was 5.978 ± 0.212 g. 2.2.2. High concentration NaOH solution immersing pretreatment (B) Ten grams dry sweet sorghum bagasse was slurried with 100 mL 20% (w/v) sodium hydroxide solution for 5 min, and then stood for 2 h in a 500 mL flask with a silicone stopple. The residues were centrifuged and washed with distilled water until neutral pH was achieved and dried at 60 °C. The yield of product was 1.642 ± 0.141 g. 2.2.3. Dilute NaOH solution autoclaving and H2O2 immersing pretreatment (C) Ten grams dry sweet sorghum bagasse was slurried with 100 mL 2% (w/v) sodium hydroxide solution for 5 min in a 500 mL flask with a silicone stopple, and then autoclaved at 121 °C for 60 min. The 5% (w/v) hydrogen peroxide was mixed into the pretreated slurry after cooling down to room temperature. The mixture was kept airtight and in dark place for 24 h. The residues were centrifuged and washed with distilled water until neutral pH was achieved and dried at 60 °C. The yield of product was 5.432 ± 0.038 g. 2.2.4. Alkaline peroxide pretreatment (D) Ten grams dry sweet sorghum bagasse was slurried with 100 mL 2% (w/v) sodium hydroxide solution for 5 min, and then
stood for 2 h in a 500 mL flask with a silicone stopple. The 5% (w/v) hydrogen peroxide was mixed into the pretreated slurry. The mixture was kept airtight and in dark place for 24 h. The residues were centrifuged and washed with distilled water until neutral pH was achieved and dried at 60 °C. The yield of product was 6.856 ± 0.014 g. 2.2.5. Autoclaving pretreatment (E) Ten grams dry sweet sorghum bagasse was slurried with 100 mL distilled water for 5 min in a 500 mL flask with a silicone stopple, and then autoclaved at 121 °C for 60 min. After cooling down to room temperature, the residues were centrifuged and washed with distilled water until neutral pH was achieved and dried at 60 °C. The yield of product was 8.824 ± 0.156 g. 2.3. Enzymatic assay and hydrolysis A commercial cellulase (Celluclast 1.5L, Sigma Aldrich) supplemented with b-glucosidase (Novzymes 188, Denmark) was used for enzymatic hydrolysis of the sweet sorghum bagasse. The activities of Celluclast 1.5L and b-glucosidase were 45.8 FPU/mL and 302.1 U/mL, respectively. One unit of cellulase activity is defined as the amount of the enzyme that releases 1 mg of glucose per minute in the reaction mixture at 50 °C and pH 4.8. One unit of b-glucosidase activity is defined as the amount of the enzyme that releases 1 lmol of p-nitrophenol per minute in the reaction mixture at 50 °C and pH 4.8 (Saha and Cotta, 2006). The mixture was hydrolyzed in sodium citrate buffer (50 mM, pH 4.8) with the substrate loading of 2%. The enzyme loadings of Celluclast 1.5L and b-glucosidase were 20 FPU/g dry biomass and 40 IU/g dry biomass, respectively. The hydrolysis was conducted in a shaker at 50 °C. Samples (1 mL) were withdrawn at 12, 24, 48, 72 and 96 h and centrifuged at 10,000 rpm for 10 min, and the supernatants were preserved at 20 °C until they were used for sugar analysis. 2.4. Microorganism and batch fermentation Active dry yeast was bought from Angel Yeast Company of Hubei Province in China for ethanol fermentation. The medium was as follows: glucose 50 g/L, yeast 5 g/L, peptone 5 g/L, MgSO47H2O 1 g/ L, K2HPO4 1 g/L. 6 M HCl or NaOH solution was used for pH control. The medium was autoclaved at 121 °C for 20 min. The hydrolyzate was sterilized by autoclaving at 121 °C for 20 min before it was inoculated with the yeast medium at the volume ratio of 1:10 of the fermentation broth aseptically. The fermentation tests were conducted at 30 ± 0.5 °C in a shaker at 150 rpm. 2.5. Analytical methods The contents of cellulose, hemicellulose and lignin were determined according to the reference (Van Soest, 1963). The sugars in hydrolyzate including glucose, xylose, arabinose, galactose and mannose were measured with a HPLC system (Shimadzu LC-10A, Japan) with an Aminex HPX-87P column (7.8 mm I.D. 30 cm, Bio-Rad, USA) (Cara et al., 2007). Deionized water was used as the mobile phase with a flow rate of 1 mL/min and column temperature of 80 °C. The activities of the cellulase and b-glucosidase were measured according to the references (Xu et al., 2010). Ethanol concentration was analyzed by gas chromatography (Agilent 7890A GC system, USA) with a flame ionization detector and isopropanol was used as an internal standard (Liu and Shen, 2008). A scanning electron microscope (SEM, Siron 200, FEI Company, USA) was used to detect the microscopic structure of the sweet sorghum bagasse samples. The Fourier Transform Infrared Raman Spectroscopy (FTIR, EQUINOX 55, BRUKER Company, Germany)
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was recorded between 400 and 4000 cm1. Discs were prepared with 2 mg dried sample and 200 mg KBr (Zhang et al., 2011). The cellulose hydrolysis yield, total sugar yield and dry matter loss are defined as following three equations (Wu et al., 2011).
R1 ¼ C 1 =ðm W 1:11Þ 100ð1ÞR2 ¼ C 2 =ðm 100Þð2ÞR3 ¼ ðm0 mt Þ=m0 100ð3Þ where R1 is cellulose hydrolysis yield, %. C1 is the glucose mass in the hydrolyzate, g. m is the dry sweet sorghum bagasse mass, g. W is the cellulose content in dry sweet sorghum bagasse, %. 1.11 in Eq. (1) is the theoretical conversion from cellulose to glucose. R2 is the total sugar yield, g sugar/100 g dry matter. C2 is the total sugar mass in the hydrolyzate, g. R3 is the dry matter loss, %. m0 is the dry sweet sorghum bagasse mass before pretreatment, g. mt is the dry sweet sorghum bagasse after pretreatment, g. 2.6. Statistical analysis Data were analyzed for statistical significance by a one-way analysis of variance (ANOVA). A 5% probability level (p = 0.05) was used to accept or reject the null hypothesis. Duncan’s multiple range tests at the level of 5% were used to analyze the significances of different pretreatment methods. In this study, means in the tables followed by the same letter with a same column means not significantly different using Duncan’s multiple range tests at the level of 5% (Duncan, 1955). 3. Results and discussion 3.1. The effects of different pretreatment methods on the composition of sweet sorghum bagasse The aim of pretreatments was to change raw material properties, remove or dissolve lignin and hemicellulose and reduce the crystallinity of cellulose (Kumar et al., 2009). To be specific, with perfect pretreatment, the lignin will be mostly degraded while the cellulose and hemicellulose will be retained. The best pretreatment method and condition usually depend mainly on the type of lignocelluloses (Taherzadeh and Karimi, 2008). The dry matter loss has also been applied to evaluate the pretreatment effect in this study. The less dry matter loses, the more potential fermentation substrate is retained (Zhu et al., 2005). Table 1 shows the main composition, acid detergent lignin (ADL) removal and dry matter loss of sweet sorghum bagasse. The results of Table 1 showed that the orders of cellulose content, hemicellulose content, ADL content, ADL removal and dry matter loss were C > A > D > B > E > control, control > E > D > A > B > C, control > E > B > D > A > C, C > A > D > B > E and B > C > A > D > E, respectively. As it could be seen from Table 1, the cellulose content of the bagasse pretreated by method C was the highest one, while its ADL content and hemicellulose content were the lowest one. Dilute NaOH pretreatment of lignocellulose materials has been
found to cause swelling, leading to an increase in internal surface area of the sweet sorghum bagasse and disruption of the lignin structure. Millet reported (1976) that the NaOH pretreatment could decrease lignin content from 24% to 55%. But no effect was observed for softwoods with lignin content greater than 25% at the normal ambient conditions. Elevated temperatures can enhance lignin removal (Taherzadeh and Karimi, 2008). Silverstein et al. (2007) also reported that 2% NaOH in 90 min at 121 °C was the best pretreatment condition, resulting in 65% of delignification. In this study, both methods A and C were conducted at 121 °C, and the pretreatment time was 60 min, which resulted in more than 80% of ADL removal. Thus lignin removal increases enzyme effectiveness by eliminating nonproductive adsorption sites and by increasing access to cellulose and hemicellulose (Kumar et al., 2009). On the other hand, sweet sorghum bagasse pretreated by method A has higher cellulose content and lower ADL lignin than the others except method C. However, the hemicellulose content with method A was significantly higher than method C (p < 0.05). Meantime, the dry matter losses of bagasse pretreated by methods A and C were 40.75% and 46.10%, respectively. Both of which belonged to the medium level among all the pretreatment methods. The pretreatment methods A and C led to a comparatively low dry matter loss but significantly reduced the ADL lignin content and increased the cellulose content, which meant 2% sodium hydroxide pretreatment under autoclaving condition had shown sound effectiveness. The dilute NaOH pretreatment was beneficial for the follow-up enzymatic hydrolysis of the bagasse, which was supported by the results of the references (Wu et al., 2011; Curreli et al., 1997). By comparing methods A and C, it can be deduced that applying hydrogen peroxide during alkaline pretreatment will improve the effectiveness of hemicellulose and ADL lignin removal and cellulose retaining. But the cost of applying hydrogen peroxide is the increase of dry matter loss. The ANOVA showed that there was no significant differences (p > 0.05) in terms of the hemicellulose content between method B and C. Therefore, applying considerable amount of sodium hydrogen will do the same work in degrading and retaining hemicellulose as using small quantity of sodium hydrogen under autoclaving condition plus hydrogen peroxide immersing. However, the ADL lignin content of bagasse pretreated by method B was only superior to the control, which would definitely affect the enzyme hydrolysis process. In addition, the dry matter loss of method B strikingly came up to 83.7%, which would be disadvantageous for potential productivity of the follow-up fermentation. Also, this means that the concentration of 20% NaOH was too high for pretreatment due to the great loss in dry matter. Method D could be considered as the moderate method because of the middle level of its cellulose, hemicellulose, ADL lignin content and the dry matter loss. Compared with method C, method D pretreated raw material in rather low temperature and pressure. So it can be concluded that high temperature and pressure during oxydic alkaline pretreatment will be beneficial for retaining cellulose, removing hemicellulose and ADL lignin, but not good for retaining dry matter. On the other
Table 1 The main composition, ADL removal and dry matter loss of sweet sorghum bagasse. No. Control A B C D E A B
Cellulose (%) f,B
49.78 ± 0.86 78.44 ± 0.35b 69.90 ± 0.09d 82.08 ± 0.48a 72.45 ± 0.15c 54.40 ± 0.27e
Hemicellulose (%) a
27.72 ± 0.08 15.09 ± 0.91d 13.94 ± 0.35d 9.45 ± 0.57e 17.52 ± 0.91c 22.44 ± 0.77b
ADLA (%)
ADL removal (%)
Dry matter loss (%)
10.83 ± 0.18a 1.68 ± 0.16d 7.50 ± 0.29b 0.97 ± 0.12e 2.29 ± 0.01c 10.71 ± 0.06a
Null 84.52 ± 1.24b 30.82 ± 1.48d 91.02 ± 0.98a 78.84 ± 0.23c 1.16 ± 1.11e
Null 40.75 ± 2.19c 83.70 ± 1.56a 46.10 ± 0.57b 32.25 ± 0.21d 13.00 ± 1.70e
Acid detergent lignin. Means in the tables followed by the same letter with a same column means not significantly different using Duncan’s multiple range tests at the level of 5%.
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hand, when methods A and D are compared, it can be deduced that method A is more effective than method D in retaining cellulose and removing hemicellulose and ADL lignin than hydrogen peroxide. The lowest dry matter loss appeared in method E, but ANOVA showed that compared with control, there was no significant difference on ADL lignin removal (p > 0.05). Liquid hot-water (LHW) pretreatment is an environmental friendly pretreatment method with no addition of chemicals. Dien et al. (2006) reported that 75% of the xylan was dissolved at 160 °C, for 20 min, and higher temperatures, e.g. 220 °C, which can dissolve hemicellulose completely and remove lignin partially with 2 min (Dien et al., 2006; Sreenath et al., 1999). Laser et al. (2002) also showed that most hemicellulose was removed at 170–230 °C for 1–46 min. The pretreatment temperature of method E in this study was 121 °C, which is not sufficient to dissolve the hemicellulose and remove the lignin. Therefore, cellulose and hemicellulose might still be bundled by a great amount of lignin, which would harm the follow-up enzyme hydrolysis and ethanol production (Hendriks and Zeeman, 2009). So when compared with method E and control, it is clear that mere high temperature and high pressure will do limited work on effective pretreatment without alkali being involved. In brief, from the aspect of contents of cellulose, hemicellulose, ADL lignin, and the dry matter loss, methods A and C would be advisable as the pretreatment methods before enzymatic hydrolysis and ethanol fermentation. On the other hand, considering that not all of the yeast for ethanol production can utilize pentose such as xylose. And in such condition, hemicellulose would turn out to be another physical barrier which surrounds the cellulose fibers and can protect the cellulose from enzymatic attack (Wu et al., 2011; Curreli et al., 1997). Therefore, method C would be the best one for the follow-up enzymatic hydrolysis and ethanol formation.
3.2. The effects of different pretreatment methods on sugar concentration
16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
3.2.2. The effects of different pretreatment methods on the xylose concentration Fig. 2 shows the effects of different pretreatment methods on xylose concentration during the enzymatic hydrolysis. As it could be seen from Fig. 2, the variation tendency of xylose concentration in the hydrolyzate was similar to that of glucose concentration, but the xylose concentration was much lower than the glucose concentration in all the pretreatment samples. This was because hemicellulose content in both pretreated and unpretreated bagasse was lower than the cellulose content. Generally speaking, the majority of hydrolyzate of hemicellulose in ligno-cellulosic waste is xylose, and there are minor amounts of arabinose in the hydrolyzate of the bagasse (Taherzadeh and Karimi, 2008). In this sense, the hydrolysis of hemicellulose is related to the xylose concentration in the hydrolyzate. The order of final xylose concentration in hydrolyzate was A > D > C > B > E > control. According to Table 1, there was more hemicellulose while comparatively less lignin remained in
6
Control A B C D E
0
12
24
36
48
60
72
84
96
Hydrolysis time (h) Fig. 1. The effects of different pretreatment methods on glucose concentration during the enzymatic hydrolysis.
Concentration of xylose (mg/mL)
Concentration of glucose (mg/mL)
3.2.1. The effects of different pretreatment methods on glucose concentration Fig. 1 shows the effects of different pretreatment methods on glucose concentration during the enzymatic hydrolysis. As it could be seen from Fig. 1, the glucose concentration in the hydrolyzate increased as the hydrolysis time prolonged. The ANOVA analysis showed that the glucose concentration in the hydrolyzate for all pretreatment methods and control reached a plateau within 24 h (p > 0.05), which indicated that all the glucose production potential had been achieved in this short period (Saha and Cotta, 2006). The order of glucose concentration in the hydrolyzate after 96 h with
different pretreatment methods was C > B > A > D > E > control. Specifically, there was no significant difference of the glucose concentration between method E and the control after hydrolysis for 24 h, although the time for method E to achieve constant concentration of glucose was much shorter than the control. With regard to method C, the glucose concentration was the highest one among all samples after 12 h, which indicated that the pretreatment method C was the most effective one on converting the cellulose in sweet sorghum bagasse to glucose in these five pretreatment methods. The final glucose concentration in method C was up to 14.16 mg/mL, which was 9.8 times as much as the control. The glucose concentration in the hydrolyzate of bagasse pretreated with method A was higher than method B during the first 24 h, but the trend was reverse in the following 24 h. It demonstrated that the pretreatment with low concentration of sodium hydrogen plus autoclaving was much better than mere high concentration of alkali pretreatment for increasing the glucose concentration in hydrolyzate during 48 h. Although the glucose concentration in the hydrolysis pretreated by method D was almost constant after 12 h, the glucose concentration in method D was 26.5% lower than method C. This proved once again that it is much better for the cellulose hydrolysis to use relatively high temperature and pressure than the normal temperature and pressure during the pretreatment process. Both method C and D used alkaline hydrogen peroxide, which is good for improving the cellulose conversion by the removal of hemicellulose and lignin and increasing the accessibility of cellulose (Sreenath et al., 1999). In short, pretreatment method C yielded the highest glucose concentration in the hydrolyzate.
5 4
Control A B C D E
3 2 1 0 0
12
24
36 48 60 72 Hydrolysis time (h)
84
96
Fig. 2. The effects of different pretreatment methods on xylose concentration during the enzymatic hydrolysis.
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methods A and D than the others, except for method C. And both the hemicellulose and ADL lignin contents of method A were less than method D. But according to Fig. 2, firstly, method A could release xylose much faster than method D, and secondly, the xylose concentration in the hydrolyzate with pretreatment method A was the highest one. So lignin amount left after pretreatment is key factor for hemicellulose degrading during the enzymatic hydrolysis in the whole process. The same conclusion could also be deduced from the results of method B and C. Besides, the xylose concentration in method E was close to the control, and both method E and the control had less xylose in the hydrolyzate. However, both of which had more hemicellulose. This indicated that less hemicellulose in the bagasse of method E and control was hydrolyzed, because much of the cellulose and hemicellulose is unreachable for enzyme under the wrap of remained lignin (Beukes and Pletschke, 2011). Accordingly, xylose concentration with method A was the highest one in all pretreatment methods due to the reasons that method A yields a relatively high content of hemicellulose and low content of ADL lignin. 3.3. The effects of different pretreatment methods on enzymatic hydrolysis and ethanol fermentation of sweet sorghum bagasse Table 2 shows the effects of different pretreatment methods on cellulose hydrolysis yield, total sugar yield and ethanol concentration of sweet sorghum bagasse. The cellulose hydrolysis yield implies the efficiency of converting cellulose to glucose. The order of cellulose hydrolysis yield of sweet sorghum bagasse with different pretreatment methods for 96 h hydrolysis was C > B > A > D > control > E. This order was almost the same as that of glucose concentration in the hydrolyzate after 96 h. It indicated that alkaline pretreatment was good for improving the cellulose conversion. The cellulose hydrolysis yield of method C was 74.29%, which was 5.88 times higher than that of the control. It was higher than the result (70%) obtained from the steam pretreatment of Zhang’s research (Zhang et al., 2011). That may be due to the reasons that the content of hemicellulose and lignin was lower in the pretreated bagasse, and more cellulose was converted to glucose. Different pretreatment conditions can result in different pretreatment effects. In low alkaline concentration processes, with the concentration of 0.5– 4% NaOH at high temperature and pressure, the structure of lignocellulose can be destroyed in the pretreatment process while NaOH pretreatment at high temperature, the majority of the lignin and hemicellulose can be removed from the solid phase (Mirahmadi et al., 2010). On the other hand, the high concentration NaOH pretreatment (6–20%) is usually used at ambient conditions. Lignin was not significantly removed from the biomass (Mirahmadi et al., 2010). The ANOVA showed there was no significant difference of the cellulose hydrolysis yield between method E and the control. Also, it showed that mere autoclaving with distilled water cannot significantly improve the cellulose hydrolysis yield. Method E in this study was not similar to LHW pretreatment, because the pretreatment temperature (121 °C) of method E in this study was lower than the LHW pretreatment (160–260 °C). Therefore, it was not enough for method E to dissolve the hemicellulose and remove the lignin
for improving the cellulose hydrolysis yield (Sreenath et al., 1999). Totally, the pretreatment method C was the most effective one on increasing the cellulose hydrolysis yield in five pretreatment methods. During the enzymatic hydrolysis of sweet sorghum bagasse, the cellulose was converted to glucose, and the hemicellulose was converted to xylose, arabinose, mannose, galactose and other sugars. Table 3 shows the sugar concentration in the hydrolyzate after enzymatic hydrolysis for 96 h. As it could be seen from Table 3, there were minor amounts of arabinose in the hydrolyzate of the bagasse pretreated by methods A, B, C and D, while no arabinose was detected in the hydrolyzate of bagasse pretreated by method E and the control. The order of total sugar concentration in the hydrolyzate is C > A > D > B > E > control. According to Table 2, the order of total sugar yield of different pretreatment method was C > A > D > B > E > control. This order was exactly the same as that of cellulose contents after pretreatments. The total sugar yield with the method C was 9.54 times higher than the control. It is indicated that the total sugar yield is relevant to the residual cellulose content after pretreatment. Besides, total sugar yields of method A, D, and B were all more than 69.61 g sugar/100 g dry matter. Thus pretreatment method A, B and D can effectively improve the total sugar yield of sweet sorghum bagasse compared to others. The total sugar yield of method E was only 10.12 g sugar/100 g dry matter and there was no significant difference between method E and the control. Because of the addition of 2% NaOH solution, most of the hemicellulose and lignin in the bagasse with pretreatment method C, A and D were removed and the cellulose can be more easily converted to glucose by cellulase (Taherzadeh and Karimi, 2008). As a result, the total sugar yield was increased. The total sugar yield of bagasse pretreated by method B (69.61 g sugar/100 g dry matter) showed that relatively high concentration of NaOH solution (20%) had positive effect on the saccharification of sweet sorghum bagasse. But it had relatively high dry matter loss according to Table 1 (83.70%) which was not an ideal pretreatment method compared with the others. The objectives of sweet sorghum bagasse pretreatment were to improve the cellulose hydrolysis yield and total sugar yield, and to produce more glucose for the follow-up ethanol fermentation. As can be seen from Table 2, the order of the ethanol concentration in fermentation broth is C > B > A > D > E > control. This order was the same as those of glucose concentration in the hydrolyzate and cellulose hydrolysis yield. The ethanol concentration with the method C was 19.13 times higher than the control. So it can be inferred that the amount of cellulose content remained in the pretreatment residue may be a kind of criterion for telling the cellulose hydrolysis yield as well as the amount of ethanol concentration in the broth after fermentation. Due to high cellulose hydrolysis yield and high total sugar yield, the ethanol concentration of method C inevitably was the highest one in all methods. 3.4. SEM photos analysis Scanning electron microscope photos of unpretreated and pretreated sweet sorghum bagasse is shown in Supplemental Fig. 1.
Table 2 The effects of different pretreatment methods on cellulose hydrolysis yield, total sugar yield and ethanol concentration of sweet sorghum bagasse.
A
No.
Cellulose hydrolysis yield (%)
Total sugar yield (g sugar/100 g dry matter)
Ethanol concentration (g/L)
Control A B C D E
12.64 ± 0.19e,A 69.94 ± 0.93c 72.89 ± 0.12b 74.29 ± 0.81a 62.46 ± 0.56d 11.78 ± 0.19e
9.53 ± 0.02e 86.83 ± 0.68b 69.61 ± 0.90d 90.94 ± 0.39a 76.06 ± 0.17c 10.12 ± 0.03e
0.32 ± 0.01e 5.55 ± 0.23b 5.73 ± 0.06b 6.12 ± 0.06a 4.93 ± 0.16c 0.89 ± 0.04d
Means in the tables followed by the same letter with a same column means not significantly different using Duncan’s multiple range tests at the level of 5%.
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Table 3 The sugar concentration in the hydrolyzate after enzymatic hydrolysis for 96 h. No.
Control A B C D E A
Sugar concentration (mg/mL) Glucose
Xylose
Arabinose
Mannose
Galactose
Total sugar
1.45 ± 0.00 12.55 ± 0.41 12.45 ± 0.20 14.16 ± 0.00 10.41 ± 0.03 1.46 ± 0.03
0.53 ± 0.00 4.98 ± 0.15 2.56 ± 0.11 4.57 ± 0.00 4.98 ± 0.06 0.62 ± 0.03
NDA 0.35 ± 0.00 0.30 ± 0.00 0.28 ± 0.01 0.36 ± 0.00 ND
ND ND ND ND ND ND
ND ND ND ND ND ND
1.97 ± 0.00 17.88 ± 0.56 15.30 ± 0.09 19.01 ± 0.01 15.75 ± 0.10 2.08 ± 0.00
ND means not detected.
The result of Supplemental Fig. 1 indicated that there was an apparent net structure in sweet sorghum bagasse pretreated by method E and the control. This complex net structure in the bagasse will restrict the cellulose being attacked by cellulase (Fan et al., 1980), while it cannot be found in other samples. Autoclaving pretreatment of method E changed the structure a little, but the net structure could not be changed constitutionally. The alkali autoclaving pretreatment such as methods A and C could degrade the lignin and hemicellulose in the bagasse. The cellulose hydrolysis yield is closely related to the removal of hemicellulose and lignin (Yu et al., 2010). The structure of bagasse pretreated by method B using 20% NaOH solution was destroyed thoroughly because relatively high concentration of NaOH could dissolve the hemicellulose. The SEM photo of the bagasse pretreated by method D seemed different from those of method A, B and C, and it showed a smooth surface of the bagasse. This suggested that the pretreatment temperature or time might be not enough to destroy the bagasse thoroughly. Also, bigger pores could be found in bagasse pretreated by method C and smaller pores could found in bagasse pretreated by method D. Both of which were applied hydrogen peroxide. The analysis of the SEM photos indicated that there were distinct changes in the bagasse pretreated by method A, B, C and D. This was also corresponding to the analysis of the cellulose hydrolysis yield and total sugar yield.
lulose hydrolysis yield, total sugar yield and ethanol concentration were 74.29%, 90.94 g sugar/100 g dry matter and 6.12 g/L, respectively, which were 5.88, 9.54 and 19.13 times higher than the control. Some significant structure and chemical bonds changes after pretreatment were found. Cellulose content and total sugar yield were related to each other, so were glucose concentration, cellulose hydrolysis yield and ethanol concentration. Acknowledgements The authors are grateful for the support provided by the Ministry of Agriculture of the People’s Republic of China under the 948 Project entitled ‘‘Introduce and industrialization of advanced agriculture biomass energy technologies’’ (Grant No. 2008 G2) and Liaoning Natural Science Foundation (Grant No. 20092031). In addition, Yue Zhang from University of Southampton of UK is greatly acknowledged for her valuable suggestion and correction of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2012.02.034. References
3.5. FTIR analysis Supplemental Fig. 2 shows the FTIR spectra of untreated and pretreated sweet sorghum bagasse. As can be seen from Supplemental Fig. 2, there were no new peaks in the spectra of unpretreated and pretreated sweet sorghum bagasse, but there were some differences in parts of peak transmittance. The absorption at 3400 cm1 represents the stretching of hydroxyl and phenol in the bagasse. After pretreatment, the absorption reduced, which indicated that less hydroxyl and phenol existed compared to the control. The absorption at 2910 cm1 represents the stretching of –CH3 and –CH2. The transmittances of the bagasse pretreated by method A, B and C were less than others, which were indicators of fracture of carbon chains. The absorption at 1630 cm1 represents the bending mode stretching of the absorbed water and stretching of C@O in lignin (Zhang et al., 2011). The strong absorption at 1057 cm1 represents the stretching of C–O in cellulose and hemicellulose (Oh et al., 2005; Cao and Tan, 2004). The transmittances of the bagasse pretreated by method C, B and A were less than others, which are indicators of the structural destroy of the bagasse after pretreatment. 4. Conclusions In this study, dilute NaOH solution autoclaving and H2O2 immersing pretreatment was considered as the most suitable method for sweet sorghum bagasse pretreatment. The highest cel-
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