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Enhanced enzymatic cellulose hydrolysis by subcritical carbon dioxide pretreatment of sugarcane bagasse
Bioresource Technology 158 (2014) 161–165
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Enhanced enzymatic cellulose hydrolysis by subcritical carbon dioxide pretreatment of sugarcane bagasse Hongdan Zhang, Shubin Wu ⇑ State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, PR China
h i g h l i g h t s A subcritical CO2 pretreatment was investigated to improve the glucose yield. Liquid fractions and solid residues were thoroughly analyzed. The enzymatic hydrolysis was affected by different pretreatment conditions. The enhanced digestibilities were confirmed by XRD, FTIR, SEM, and TGA analyses. The proposed process can be a contribution to develop a biorefinery.
a r t i c l e
i n f o
Article history: Received 22 December 2013 Received in revised form 5 February 2014 Accepted 8 February 2014 Available online 15 February 2014 Keywords: Sugarcane bagasse Subcritical CO2 pretreatment Glucose Enzymatic hydrolysis
a b s t r a c t Most biomass pretreatment processes for sugar production are run at low-solid concentration (<10 wt.%). Subcritical carbon dioxide (CO2) could provide a more sustainable pretreatment medium while using relative high-solid contents (15 wt.%). The effects of subcritical CO2 pretreatment of sugarcane bagasse to the solid and glucan recoveries at different pretreatment conditions were investigated. Subsequently, enzymatic hydrolysis at different hydrolysis time was applied to obtain maximal glucose yield, which can be used for ethanol fermentation. The maximum glucose yield in enzyme hydrolyzate reached 38.5 g based on 100 g raw material after 72 h of enzymatic hydrolysis, representing 93.0% glucose in sugarcane bagasse. The enhanced digestibilities of subcritical CO2 pretreated sugarcane bagasse were due to the removal of hemicellulose, which were confirmed by XRD, FTIR, SEM, and TGA analyses. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Global energy inevitable depletion has generated strong interest in the development of more sustainable transportation fuels and renewable carbon-based raw materials, such as those produced from biomass (Dodds and Gross, 2007; Somerville, 2006). Sugarcane bagasse was used for the bioconversion of lignocellulosic biomass to bio-fuels (such as ethanol and biodiesel) due to its availability, abundance, and renewability (Pandey et al., 2000; Rabelo et al., 2009). The abundant cellulose in sugarcane bagasse could be hydrolyzed to glucose by enzyme for subsequent ethanolic fermentation (Martin et al., 2002). The coexistence of hemicellulose and lignin with cellulose make the enzymatic hydrolysis of cellulose difficult and tedious (Kim and Hong, 2001; Zheng et al., 1998). And a pretreatment process is essentially required to break it down (Cuvilas ⇑ Corresponding author. Tel.: +86 20 22236808; fax: +86 20 87518783. E-mail address: [email protected] (S. Wu). http://dx.doi.org/10.1016/j.biortech.2014.02.030 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
and Yang, 2012). The pretreatments using acid or base solutions can achieve high reaction rates and significantly improve cellulose hydrolysis but they require corrosion resistant equipment (Sun and Cheng, 2002; Wei et al., 2012). Dilute NaOH treatment of lignocellulosic materials caused disruption of the lignin structure, separation of linkages between lignin and carbohydrates, the digestibility of NaOH-treated hardwood increased with the decrease of lignin content, but they require chemicals which are not friendly to the environment (Esteghlalian et al., 1997). The hot water treatment produces digestible cellulose and needs high energy for operation (Zhang et al., 2013). The cost of ammonia fiber expansion (AFEX) is low, but the degradation and loss of hemicellulose, and the requirement of water to wash pretreated solids resist the development of it (Krishnan et al., 2010). As a consequence, the search for new methods that are less toxic with low-cost are still being actively pursued (Muhammad et al., 2013). Supercritical CO2 which has been mostly used as an extraction solvent (Kim and Hong, 2000), is being considered for pretreatment process due to its attractive properties such as nontoxicity, ready
162
H. Zhang, S. Wu / Bioresource Technology 158 (2014) 161–165
availability, low-cost consumption. Furthermore, CO2 has a low critical temperature (31.1 °C) and pressure (7.36 MPa), can form weak acid catalyst with water (CO2 + H2O H2CO3 H+ + HCO3
2H+ + CO32) (Muzafera et al., 2007; Luterbacher et al., 2012), which helps degrade cellulose or/and lignin, increase the accessible surface area of lignocellulose, and improve biomass digestibility at enzymatic hydrolysis stage (Gao et al., 2002; Narayanaswamy et al., 2011). Many of the current efforts are directed at the supercritical CO2 pretreatment of biomass material. Narayanaswamy et al. (2011) investigated that the largest glucose yield reached 30 g/100 g dry corn stover during enzymatic hydrolysis after supercritical CO2 pretreatment, which was achieved at 24.1 MPa and 150 °C for 60 min. When the cellulosic materials were treated with supercritical CO2, the capability of cellulose hydrolysis were enhanced (Schacht et al., 2008). Compared with supercritical CO2 pretreatment, the demands of apparatus to the subcritical CO2 process is easy to realize, and the improved glucose yield from sugarcane bagasse with subcritical CO2 pretreatment (<7.36 MPa) has yet not been reported. Based on the above viewpoint, subcritical CO2 pretreatment of sugarcane bagasse was proposed to improve glucose yield during enzymatic hydrolysis. Firstly, the solid yield and glucan recovery under different pretreatment conditions (CO2 pressure, pretreatment time and temperature) were investigated. Subsequently, enzymatic hydrolysis under different length of time were carried out to evaluate the efficiency of subcritical CO2 pretreatment process. Furthermore, we also detected the changes of raw material, pretreated, and enzymolysis residue samples (5 MPa CO2, 100 min time and 180 °C pretreatment temperature) in the crystallinity by X-ray diffraction (XRD), in functional groups by Fourier transform infrared spectroscopy (FTIR), in surface structure by Scanning electron microscopy (SEM), and in thermochemical properties by Thermogravimetric analysis (TGA) to provide a mechanism for subcritical CO2 pretreatment. 2. Methods 2.1. Raw material Sugarcane bagasse were supplied by a company located in Guangdong, China and cut with a laboratory mill (XuLang Machinery, Guangzhou, China) to a particle size <1 mm. Then the samples were air-dried for further use. The chemical composition of the raw material (on a dry weight basis) was 37.2 ± 1.1% glucan, 24.6 ± 0.1% xylan, 1.5 ± 0.2% arabinan, 0.6 ± 0.1% galactan, 22.6 ± 0.9% acidinsoluble lignin (AIL), 2.2 ± 0.2% acid-soluble lignin (ASL) and 4.6 ± 0.6% ash. CO2 was purchased from Guangzhou Junduo Gases Co. (Guangzhou, China) and was purified to 99.9% purity using an activated carbon column. 2.2. Subcritical CO2 pretreatment Subcritical CO2 pretreatment was carried out in a stirred 1L Parr reactor. The pretreatment were performed at various pressures (0–7 MPa), time periods (20–100 min), and temperatures (140– 180 °C) with the 15% (w/v) solid to liquid ratio. Forty-five grams of dry sugarcane bagasse and 300 mL of de-ionized water were added to the reactor. Then a certain amount of CO2 was loaded into the reactor to reach the given pressure though a pressure regulator. The agitation was set at 300 rpm to mix biomass with CO2 during the reacting process. When the reaction process was finished, the reactor was immediately cooled down by cooling water (until below 40 °C). Then the pressure was released instantaneously using a quick release ball valve. The pretreated solution was then
separated by filtration. The solid yield and glucan content in the pretreated solids were analyzed. Then the solid fraction was stored in refrigerator for further enzymatic hydrolysis. 2.3. Enzymatic hydrolysis The enzymatic hydrolysis was then performed in 500 mL flasks using 0.05 mol/L acetic acid sodium acetate buffer (pH 4.8) and 2% dry matter (w/w) at 50 °C on an shaker at 150 rpm for 6 h, 12 h, 24 h, 48 h and 72 h. Cellulase was purchased from Genencor (Shanghai, China), with a filter paper activity of 20 FPU/mL. The cellulase enzymes were added at the loading of 20 FPU/g dry substrate for all hydrolysis experiments. At the same time, two drops of acetic ether were added to inhibit microbial contamination or growth. After the enzymatic hydrolysis process was completed, glucose in enzyme hydrolyzate were analyzed by IC system. 2.4. Characterization 2.4.1. Analysis methods The chemical composition of sugarcane bagasse and pretreated solids were determined using the standard provided by the National Renewable Energies Laboratory in USA (Sluiter et al., 2006). All liquid products from the enzyme hydrolysate were diluted appropriately with the de-ionized water and then filtered using a 0.22 um filter. The concentration of glucose was quantitatively analyzed at 30 °C by IC system (Dionex ICS-3000) with a CarboPac PA20 column. 2.4.2. Crystallinity measurements The crystallinity of untreated, pretreated solid and enzymatic residue (5 MPa CO2, 100 min time and 180 °C pretreatment temperature) were determined by X-ray diffraction using a Bruker D8-ADVANCE (German) with Ni-filtered and Cu radiation (k = 0.15418 nm). The scattering angle (2h) was from 10 to 60° in steps of 0.04° at time intervals of 0.2 s. The crystalline index (CrI) was determined based on the formula by Segal et al. (1959) as follows.
CrI ¼ ½ðI002 Iam Þ=I002 100% in which I002 is the scattered intensity at the main peak around 22.5°; where Iam is the scattered intensity due to the amorphous portion evaluated as the minimum intensity between the main and secondary (the broad peak at 18.0°) peaks. 2.4.3. Fourier Transform Infrared Spectroscopy (FT-IR) FT-IR was recorded from a FT-IR spectrophotometer (Tensor 27, Bruker, Germany). The samples (KBr pellets) for analyses were prepared by mixing 2 mg material powder with 200 mg KBr. Thirtytwo scans were taken from 4000 to 400 cm1. 2.4.4. Scanning Electron Microscopy (SEM) analysis Scanning electron microscopy (SEM) of the three samples was carried out with a HITACHI S-3700N (Japan) instrument at 10 kV. Each sample was sputter-coated with gold for 180 s prior to the observation. All observations were utilized at 2000 magnification. 2.4.5. Thermogravimetric analysis Thermogravimetric analysis (TGA) was carried out on a TA Q500 instrument (America). Dynamic TG scans were conducted in a temperature ranging from 30 to 700 °C at a heating rate of 10 °C/min. The experiments were carried out under 25 ml/min nitrogen atmosphere. About 5–7 mg samples were used.
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at various pretreatment conditions were investigated and summarized in Fig. 1A–C. In the respective figures, the enzymatic hydrolysis of raw sugarcane bagasse biomass at different hydrolysis time were also displayed for comparison. From the Figures, it could know that the enzymatic digestibilities were low, the glucose yields were below 10% for a 72 h hydrolysis time, which agreed with the saccharification behavior of eucalyptus flour without pretreatment (Teramoto et al., 2008). Fig. 1A showed the relationship between pretreatment pressure and the glucose yield of enzymatic hydrolysis at different hydrolysis time. It can be seen from it that increasing hydrolysis time from 6 h to 72 h led to just a 21.4–46.4% increase of glucose yield without subcritical CO2 except for hot water. The glucose yield of pretreated sugarcane bagasse after 6 h and 72 h enzymatic hydrolysis at 5 MPa CO2 was 1.7-fold, and 1.5-fold, as great as that at 0 MPa CO2, respectively. The dramatic increase indicated that even though higher pretreatment pressure decreased the glucan recovery during the pretreatment process, it also had a positive effect on enzymatic hydrolysis, accounted by the fact that higher pretreatment pressure could result in the more degradation of hemicelluloses and the increasing accessibility of the cellulose (Kim and Hong, 2001). Glucose yield reached a maximum of 46.4%, 55.2%, 67.7%, 71.3%, and 71.1% at 72 h with pretreatment pressures of 0, 1, 3, 5, and 7 MPa, respectively. From them we can know that the glucose yield increased with the increment of pretreatment pressure ranging from 0 to 5 MPa, showed little change at 7 MPa. The results demonstrated that pretreatment pressure played an active role in improving the glucose yield of enzymatic hydrolysis, the similar situation was also observed by Zheng et al. (1998). Therefore, a pressure of 5 MPa was considered suitable for this sugarcane bagasse pretreatment method. Fig. 1B illustrated the relationship between the pretreatment time and glucose yield of pretreated sugarcane bagasse from 6 h to 72 h of enzymatic hydrolysis. Pretreatment was carried out for various time durations at constant pressure (5 MPa), temperature (160 °C) and a relative high solid load of 15%. After 72 h hydrolysis time, the glucose yield for 20 min pretreatment time was 49.7%, whereas the glucose yield of 40 and 60 min pretreated time were 72.3% and 71.3%, respectively. Thus, pretreatment duration increasing from 40 to 60 min failed to significantly affect glucose yield. Further increasing the pretreated time had also improvement on the glucose yield. 100 min pretreated time resulted in a 86.70% of glucose yield, which is considerably higher than 80 min pretreated time (78.4%). This trend suggested that the longer pretreatment time, the easier accessibility of enzyme to cellulose, and the higher glucose yield. Hence, 100 min was regarded as the relatively favorable pretreatment residence time, as longer
3. Results and discussion 3.1. Effect of subcritical CO2 pretreatment on glucan recovery of pretreated solids As the recalcitrant structure of lignocellulosic biomass inhibits the enzyme hydrolysis, pretreatment should be applied to overcome its recalcitrance. In general, pretreatment breaks down the linkages among three components (cellulose, hemicellulose, lignin) or/and releases some of them, which makes the cellulose more accessibility to the enzyme. However, the glucan, as the reactant of subsequent enzymolysis, were required to be preserved as much as possible after pretreatment. Therefore, it is necessary to investigate the effect of subcritical CO2 pretreatment on glucan recoveries of pretreated solids. So the glucan recoveries and glucan contents of pretreated solids, as well as the solid recoveries of raw material after different pretreatment were summarized in Table 1. It can be seen from Table 1 that the solid recoveries of pretreated material ranged from 67.5% to 86.1%, which implied that mass loss could be found at different pretreated conditions. The results indicated that some constituents in the feedstock were removed because of the weak acid conditions formed by subcritical CO2 and water during pretreatment. Moreover, all the glucan content of pretreated solids had a relative increase as compared with the raw material (37.2%). This phenomenon could also be explained that the large amorphous hemicellulose in biomass material was removed during pretreatment, so the relative content of glucan were increased. For glucan recovery, when the pretreatment pressure increased from 0 to 7 MPa, it decreased from 98.3% to 94.1%. It was also found that pretreatment time showed no significant effect on the glucan recovery at the same pretreatment pressure and temperature (5 MPa and 160 °C), and the glucan recovery was around 95.4–96.5%. The maximal glucan recovery (98.2%) was obtained when the pretreatment temperature was rose to 180 °C. This could be explained that hemicellulose and lignin may solubilise at about 180 °C under neutral conditions (Gao et al., 2010), as the amorphous hemicellulose were degraded, the left cellulose were covered by solvable lignin, which resisted the continuous degradation of cellulose during subcritical CO2 pretreatment. 3.2. Effect of subcritical CO2 pretreatment on enzymatic hydrolysis The high glucan recovery during subcritical CO2 pretreatment would not represent a final high glucose yield because of the effect of enzymatic digestibility. Therefore, differences in the enzymatic hydrolysis of sugarcane bagasse pretreated with subcritical CO2
Table 1 Recoveries of solid and glucan, and glucan content in pretreated solids at varying subcritical CO2 pretreatment conditions. Pressure/MPa
Temperature/°C
Time/min
Solid recovery (%)
Glucan content (%)
Glucan recovery (%)
0 1 3 5 7
160
60
78.6 76.1 74.9 72.4 72.0
46.5 ± 1.2 47.2 ± 1.1 48.0 ± 0.5 49.0 ± 1.1 48.6 ± 0.4
98.3 96.6 96.6 95.4 94.1
5
20 40 60 80 100
160
80.4 74.5 72.4 71.6 71.4
44.3 ± 0.7 47.8 ± 1.0 49.0 ± 1.1 49.7 ± 0.2 50.3 ± 0.4
95.7 95.7 95.4 95.7 96.5
5
100
140 150 160 170 180
86.1 78.8 71.4 67.8 67.5
41.8 ± 0.3 46.0 ± 0.6 50.3 ± 0.4 53.5 ± 0.6 54.1 ± 0.6
96.7 97.4 96.5 97.5 98.2
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80 material 3Mpa
70
0Mpa 5Mpa
1Mpa 7Mpa
(A) Glucose yield (%)
60 50 40 30 20 10 0 0
1
20
30
40
50
60
70
80
60
70
80
Hydrolysis time (h) 100 material 60min
90
Glucose yield (%)
80
20min 80min
40min 100min
(B)
70 60 50 40 30 20 10
180 °C, and/or the enzymatic hydrolysis increased from 6 to 72 h. After 72 h hydrolysis, the glucose yield reached 37.2% at relative low pretreated temperature of 140 °C. When the pretreatment temperature increased to 160 °C, the glucose yield increased sharply to 86.7%, which was 2.3-fold as great as that at 140 °C. As the pretreatment temperature went on increasing, the glucose yield also increased gradually, and 93.0% of glucose yield cold be obtained at 180 °C, representing 38.5 g glucose based on 100 g raw material. Thus, pretreatment temperature is one of most important factors in increasing the enzymatic hydrolysis efficiency of sugarcane bagasse. This phenomenon could be concluded that due to the more removal of hemicellulose with higher pretreatment temperature (more severe pretreatment conditions), more cellulose were exposed to the cellulose (Donohoe et al., 2008). Higher temperatures could improve glucose yield, however, higher temperatures led to the consumption of higher energy and the formation of inhibitors for fermentation such as furfural and hydroxymethylfurfural (Palmqvist and Hahn-Hagerdal, 2000; Alvira et al., 2010). Hence, 180 °C was recommended as the optimum pretreatment temperature for glucose yield of sugarcane bagasse. Fig. 1C also showed a comparison of enzymatic hydrolysis of subcritical CO2 (5 MPa) and supercritical CO2 (8.3 MPa) pretreatment of sugarcane bagasse at constant temperature (180 °C) and time (100 min). After a 6 h hydrolysis time, the glucose yield reached 68.3% and 63.2%, respectively. As the hydrolysis time prolonged, the glucose yield increased gradually. About 93.0% (subcritical CO2 pretreatment) and 94.7% (supercritical CO2 pretreatment) of glucose could be obtained after 72 h hydrolysis time. The results indicated that increasing CO2 pressure from 5 MPa (subcritical) to 8.3 MPa (supercritical) improved glucose yield by only 0.7 g based on 100 g raw material. This meant that subcritical CO2 pretreatment was an effective way to obtain glucose in enzymatic hydrolysis.
0 0
1
20
30
40
50
3.3. The different composition of different pretreated samples
Hydrolysis time (h) 110 100
140oC 180oC
material 170oC
After subcritical CO2 pretreatment and enzymatic hydrolysis, the chemical composition of samples would be different from the raw material. And the composition of raw material, pretreated solid, and enzymolysis residue at pretreatment conditions of 5 MPa CO2, 100 min time and 180 °C were presented in Fig. 2. For glucan, the content increased from 37.2% (raw material) to 54.1% after pretreatment. At the same time, the xylan content decreased from 24.6% to 1.8%. This phenomenon could be concluded that the
150oC 160oC supercritical CO2
(C)
80 70 60 50 40
80
30 20 10 0
0
1
20
30
40
50
60
70
80
Hydrolysis time (h) Fig. 1. Effect of pretreatment conditions on the enzymatic hydrolysis of sugarcane bagasse. (A) effect of pretreatment pressure on the yield of glucose; (B) effect of pretreatment time on the yield of glucose; (C) effect of pretreatment temperature on the yield of glucose.
pretreatment time was disadvantageous to the recovery of glucan and energy conservation (Chen and Fu, 2013). The effects of subcritical CO2 pretreatment temperature on the enzymatic hydrolysis of sugarcane bagasse were shown in Fig. 1C. It can be seen from Fig. 1C that, the glucose yield increased gradually as the pretreatment temperature rose from 140 to
The composition of samples (%)
Glucose yield (%)
90
70
glucan xylan lignin
60 50 40 30 20 10 0
raw material
pretreated solid
enzymatic residue
Fig. 2. The different composition of raw material, pretreated solid, and enzymolysis residue.
H. Zhang, S. Wu / Bioresource Technology 158 (2014) 161–165
relative glucan content increased due to the decrease of xylan (the degradation of hemicellulose) during subcritical CO2 pretreatment. After enzymatic hydrolysis, the contents of glucan and xylan decreased sharply to 3.5% and 0.2%, respectively. The results could be ascribed to the degradation of most cellulose and little hemicellulose during enzymatic hydrolysis. Though little lignin were degraded during pretreatment and enzymatic hydrolysis, the lignin content increased gradually, reached 78.2% after enzymatic hydrolysis, this meant that about 95.6% of lignin (21.6 g per 100 g raw material) could be recovered after enzymatic hydrolysis. 3.4. The characterization of different pretreated samples The changes of raw material, pretreated, and enzymolysis residue samples (5 MPa CO2, 100 min time and 180 °C pretreatment temperature) in the crystallinity by XRD, in functional groups by FTIR, in surface structure by SEM, and in thermochemical properties by TGA were provided in supplement file. 4. Conclusions The subcritical CO2 pretreatment on sugarcane bagasse enhanced enzymatic hydrolysis considerably. The pretreatment performed at 5 MPa CO2, 100 min, and 180 °C resulted in 38.5 g per 100 g raw material after 72 h hydrolysis time, representing 93.0% glucose in sugarcane bagasse. XRD and FTIR analyses indicated changes in crystallinity and functional groups of raw material, pretreated solid and enzymatic residue. SEM showed numerous cracks and fragements in pretreated material. An thermal stability increase was observed by TGA. So, subcritical CO2 pretreatment could be successfully applied to sugarcane bagasse to improve the glucose yield during enzymatic hydrolysis. Acknowledgements This work was supported by the National High Technology Research and Development Program of China (No. 2012AA101806), National Key Basic Research Program of China (No. 2013CB228101), the National Natural Science Foundation of China (Nos. 31270625 and 21176095). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014. 02.030. References Alvira, P., Tomas-pejo, E., Ballesteros, M., Negro, M.J., 2010. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis. A review. Bioresour. Technol. 101, 4851–4861.
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Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Enhanced enzymatic cellulose hydrolysis by subcritical carbon dioxide pretreatment of sugarcane bagasse Hongdan Zhang, Shubin Wu ⇑ State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, PR China
h i g h l i g h t s A subcritical CO2 pretreatment was investigated to improve the glucose yield. Liquid fractions and solid residues were thoroughly analyzed. The enzymatic hydrolysis was affected by different pretreatment conditions. The enhanced digestibilities were confirmed by XRD, FTIR, SEM, and TGA analyses. The proposed process can be a contribution to develop a biorefinery.
a r t i c l e
i n f o
Article history: Received 22 December 2013 Received in revised form 5 February 2014 Accepted 8 February 2014 Available online 15 February 2014 Keywords: Sugarcane bagasse Subcritical CO2 pretreatment Glucose Enzymatic hydrolysis
a b s t r a c t Most biomass pretreatment processes for sugar production are run at low-solid concentration (<10 wt.%). Subcritical carbon dioxide (CO2) could provide a more sustainable pretreatment medium while using relative high-solid contents (15 wt.%). The effects of subcritical CO2 pretreatment of sugarcane bagasse to the solid and glucan recoveries at different pretreatment conditions were investigated. Subsequently, enzymatic hydrolysis at different hydrolysis time was applied to obtain maximal glucose yield, which can be used for ethanol fermentation. The maximum glucose yield in enzyme hydrolyzate reached 38.5 g based on 100 g raw material after 72 h of enzymatic hydrolysis, representing 93.0% glucose in sugarcane bagasse. The enhanced digestibilities of subcritical CO2 pretreated sugarcane bagasse were due to the removal of hemicellulose, which were confirmed by XRD, FTIR, SEM, and TGA analyses. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Global energy inevitable depletion has generated strong interest in the development of more sustainable transportation fuels and renewable carbon-based raw materials, such as those produced from biomass (Dodds and Gross, 2007; Somerville, 2006). Sugarcane bagasse was used for the bioconversion of lignocellulosic biomass to bio-fuels (such as ethanol and biodiesel) due to its availability, abundance, and renewability (Pandey et al., 2000; Rabelo et al., 2009). The abundant cellulose in sugarcane bagasse could be hydrolyzed to glucose by enzyme for subsequent ethanolic fermentation (Martin et al., 2002). The coexistence of hemicellulose and lignin with cellulose make the enzymatic hydrolysis of cellulose difficult and tedious (Kim and Hong, 2001; Zheng et al., 1998). And a pretreatment process is essentially required to break it down (Cuvilas ⇑ Corresponding author. Tel.: +86 20 22236808; fax: +86 20 87518783. E-mail address: [email protected] (S. Wu). http://dx.doi.org/10.1016/j.biortech.2014.02.030 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
and Yang, 2012). The pretreatments using acid or base solutions can achieve high reaction rates and significantly improve cellulose hydrolysis but they require corrosion resistant equipment (Sun and Cheng, 2002; Wei et al., 2012). Dilute NaOH treatment of lignocellulosic materials caused disruption of the lignin structure, separation of linkages between lignin and carbohydrates, the digestibility of NaOH-treated hardwood increased with the decrease of lignin content, but they require chemicals which are not friendly to the environment (Esteghlalian et al., 1997). The hot water treatment produces digestible cellulose and needs high energy for operation (Zhang et al., 2013). The cost of ammonia fiber expansion (AFEX) is low, but the degradation and loss of hemicellulose, and the requirement of water to wash pretreated solids resist the development of it (Krishnan et al., 2010). As a consequence, the search for new methods that are less toxic with low-cost are still being actively pursued (Muhammad et al., 2013). Supercritical CO2 which has been mostly used as an extraction solvent (Kim and Hong, 2000), is being considered for pretreatment process due to its attractive properties such as nontoxicity, ready
162
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availability, low-cost consumption. Furthermore, CO2 has a low critical temperature (31.1 °C) and pressure (7.36 MPa), can form weak acid catalyst with water (CO2 + H2O H2CO3 H+ + HCO3
2H+ + CO32) (Muzafera et al., 2007; Luterbacher et al., 2012), which helps degrade cellulose or/and lignin, increase the accessible surface area of lignocellulose, and improve biomass digestibility at enzymatic hydrolysis stage (Gao et al., 2002; Narayanaswamy et al., 2011). Many of the current efforts are directed at the supercritical CO2 pretreatment of biomass material. Narayanaswamy et al. (2011) investigated that the largest glucose yield reached 30 g/100 g dry corn stover during enzymatic hydrolysis after supercritical CO2 pretreatment, which was achieved at 24.1 MPa and 150 °C for 60 min. When the cellulosic materials were treated with supercritical CO2, the capability of cellulose hydrolysis were enhanced (Schacht et al., 2008). Compared with supercritical CO2 pretreatment, the demands of apparatus to the subcritical CO2 process is easy to realize, and the improved glucose yield from sugarcane bagasse with subcritical CO2 pretreatment (<7.36 MPa) has yet not been reported. Based on the above viewpoint, subcritical CO2 pretreatment of sugarcane bagasse was proposed to improve glucose yield during enzymatic hydrolysis. Firstly, the solid yield and glucan recovery under different pretreatment conditions (CO2 pressure, pretreatment time and temperature) were investigated. Subsequently, enzymatic hydrolysis under different length of time were carried out to evaluate the efficiency of subcritical CO2 pretreatment process. Furthermore, we also detected the changes of raw material, pretreated, and enzymolysis residue samples (5 MPa CO2, 100 min time and 180 °C pretreatment temperature) in the crystallinity by X-ray diffraction (XRD), in functional groups by Fourier transform infrared spectroscopy (FTIR), in surface structure by Scanning electron microscopy (SEM), and in thermochemical properties by Thermogravimetric analysis (TGA) to provide a mechanism for subcritical CO2 pretreatment. 2. Methods 2.1. Raw material Sugarcane bagasse were supplied by a company located in Guangdong, China and cut with a laboratory mill (XuLang Machinery, Guangzhou, China) to a particle size <1 mm. Then the samples were air-dried for further use. The chemical composition of the raw material (on a dry weight basis) was 37.2 ± 1.1% glucan, 24.6 ± 0.1% xylan, 1.5 ± 0.2% arabinan, 0.6 ± 0.1% galactan, 22.6 ± 0.9% acidinsoluble lignin (AIL), 2.2 ± 0.2% acid-soluble lignin (ASL) and 4.6 ± 0.6% ash. CO2 was purchased from Guangzhou Junduo Gases Co. (Guangzhou, China) and was purified to 99.9% purity using an activated carbon column. 2.2. Subcritical CO2 pretreatment Subcritical CO2 pretreatment was carried out in a stirred 1L Parr reactor. The pretreatment were performed at various pressures (0–7 MPa), time periods (20–100 min), and temperatures (140– 180 °C) with the 15% (w/v) solid to liquid ratio. Forty-five grams of dry sugarcane bagasse and 300 mL of de-ionized water were added to the reactor. Then a certain amount of CO2 was loaded into the reactor to reach the given pressure though a pressure regulator. The agitation was set at 300 rpm to mix biomass with CO2 during the reacting process. When the reaction process was finished, the reactor was immediately cooled down by cooling water (until below 40 °C). Then the pressure was released instantaneously using a quick release ball valve. The pretreated solution was then
separated by filtration. The solid yield and glucan content in the pretreated solids were analyzed. Then the solid fraction was stored in refrigerator for further enzymatic hydrolysis. 2.3. Enzymatic hydrolysis The enzymatic hydrolysis was then performed in 500 mL flasks using 0.05 mol/L acetic acid sodium acetate buffer (pH 4.8) and 2% dry matter (w/w) at 50 °C on an shaker at 150 rpm for 6 h, 12 h, 24 h, 48 h and 72 h. Cellulase was purchased from Genencor (Shanghai, China), with a filter paper activity of 20 FPU/mL. The cellulase enzymes were added at the loading of 20 FPU/g dry substrate for all hydrolysis experiments. At the same time, two drops of acetic ether were added to inhibit microbial contamination or growth. After the enzymatic hydrolysis process was completed, glucose in enzyme hydrolyzate were analyzed by IC system. 2.4. Characterization 2.4.1. Analysis methods The chemical composition of sugarcane bagasse and pretreated solids were determined using the standard provided by the National Renewable Energies Laboratory in USA (Sluiter et al., 2006). All liquid products from the enzyme hydrolysate were diluted appropriately with the de-ionized water and then filtered using a 0.22 um filter. The concentration of glucose was quantitatively analyzed at 30 °C by IC system (Dionex ICS-3000) with a CarboPac PA20 column. 2.4.2. Crystallinity measurements The crystallinity of untreated, pretreated solid and enzymatic residue (5 MPa CO2, 100 min time and 180 °C pretreatment temperature) were determined by X-ray diffraction using a Bruker D8-ADVANCE (German) with Ni-filtered and Cu radiation (k = 0.15418 nm). The scattering angle (2h) was from 10 to 60° in steps of 0.04° at time intervals of 0.2 s. The crystalline index (CrI) was determined based on the formula by Segal et al. (1959) as follows.
CrI ¼ ½ðI002 Iam Þ=I002 100% in which I002 is the scattered intensity at the main peak around 22.5°; where Iam is the scattered intensity due to the amorphous portion evaluated as the minimum intensity between the main and secondary (the broad peak at 18.0°) peaks. 2.4.3. Fourier Transform Infrared Spectroscopy (FT-IR) FT-IR was recorded from a FT-IR spectrophotometer (Tensor 27, Bruker, Germany). The samples (KBr pellets) for analyses were prepared by mixing 2 mg material powder with 200 mg KBr. Thirtytwo scans were taken from 4000 to 400 cm1. 2.4.4. Scanning Electron Microscopy (SEM) analysis Scanning electron microscopy (SEM) of the three samples was carried out with a HITACHI S-3700N (Japan) instrument at 10 kV. Each sample was sputter-coated with gold for 180 s prior to the observation. All observations were utilized at 2000 magnification. 2.4.5. Thermogravimetric analysis Thermogravimetric analysis (TGA) was carried out on a TA Q500 instrument (America). Dynamic TG scans were conducted in a temperature ranging from 30 to 700 °C at a heating rate of 10 °C/min. The experiments were carried out under 25 ml/min nitrogen atmosphere. About 5–7 mg samples were used.
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at various pretreatment conditions were investigated and summarized in Fig. 1A–C. In the respective figures, the enzymatic hydrolysis of raw sugarcane bagasse biomass at different hydrolysis time were also displayed for comparison. From the Figures, it could know that the enzymatic digestibilities were low, the glucose yields were below 10% for a 72 h hydrolysis time, which agreed with the saccharification behavior of eucalyptus flour without pretreatment (Teramoto et al., 2008). Fig. 1A showed the relationship between pretreatment pressure and the glucose yield of enzymatic hydrolysis at different hydrolysis time. It can be seen from it that increasing hydrolysis time from 6 h to 72 h led to just a 21.4–46.4% increase of glucose yield without subcritical CO2 except for hot water. The glucose yield of pretreated sugarcane bagasse after 6 h and 72 h enzymatic hydrolysis at 5 MPa CO2 was 1.7-fold, and 1.5-fold, as great as that at 0 MPa CO2, respectively. The dramatic increase indicated that even though higher pretreatment pressure decreased the glucan recovery during the pretreatment process, it also had a positive effect on enzymatic hydrolysis, accounted by the fact that higher pretreatment pressure could result in the more degradation of hemicelluloses and the increasing accessibility of the cellulose (Kim and Hong, 2001). Glucose yield reached a maximum of 46.4%, 55.2%, 67.7%, 71.3%, and 71.1% at 72 h with pretreatment pressures of 0, 1, 3, 5, and 7 MPa, respectively. From them we can know that the glucose yield increased with the increment of pretreatment pressure ranging from 0 to 5 MPa, showed little change at 7 MPa. The results demonstrated that pretreatment pressure played an active role in improving the glucose yield of enzymatic hydrolysis, the similar situation was also observed by Zheng et al. (1998). Therefore, a pressure of 5 MPa was considered suitable for this sugarcane bagasse pretreatment method. Fig. 1B illustrated the relationship between the pretreatment time and glucose yield of pretreated sugarcane bagasse from 6 h to 72 h of enzymatic hydrolysis. Pretreatment was carried out for various time durations at constant pressure (5 MPa), temperature (160 °C) and a relative high solid load of 15%. After 72 h hydrolysis time, the glucose yield for 20 min pretreatment time was 49.7%, whereas the glucose yield of 40 and 60 min pretreated time were 72.3% and 71.3%, respectively. Thus, pretreatment duration increasing from 40 to 60 min failed to significantly affect glucose yield. Further increasing the pretreated time had also improvement on the glucose yield. 100 min pretreated time resulted in a 86.70% of glucose yield, which is considerably higher than 80 min pretreated time (78.4%). This trend suggested that the longer pretreatment time, the easier accessibility of enzyme to cellulose, and the higher glucose yield. Hence, 100 min was regarded as the relatively favorable pretreatment residence time, as longer
3. Results and discussion 3.1. Effect of subcritical CO2 pretreatment on glucan recovery of pretreated solids As the recalcitrant structure of lignocellulosic biomass inhibits the enzyme hydrolysis, pretreatment should be applied to overcome its recalcitrance. In general, pretreatment breaks down the linkages among three components (cellulose, hemicellulose, lignin) or/and releases some of them, which makes the cellulose more accessibility to the enzyme. However, the glucan, as the reactant of subsequent enzymolysis, were required to be preserved as much as possible after pretreatment. Therefore, it is necessary to investigate the effect of subcritical CO2 pretreatment on glucan recoveries of pretreated solids. So the glucan recoveries and glucan contents of pretreated solids, as well as the solid recoveries of raw material after different pretreatment were summarized in Table 1. It can be seen from Table 1 that the solid recoveries of pretreated material ranged from 67.5% to 86.1%, which implied that mass loss could be found at different pretreated conditions. The results indicated that some constituents in the feedstock were removed because of the weak acid conditions formed by subcritical CO2 and water during pretreatment. Moreover, all the glucan content of pretreated solids had a relative increase as compared with the raw material (37.2%). This phenomenon could also be explained that the large amorphous hemicellulose in biomass material was removed during pretreatment, so the relative content of glucan were increased. For glucan recovery, when the pretreatment pressure increased from 0 to 7 MPa, it decreased from 98.3% to 94.1%. It was also found that pretreatment time showed no significant effect on the glucan recovery at the same pretreatment pressure and temperature (5 MPa and 160 °C), and the glucan recovery was around 95.4–96.5%. The maximal glucan recovery (98.2%) was obtained when the pretreatment temperature was rose to 180 °C. This could be explained that hemicellulose and lignin may solubilise at about 180 °C under neutral conditions (Gao et al., 2010), as the amorphous hemicellulose were degraded, the left cellulose were covered by solvable lignin, which resisted the continuous degradation of cellulose during subcritical CO2 pretreatment. 3.2. Effect of subcritical CO2 pretreatment on enzymatic hydrolysis The high glucan recovery during subcritical CO2 pretreatment would not represent a final high glucose yield because of the effect of enzymatic digestibility. Therefore, differences in the enzymatic hydrolysis of sugarcane bagasse pretreated with subcritical CO2
Table 1 Recoveries of solid and glucan, and glucan content in pretreated solids at varying subcritical CO2 pretreatment conditions. Pressure/MPa
Temperature/°C
Time/min
Solid recovery (%)
Glucan content (%)
Glucan recovery (%)
0 1 3 5 7
160
60
78.6 76.1 74.9 72.4 72.0
46.5 ± 1.2 47.2 ± 1.1 48.0 ± 0.5 49.0 ± 1.1 48.6 ± 0.4
98.3 96.6 96.6 95.4 94.1
5
20 40 60 80 100
160
80.4 74.5 72.4 71.6 71.4
44.3 ± 0.7 47.8 ± 1.0 49.0 ± 1.1 49.7 ± 0.2 50.3 ± 0.4
95.7 95.7 95.4 95.7 96.5
5
100
140 150 160 170 180
86.1 78.8 71.4 67.8 67.5
41.8 ± 0.3 46.0 ± 0.6 50.3 ± 0.4 53.5 ± 0.6 54.1 ± 0.6
96.7 97.4 96.5 97.5 98.2
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80 material 3Mpa
70
0Mpa 5Mpa
1Mpa 7Mpa
(A) Glucose yield (%)
60 50 40 30 20 10 0 0
1
20
30
40
50
60
70
80
60
70
80
Hydrolysis time (h) 100 material 60min
90
Glucose yield (%)
80
20min 80min
40min 100min
(B)
70 60 50 40 30 20 10
180 °C, and/or the enzymatic hydrolysis increased from 6 to 72 h. After 72 h hydrolysis, the glucose yield reached 37.2% at relative low pretreated temperature of 140 °C. When the pretreatment temperature increased to 160 °C, the glucose yield increased sharply to 86.7%, which was 2.3-fold as great as that at 140 °C. As the pretreatment temperature went on increasing, the glucose yield also increased gradually, and 93.0% of glucose yield cold be obtained at 180 °C, representing 38.5 g glucose based on 100 g raw material. Thus, pretreatment temperature is one of most important factors in increasing the enzymatic hydrolysis efficiency of sugarcane bagasse. This phenomenon could be concluded that due to the more removal of hemicellulose with higher pretreatment temperature (more severe pretreatment conditions), more cellulose were exposed to the cellulose (Donohoe et al., 2008). Higher temperatures could improve glucose yield, however, higher temperatures led to the consumption of higher energy and the formation of inhibitors for fermentation such as furfural and hydroxymethylfurfural (Palmqvist and Hahn-Hagerdal, 2000; Alvira et al., 2010). Hence, 180 °C was recommended as the optimum pretreatment temperature for glucose yield of sugarcane bagasse. Fig. 1C also showed a comparison of enzymatic hydrolysis of subcritical CO2 (5 MPa) and supercritical CO2 (8.3 MPa) pretreatment of sugarcane bagasse at constant temperature (180 °C) and time (100 min). After a 6 h hydrolysis time, the glucose yield reached 68.3% and 63.2%, respectively. As the hydrolysis time prolonged, the glucose yield increased gradually. About 93.0% (subcritical CO2 pretreatment) and 94.7% (supercritical CO2 pretreatment) of glucose could be obtained after 72 h hydrolysis time. The results indicated that increasing CO2 pressure from 5 MPa (subcritical) to 8.3 MPa (supercritical) improved glucose yield by only 0.7 g based on 100 g raw material. This meant that subcritical CO2 pretreatment was an effective way to obtain glucose in enzymatic hydrolysis.
0 0
1
20
30
40
50
3.3. The different composition of different pretreated samples
Hydrolysis time (h) 110 100
140oC 180oC
material 170oC
After subcritical CO2 pretreatment and enzymatic hydrolysis, the chemical composition of samples would be different from the raw material. And the composition of raw material, pretreated solid, and enzymolysis residue at pretreatment conditions of 5 MPa CO2, 100 min time and 180 °C were presented in Fig. 2. For glucan, the content increased from 37.2% (raw material) to 54.1% after pretreatment. At the same time, the xylan content decreased from 24.6% to 1.8%. This phenomenon could be concluded that the
150oC 160oC supercritical CO2
(C)
80 70 60 50 40
80
30 20 10 0
0
1
20
30
40
50
60
70
80
Hydrolysis time (h) Fig. 1. Effect of pretreatment conditions on the enzymatic hydrolysis of sugarcane bagasse. (A) effect of pretreatment pressure on the yield of glucose; (B) effect of pretreatment time on the yield of glucose; (C) effect of pretreatment temperature on the yield of glucose.
pretreatment time was disadvantageous to the recovery of glucan and energy conservation (Chen and Fu, 2013). The effects of subcritical CO2 pretreatment temperature on the enzymatic hydrolysis of sugarcane bagasse were shown in Fig. 1C. It can be seen from Fig. 1C that, the glucose yield increased gradually as the pretreatment temperature rose from 140 to
The composition of samples (%)
Glucose yield (%)
90
70
glucan xylan lignin
60 50 40 30 20 10 0
raw material
pretreated solid
enzymatic residue
Fig. 2. The different composition of raw material, pretreated solid, and enzymolysis residue.
H. Zhang, S. Wu / Bioresource Technology 158 (2014) 161–165
relative glucan content increased due to the decrease of xylan (the degradation of hemicellulose) during subcritical CO2 pretreatment. After enzymatic hydrolysis, the contents of glucan and xylan decreased sharply to 3.5% and 0.2%, respectively. The results could be ascribed to the degradation of most cellulose and little hemicellulose during enzymatic hydrolysis. Though little lignin were degraded during pretreatment and enzymatic hydrolysis, the lignin content increased gradually, reached 78.2% after enzymatic hydrolysis, this meant that about 95.6% of lignin (21.6 g per 100 g raw material) could be recovered after enzymatic hydrolysis. 3.4. The characterization of different pretreated samples The changes of raw material, pretreated, and enzymolysis residue samples (5 MPa CO2, 100 min time and 180 °C pretreatment temperature) in the crystallinity by XRD, in functional groups by FTIR, in surface structure by SEM, and in thermochemical properties by TGA were provided in supplement file. 4. Conclusions The subcritical CO2 pretreatment on sugarcane bagasse enhanced enzymatic hydrolysis considerably. The pretreatment performed at 5 MPa CO2, 100 min, and 180 °C resulted in 38.5 g per 100 g raw material after 72 h hydrolysis time, representing 93.0% glucose in sugarcane bagasse. XRD and FTIR analyses indicated changes in crystallinity and functional groups of raw material, pretreated solid and enzymatic residue. SEM showed numerous cracks and fragements in pretreated material. An thermal stability increase was observed by TGA. So, subcritical CO2 pretreatment could be successfully applied to sugarcane bagasse to improve the glucose yield during enzymatic hydrolysis. Acknowledgements This work was supported by the National High Technology Research and Development Program of China (No. 2012AA101806), National Key Basic Research Program of China (No. 2013CB228101), the National Natural Science Foundation of China (Nos. 31270625 and 21176095). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014. 02.030. References Alvira, P., Tomas-pejo, E., Ballesteros, M., Negro, M.J., 2010. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis. A review. Bioresour. Technol. 101, 4851–4861.
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