- Email: [email protected]
Comparison of sodium hydroxide and calcium hydroxide pretreatments on the enzymatic hydrolysis and lignin recovery of sugarcane bagasse
Accepted Manuscript Comparison of sodium hydroxide and calcium hydroxide pretreatments on the enzymatic hydrolysis and lignin recovery of sugarcane bagasse Menglei Chang, Denian Li, Wen Wang, Dongchu Chen, Yuyuan Zhang, Huawen Hu, Xiufang Ye PII: DOI: Reference:
S0960-8524(17)31406-2 http://dx.doi.org/10.1016/j.biortech.2017.08.101 BITE 18710
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
14 July 2017 14 August 2017 16 August 2017
Please cite this article as: Chang, M., Li, D., Wang, W., Chen, D., Zhang, Y., Hu, H., Ye, X., Comparison of sodium hydroxide and calcium hydroxide pretreatments on the enzymatic hydrolysis and lignin recovery of sugarcane bagasse, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.08.101
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Comparison of sodium hydroxide and calcium hydroxide pretreatments on the enzymatic hydrolysis and lignin recovery of sugarcane bagasse Menglei Changa, Denian Lib, Wen Wangb,* Dongchu Chena, Yuyuan Zhanga, Huawen Hua, Xiufang Yea a
College of Materials Science and Energy Engineering, Foshan University, Foshan
528000, Guangdong, P. R. China b
Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences; CAS Key
Laboratory of Renewable Energy; Guangdong Provincial Key Laboratory of New and Renewable Energy Research and Development; Guangzhou 510640, Guangdong, P. R. China Abstract: Sodium hydroxide (NaOH) and calcium hydroxide (Ca(OH) 2) respectively dissolved in water and 70% glycerol were applied to treat sugarcane bagasse (SCB) under the condition of 80 ºC for 2 h. NaOH solutions could remove more lignin and obtain higher enzymatic hydrolysis efficiency of SCB than Ca(OH) 2 solutions. Compared with the alkali-water solutions, the enzymatic hydrolysis of SCB treated in NaOH-glycerol solution decreased, while that in Ca(OH) 2-glycerol solution increased. The lignin in NaOH-water pretreatment liquor could be easily recovered by calcium chloride (CaCl2) at room temperature, but that in Ca(OH)2-water pretreatment liquor couldn’t. NaOH pretreatment is more suitable for facilitating enzymatic hydrolysis and lignin recovery of SCB than Ca(OH)2 pretreatment.
*
Corresponding author, Email: [email protected]; Tel.: 86-20-37029703 1
Keywords: Sugarcane bagasse; Alkaline pretreatment; Calcium chloride; Enzymatic hydrolysis; Lignin recovery 1. Introduction Pretreatment is a fundamental step for refining biofuels and bio-based products from the lignocellulosic biomass as the bioconversion of cellulose into fermentable monosaccharides (Akhtar et al., 2016). It breaks up the tight structure of cell wall formed by cellulose, hemicellulose and lignin to improve the enzymatic saccharification efficiency of cellulose and/or hemicellulose (Silveira et al., 2015). Alkaline pretreatment, a kind of pretreatment methods, possesses some desirable features. It mainly removes lignin and can be carried out at milder condition than several other pretreatments like hydrothermal pretreatments, etc. (Kim et al., 2016). The hazard of the alkaline pretreatment liquor to environment could be relieved with the development of several technologies such as adsorption, precipitation, ultrafiltration, bioaugmentation, electrocoagulation and electrolysis (Li et al., 2015). Sodium hydroxide (NaOH) and calcium hydroxide (Ca(OH) 2) are the common reagents to be used for alkaline pretreatment. NaOH pretreatment particularly cleaves the ether and ester bonds in lignin-carbohydrate complexes, and the ester and carbon-to-carbon bonds in lignin molecules, while Ca(OH)2 pretreatment mainly removes acetyl groups (Kim et al., 2016). NaOH is one of the strongest alkalis for effective pretreatment to attain higher enzymatic hydrolysis of cellulose than other alkalis, and has a moderate cost in comparison with potassium hydroxide (KOH) and
2
Ca(OH)2 (Kim et al., 2016; Rodrigues et al., 2016). Ca(OH) 2 has the lowest cost and can be recovered in the form of calcium carbonate (CaCO3) by injecting CO2 into the solution (Park et al., 2010; Rodrigues et al., 2016). Ca(OH) 2 pretreatment is safer to operate than NaOH pretreatment due to its weaker caustic feature. Compared with NaOH pretreatment, the enzymatic hydrolysis efficiencies of sweet sorghum bagasse and catalpa sawdust treated by Ca(OH)2 were higher (Yan et al., 2015; Jin et al., 2016), while those of Ca(OH)2-treated switchgrass and miscanthus were lower (Rodrigues et al., 2016). Namely, it was not sure whether NaOH or Ca(OH)2 pretreatment was the optimum option for maximally enhancing the enzymatic hydrolysis of a certain lignocellulose. This study compared the enzymatic hydrolysis of sugarcane bagasse (SCB) treated by NaOH and Ca(OH)2 solutions at the same condition. In addition, to reduce the pollution from the alkaline pretreatment liquor has been still the big challenge for the industrialization of alkaline pretreatment (Wang et al., 2016). The calcium chloride (CaCl2) addition which is simple operation and low cost was used to flocculate lignin from the two alkaline pretreatment liquors at the room temperature. The impact of CaCl2 loading on the lignin flocculation was evaluated. 2. Materials and methods 2.1. Materials SCB, the pre-dried residues provided by Fenghao Alcohol Co., Ltd. (China), was milled and screened out with 40-60 mesh sifter. After being washed for several times, the sifted SCB was oven-dried at 60 ºC to a constant mass. NaOH was respectively
3
solved in pure water and 70% glycerol to form 2% (W/V) NaOH-water and NaOH-glycerol solutions, and so did 2% Ca(OH)2-water and Ca(OH)2-glycerol solutions. The cellulase of 191.7 FPU/g powder containing 107.5 U xylanase was purchased from Imperial Jade Bio-technology Co. Ltd. (China). NaOH, Ca(OH)2, glycerol and CaCl2 were the analytical reagents. 2.2. Alkaline pretreatment SCB was respectively treated in the above alkaline water and glycerol solutions at 80 ºC for 2 h with liquid-to-solid ratio of 10:1 (Wang et al., 2016). The solid residue was separated with the pretreatment liquor through filtering and manually squeezing. After being thoroughly washed with tap water to a neutral pH, the solid residue was dried at 60 ºC to a constant mass. In addition, SCB treated in 70% glycerol at 80 ºC for 2 h was used as the control. 2.3. Enzymatic hydrolysis The pretreated SCB of 1 g was added into 100 mL conical flask containing 10 mL 0.05M acetate buffer (pH 4.8). The cellulase was loaded as 20 FPU/g cellulose. The enzymatic hydrolysis was conducted at 50 ºC, 150 rpm for 72 h. 2.4. Lignin recovery The pretreatment liquor of 5 mL was transferred into a preweighted 10 mL EP tube. The CaCl2 loadings of 0.01, 0.15, 0.25, 0.35, 0.45 and 0.5 g were respectively added into the tubes. After being shaken for a while at room temperature, the tubes were centrifuged at 10000 rpm for 5 min. The supernatants were transferred into the new
4
preweighted 10 mL EP tubes, and the precipitates were oven-dried at 60 ºC to a constant mass. The unreacted CaCl2 in the supernatants were measured by the mass of CaCO3 sediment with the addition of excess Na2CO3. The lignin recovery was calculated as the following: ignin reco ery
g
.
g
g
where 0.36 is the molecular mass ratio of calcium ion (Ca2+) to CaCl2. 2.5. Analytic methods The compositional analysis was performed according to the NREL method (Sluiter et al., 2008). The activities of cellulase and xylanase were assayed by the dinitrosalicylic acid (DNS) methods (Ghose, 1987; Ghose and Bisaria, 1987). The xylanase activity was defined as the amount (mg) of xylose released from xylan in 1 min by 1 g enzyme powder. The sugar concentrations of the hydrolysate were determined at 50 ºC by the high performance liquid chromatograph (HPLC) system (Waters 2698, USA) equipped with SH1011 column (Shodex). The mobile phase was 5 mM H2SO4 at the flow rate of 0.5 mL/min. The glycan conversion was calculated as the described equation (Wang et al., 2017) which was defined as the ratio of the mass of cellobiose, glucose and xylose detected in the hydrolysate to their theoretical release from glucan and xylan. 3. Results and discussion 3.1 Compositional analysis The 70% glycerol which was the optimum loading to remove lignin at 230 ºC for 1 h was selected (Chen, 2010). The compositions of raw and treated SCB were shown in
5
Table 1. The NaOH solutions exhibited higher capability to remove lignin than the Ca(OH)2 solutions. NaOH can be dissolved completely in the water and 70% glycerol, while Ca(OH)2 can be hardly dissolved in the water and form turbid liquid in 70% glycerol. The complete solubility of NaOH endows the stronger alkalinity of NaOH solutions to degrade lignin (Rodrigues et al., 2016). The degradation of xylan and lignin in the NaOH-water solution was more than those in NaOH-glycerol solution, while the opposite results appeared in Ca(OH)2-water and Ca(OH)2-glycerol solutions (Table 1). It is well known that the glycerol has a much higher viscosity than water, which can impede mass transfer and thus hinder the reaction between NaOH and lignin. While the glycerol has the ability to increase the solubility of Ca(OH) 2 (Sanderson et al., 1987), which can increase the alkalinity of Ca(OH)2-glycerol solution. Since 70% glycerol hardly influenced the composition of SCB at 80 ºC for 2 h, it could be inferred that the slight incremental removal of xylan and lignin in Ca(OH)2-glycerol should be ascribed to its alkalinity improvement. 3.2 Enzymatic hydrolysis The enzymatic hydrolysis of treated SCB was shown in Fig. 1. SCB treated in NaOH-water solution obtained the highest sugar concentration and glycan conversion, while SCB treated in 70% glycerol got the lowest. Corresponding to the compositional analysis, SCB treated in NaOH solutions were hydrolyzed more effectively than those treated in Ca(OH)2 solutions. It should be ascribed to the larger removal of lignin in NaOH solutions because of the hindrance of lignin on the access of cellulase to
6
cellulose (Rahikainen et al., 2013a and b). Compared with SCB treated in alkali-water solutions, the enzymatic hydrolysis efficiency of NaOH-glycerol treated SCB decreased, while that of Ca(OH)2-glycerol treated SCB increased. The interaction between alkalis and glycerol should be explored further. The optimization of Ca(OH)2-glycerol pretreatment condition would be worth to carry out because the operation of Ca(OH)2 pretreatment is relative low-cost and safe, and the glycerol can be recycled from the byproducts in the biodiesel production process. 3.3 Lignin recovery Lignin is the chief component released from lignocellulose in the alkaline pretreatment liquors (Kim et al., 2016). CaCl2 has the potential to flocculate the lignin from the alkaline pretreatment liquors (Ge et al., 2003; Piazza et al., 2017). According to Fig. 1, the lower glycan conversion of NaOH-glycerol treated SCB than that of NaOH-water treated SCB indicated the impractical application of NaOH-glycerol pretreatment. The flocculation of lignin from NaOH-glycerol pretreatment liquor was not investigated. After CaCl2 was added into the alkali-water pretreatment liquors, lignin could be flocculated from NaOH-water pretreatment liquor, but not from Ca(OH)2-water pretreatment liquor. Calcium ion (Ca2+) could replace the sodium ion (Na+) to bridge the negative charges of lignin components to flocculate lignin (Ge et al., 2003; Piazza et al., 2017). As for the Ca(OH)2-water pretreatment liquor, CaCl2 could not play the same role as for the NaOH pretreatment liquor due to no ionic replacement between Ca2+ and lignin-Ca complex formed in the pretreatment process (Yan et al.,
7
2015). For the same reason, the flocculation of lignin from Ca(OH) 2-glycerol pretreatment liquor was not explored. After centrifugation, the solid and liquid parts were obtained from the NaOH-water pretreatment liquor. The maximum lignin recovery of NaOH pretreatment liquor could be reached when the CaCl2 loading was 9%, and the pH value of the supernatant had an inappreciable decrease with the addition of CaCl2 increasing and kept around 12 (Fig. 2). The lignin sediment could be further purified to remove polysaccharides through organic solvent extraction and/or polysaccharidase enzymes hydrolysis. The supernatant obtained at 9% CaCl2 loading was reused to treat SCB under the condition of liquid-to-solid ratio of 10:1, 80 ºC for 2 h. The recovery of the solid residue was 91.1%, which was similar to 2% Ca(OH)2-water treated SCB. Maybe some reagents like glycerol should be added into the supernatant to improve its pretreatment efficiency. 4. Conclusions NaOH pretreatment was more suitable for treating SCB than Ca(OH) 2 pretreatment due to its advantage for more significant enzymatic hydrolysis efficiency and easier lignin recovery. Glycerol might be an optional promotor for Ca(OH) 2 pretreatment to improve the effective enzymatic saccharification of SCB. Acknowledgements The authors greatly appreciate the Guangdong Public Welfare Research and Capacity Building Project (2016A020221025), the National Natural Science Foundation of China (21506216), the Project Funded by Engineering Technology Center of Foshan
8
City (2014GA000355), the Engineering Project for Innovating and for Enhancing Universities of Guangdong Province (2016GCZX008), and the Key Platform Financing Programs from the Education Department of Guangdong Province (gg041002). References 1. Akhtar, N., Gupta, K., Goyal, D., Goyal, A. 2016. Recent advances in pretreatment technologies for efficient hydrolysis of lignocellulosic biomass. Environ. Prog. Sustain. Energy. 35, 489-511. 2. Chen, X. 2010. Studies on pretreamtent of wheat straw by glycerol. Dissertation, Jiangnan University. 3. Ge, J., Xu, M., Wang, S. 2003. Pretreatment of soda straw pulp black-liquor with calcium chloride. Industrial Water Treatment. 23, 37-39. 4. Ghose, T.K. 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257-268. 5. Ghose, T.K., Bisaria, V.S. 1987. Measurement of hemicellulase activities: I-xylanase. Pure Appl. Chem. 59, 1739-1752. 6. Jin, S., Zhang, G., Zhang, P., Li, F., Fan, S., Li, J. 2016. Thermo-chemical pretreatment and enzymatic hydrolysis for enhancing saccharification of catalpa sawdust. Bioresour. Technol. 205, 34-39. 7. Kim, J.S., Lee, Y.Y., Kim, T.H. 2016. A review on alkaline pretreatment technology for bioconversion of lignocellulosic biomass. Bioresour. Technol. 199, 42-48.
9
8. Li, Y., Qi, B., Luo, J., Wan, Y. 2015. Alkali recycling from rice straw hydrolyzate by ultrafiltration: fouling mechanism and pretreatment efficiency. Ind. Eng. Chem. Res. 54, 7925-7932. 9. Park, J.Y., Shiroma, R., Al-Haq, M.I., Zhang, Y., Ike, M., Arai-Sanoh, Y., Ida, A., Kondo, M., Tokuyasu, K. 2010. A novel lime pretreatment for subsequent bioethanol production from rice straw - Calcium capturing by carbonation (CaCCO) process. Bioresour. Technol. 101, 6805-6811. 10. Piazza, G.J., Lora, J.H., Garcia, R.A. 2017. Flocculation of wheat straw soda lignin by hemoglobin and chicken blood: effects of cationic polymer or calcium chloride. J. Chem. Technol. Biotechnol. 92, 793-800. 11. Rahikainen, J.L., Evans, J.D., Mikander, S., Kalliola, A., Puranen, T., Tamminen, T., Marjamaa, K., Kruus, K. 2013a. Cellulase-lignin interactions—The role of carbohydrate-binding module and pH in non-productive binding. Enzyme Microb. Technol. 53, 315-321. 12. Rahikainen, J.L., Martin-Sampedro, R., Heikkinen, H., Rovio, S., Marjamaa, K., Tamminen, T., Rojas, O.J., Kruus, K. 2013b. Inhibitory effect of lignin during cellulose bioconversion—The effect of lignin chemistry on non-productive enzyme adsorption. Bioresour. Technol. 133, 270-278. 13. Rodrigues, C.I.S., Jackson, J.J., Montross, M.D. 2016. A molar basis comparison of calcium hydroxide, sodium hydroxide, and potassium hydroxide on the pretreatment of switchgrass and miscanthus under high solids conditions. Ind.
10
Crop. Prod. 92, 165-173. 14. Sanderson, J.R., Smith, W.A., Marquis, E.T., Keating, K.P. 1987. Purification of propylene oxide by treatment with calcium hydroxide in glycerol or sugar water. US Patent 4691034. 15. Silveira, M.H.L., Morais, A.R.C., Lopes, A.M.D., Olekszyszen, D.N., Bogel-Lukasik, R., Andreaus, J., Ramos, L.P. 2015. Current pretreatment technologies for the development of cellulosic ethanol and biorefineries. ChemSusChem, 8, 3366-3390. 16. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D. 2008. Determination of structural carbohydrates and lignin in biomass. Technical Report NREL/TP-510-42618. 17. Wang, W., Chen, X., Tan, X., Wang, Q., Liu, Y., He, M., Yu, Q., Qi, W., Luo, Y., Zhuang, X., Yuan, Z. 2017. Feasibility of reusing the black liquor for enzymatic hydrolysis and ethanol fermentation. Bioresour. Technol. 228, 235-240. 18. Wang, W., Wang, Q., Tan, X., Qi, W., Yu, Q., Zhou, G., Zhuang, X., Yuan, Z. 2016. High conversion of sugarcane bagasse into monosaccharides based on sodium hydroxide pretreatment at low water consumption and wastewater generation. Bioresour. Technol. 218, 1230-1236. 19. Yan, Z., Li, J., Chang, S., Cui, T., Jiang, Y., Yu, M., Zhang, L., Zhao, G., Qi, P., Li, S. 2015. Lignin relocation contributed to the alkaline pretreatment efficiency of sweet sorghum bagasse. Fuel. 158, 152-158.
11
Table 1 Compositions of the raw and treated SCB. Fig. 1 Enzymatic hydrolysis of treated SCB at 50 ºC, 150 rpm for 72 h with cellulase loading of 20 FPU/g cellulose. Fig. 2 pH value changes and lignin recoveries from NaOH-water pretreatment liquor with CaCl2 loading increasing.
12
Table 1 SCB Raw 2% NaOH-water 2% Ca(OH)2-water 2% NaOH-glycerol 2% Ca(OH)2-glycerol 70% glycerol
Solid yield (%) 73.2 90.4 78.7 90.0 99.9
Glucan (%)
Xylan (%)
Araban (%)
Klason lignin (%)
47.5±0.83 63.5±0.46 51.4±0.37 57.7±0.40 52.5±1.93 46.5±0.05
27.2±0.38 29.0±0.81 27.3±0.00 30.5±0.82 27.4±0.06 26.4±0.10
5.1±0.04 5.4±0.31 5.3±0.01 5.4±0.04 5.2±0.00 5.0±0.09
23.4±0.37 9.7±0.10 18.7±0.20 11.0±0.00 18.3±0.05 22.6±0.12
13
Fig. 1 75
Cellobiose Arabiose
Glucose Xylose Glycan conversion
70 65
55 50
60
Sugar concentration (g/L)
60
45
55 50
40
45
35
40
30
35 25
30 25
20
20
15
15
10
10
5
5 0
NaOH-water Ca(OH) -water 2
NaOH-glycerol Ca(OH)2-glycerol
Treated SCB
14
Glycerol
0
Glycan conversion (%)
80
Fig. 2 24
Lignin recovery pH value
Lignin recovery (%) & pH value
22 20 18 16 14 12 10 8 6 4 2 0
0
1%
3%
5%
7%
CaCl2 loadings (g/mL)
15
9%
11%
13%
Highlights
NaOH solution was superior to Ca(OH)2 solution to pretreat sugarcane bagasse.
Glycerol could improve Ca(OH)2 pretreatment, but weaken NaOH pretreatment.
Lignin in NaOH pretreatment liquor could be mostly recovered by CaCl2 at mild condition.
CaCl2 could not recover lignin from Ca(OH)2 pretreatment liquor.
16
S0960-8524(17)31406-2 http://dx.doi.org/10.1016/j.biortech.2017.08.101 BITE 18710
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
14 July 2017 14 August 2017 16 August 2017
Please cite this article as: Chang, M., Li, D., Wang, W., Chen, D., Zhang, Y., Hu, H., Ye, X., Comparison of sodium hydroxide and calcium hydroxide pretreatments on the enzymatic hydrolysis and lignin recovery of sugarcane bagasse, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.08.101
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Comparison of sodium hydroxide and calcium hydroxide pretreatments on the enzymatic hydrolysis and lignin recovery of sugarcane bagasse Menglei Changa, Denian Lib, Wen Wangb,* Dongchu Chena, Yuyuan Zhanga, Huawen Hua, Xiufang Yea a
College of Materials Science and Energy Engineering, Foshan University, Foshan
528000, Guangdong, P. R. China b
Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences; CAS Key
Laboratory of Renewable Energy; Guangdong Provincial Key Laboratory of New and Renewable Energy Research and Development; Guangzhou 510640, Guangdong, P. R. China Abstract: Sodium hydroxide (NaOH) and calcium hydroxide (Ca(OH) 2) respectively dissolved in water and 70% glycerol were applied to treat sugarcane bagasse (SCB) under the condition of 80 ºC for 2 h. NaOH solutions could remove more lignin and obtain higher enzymatic hydrolysis efficiency of SCB than Ca(OH) 2 solutions. Compared with the alkali-water solutions, the enzymatic hydrolysis of SCB treated in NaOH-glycerol solution decreased, while that in Ca(OH) 2-glycerol solution increased. The lignin in NaOH-water pretreatment liquor could be easily recovered by calcium chloride (CaCl2) at room temperature, but that in Ca(OH)2-water pretreatment liquor couldn’t. NaOH pretreatment is more suitable for facilitating enzymatic hydrolysis and lignin recovery of SCB than Ca(OH)2 pretreatment.
*
Corresponding author, Email: [email protected]; Tel.: 86-20-37029703 1
Keywords: Sugarcane bagasse; Alkaline pretreatment; Calcium chloride; Enzymatic hydrolysis; Lignin recovery 1. Introduction Pretreatment is a fundamental step for refining biofuels and bio-based products from the lignocellulosic biomass as the bioconversion of cellulose into fermentable monosaccharides (Akhtar et al., 2016). It breaks up the tight structure of cell wall formed by cellulose, hemicellulose and lignin to improve the enzymatic saccharification efficiency of cellulose and/or hemicellulose (Silveira et al., 2015). Alkaline pretreatment, a kind of pretreatment methods, possesses some desirable features. It mainly removes lignin and can be carried out at milder condition than several other pretreatments like hydrothermal pretreatments, etc. (Kim et al., 2016). The hazard of the alkaline pretreatment liquor to environment could be relieved with the development of several technologies such as adsorption, precipitation, ultrafiltration, bioaugmentation, electrocoagulation and electrolysis (Li et al., 2015). Sodium hydroxide (NaOH) and calcium hydroxide (Ca(OH) 2) are the common reagents to be used for alkaline pretreatment. NaOH pretreatment particularly cleaves the ether and ester bonds in lignin-carbohydrate complexes, and the ester and carbon-to-carbon bonds in lignin molecules, while Ca(OH)2 pretreatment mainly removes acetyl groups (Kim et al., 2016). NaOH is one of the strongest alkalis for effective pretreatment to attain higher enzymatic hydrolysis of cellulose than other alkalis, and has a moderate cost in comparison with potassium hydroxide (KOH) and
2
Ca(OH)2 (Kim et al., 2016; Rodrigues et al., 2016). Ca(OH) 2 has the lowest cost and can be recovered in the form of calcium carbonate (CaCO3) by injecting CO2 into the solution (Park et al., 2010; Rodrigues et al., 2016). Ca(OH) 2 pretreatment is safer to operate than NaOH pretreatment due to its weaker caustic feature. Compared with NaOH pretreatment, the enzymatic hydrolysis efficiencies of sweet sorghum bagasse and catalpa sawdust treated by Ca(OH)2 were higher (Yan et al., 2015; Jin et al., 2016), while those of Ca(OH)2-treated switchgrass and miscanthus were lower (Rodrigues et al., 2016). Namely, it was not sure whether NaOH or Ca(OH)2 pretreatment was the optimum option for maximally enhancing the enzymatic hydrolysis of a certain lignocellulose. This study compared the enzymatic hydrolysis of sugarcane bagasse (SCB) treated by NaOH and Ca(OH)2 solutions at the same condition. In addition, to reduce the pollution from the alkaline pretreatment liquor has been still the big challenge for the industrialization of alkaline pretreatment (Wang et al., 2016). The calcium chloride (CaCl2) addition which is simple operation and low cost was used to flocculate lignin from the two alkaline pretreatment liquors at the room temperature. The impact of CaCl2 loading on the lignin flocculation was evaluated. 2. Materials and methods 2.1. Materials SCB, the pre-dried residues provided by Fenghao Alcohol Co., Ltd. (China), was milled and screened out with 40-60 mesh sifter. After being washed for several times, the sifted SCB was oven-dried at 60 ºC to a constant mass. NaOH was respectively
3
solved in pure water and 70% glycerol to form 2% (W/V) NaOH-water and NaOH-glycerol solutions, and so did 2% Ca(OH)2-water and Ca(OH)2-glycerol solutions. The cellulase of 191.7 FPU/g powder containing 107.5 U xylanase was purchased from Imperial Jade Bio-technology Co. Ltd. (China). NaOH, Ca(OH)2, glycerol and CaCl2 were the analytical reagents. 2.2. Alkaline pretreatment SCB was respectively treated in the above alkaline water and glycerol solutions at 80 ºC for 2 h with liquid-to-solid ratio of 10:1 (Wang et al., 2016). The solid residue was separated with the pretreatment liquor through filtering and manually squeezing. After being thoroughly washed with tap water to a neutral pH, the solid residue was dried at 60 ºC to a constant mass. In addition, SCB treated in 70% glycerol at 80 ºC for 2 h was used as the control. 2.3. Enzymatic hydrolysis The pretreated SCB of 1 g was added into 100 mL conical flask containing 10 mL 0.05M acetate buffer (pH 4.8). The cellulase was loaded as 20 FPU/g cellulose. The enzymatic hydrolysis was conducted at 50 ºC, 150 rpm for 72 h. 2.4. Lignin recovery The pretreatment liquor of 5 mL was transferred into a preweighted 10 mL EP tube. The CaCl2 loadings of 0.01, 0.15, 0.25, 0.35, 0.45 and 0.5 g were respectively added into the tubes. After being shaken for a while at room temperature, the tubes were centrifuged at 10000 rpm for 5 min. The supernatants were transferred into the new
4
preweighted 10 mL EP tubes, and the precipitates were oven-dried at 60 ºC to a constant mass. The unreacted CaCl2 in the supernatants were measured by the mass of CaCO3 sediment with the addition of excess Na2CO3. The lignin recovery was calculated as the following: ignin reco ery
g
.
g
g
where 0.36 is the molecular mass ratio of calcium ion (Ca2+) to CaCl2. 2.5. Analytic methods The compositional analysis was performed according to the NREL method (Sluiter et al., 2008). The activities of cellulase and xylanase were assayed by the dinitrosalicylic acid (DNS) methods (Ghose, 1987; Ghose and Bisaria, 1987). The xylanase activity was defined as the amount (mg) of xylose released from xylan in 1 min by 1 g enzyme powder. The sugar concentrations of the hydrolysate were determined at 50 ºC by the high performance liquid chromatograph (HPLC) system (Waters 2698, USA) equipped with SH1011 column (Shodex). The mobile phase was 5 mM H2SO4 at the flow rate of 0.5 mL/min. The glycan conversion was calculated as the described equation (Wang et al., 2017) which was defined as the ratio of the mass of cellobiose, glucose and xylose detected in the hydrolysate to their theoretical release from glucan and xylan. 3. Results and discussion 3.1 Compositional analysis The 70% glycerol which was the optimum loading to remove lignin at 230 ºC for 1 h was selected (Chen, 2010). The compositions of raw and treated SCB were shown in
5
Table 1. The NaOH solutions exhibited higher capability to remove lignin than the Ca(OH)2 solutions. NaOH can be dissolved completely in the water and 70% glycerol, while Ca(OH)2 can be hardly dissolved in the water and form turbid liquid in 70% glycerol. The complete solubility of NaOH endows the stronger alkalinity of NaOH solutions to degrade lignin (Rodrigues et al., 2016). The degradation of xylan and lignin in the NaOH-water solution was more than those in NaOH-glycerol solution, while the opposite results appeared in Ca(OH)2-water and Ca(OH)2-glycerol solutions (Table 1). It is well known that the glycerol has a much higher viscosity than water, which can impede mass transfer and thus hinder the reaction between NaOH and lignin. While the glycerol has the ability to increase the solubility of Ca(OH) 2 (Sanderson et al., 1987), which can increase the alkalinity of Ca(OH)2-glycerol solution. Since 70% glycerol hardly influenced the composition of SCB at 80 ºC for 2 h, it could be inferred that the slight incremental removal of xylan and lignin in Ca(OH)2-glycerol should be ascribed to its alkalinity improvement. 3.2 Enzymatic hydrolysis The enzymatic hydrolysis of treated SCB was shown in Fig. 1. SCB treated in NaOH-water solution obtained the highest sugar concentration and glycan conversion, while SCB treated in 70% glycerol got the lowest. Corresponding to the compositional analysis, SCB treated in NaOH solutions were hydrolyzed more effectively than those treated in Ca(OH)2 solutions. It should be ascribed to the larger removal of lignin in NaOH solutions because of the hindrance of lignin on the access of cellulase to
6
cellulose (Rahikainen et al., 2013a and b). Compared with SCB treated in alkali-water solutions, the enzymatic hydrolysis efficiency of NaOH-glycerol treated SCB decreased, while that of Ca(OH)2-glycerol treated SCB increased. The interaction between alkalis and glycerol should be explored further. The optimization of Ca(OH)2-glycerol pretreatment condition would be worth to carry out because the operation of Ca(OH)2 pretreatment is relative low-cost and safe, and the glycerol can be recycled from the byproducts in the biodiesel production process. 3.3 Lignin recovery Lignin is the chief component released from lignocellulose in the alkaline pretreatment liquors (Kim et al., 2016). CaCl2 has the potential to flocculate the lignin from the alkaline pretreatment liquors (Ge et al., 2003; Piazza et al., 2017). According to Fig. 1, the lower glycan conversion of NaOH-glycerol treated SCB than that of NaOH-water treated SCB indicated the impractical application of NaOH-glycerol pretreatment. The flocculation of lignin from NaOH-glycerol pretreatment liquor was not investigated. After CaCl2 was added into the alkali-water pretreatment liquors, lignin could be flocculated from NaOH-water pretreatment liquor, but not from Ca(OH)2-water pretreatment liquor. Calcium ion (Ca2+) could replace the sodium ion (Na+) to bridge the negative charges of lignin components to flocculate lignin (Ge et al., 2003; Piazza et al., 2017). As for the Ca(OH)2-water pretreatment liquor, CaCl2 could not play the same role as for the NaOH pretreatment liquor due to no ionic replacement between Ca2+ and lignin-Ca complex formed in the pretreatment process (Yan et al.,
7
2015). For the same reason, the flocculation of lignin from Ca(OH) 2-glycerol pretreatment liquor was not explored. After centrifugation, the solid and liquid parts were obtained from the NaOH-water pretreatment liquor. The maximum lignin recovery of NaOH pretreatment liquor could be reached when the CaCl2 loading was 9%, and the pH value of the supernatant had an inappreciable decrease with the addition of CaCl2 increasing and kept around 12 (Fig. 2). The lignin sediment could be further purified to remove polysaccharides through organic solvent extraction and/or polysaccharidase enzymes hydrolysis. The supernatant obtained at 9% CaCl2 loading was reused to treat SCB under the condition of liquid-to-solid ratio of 10:1, 80 ºC for 2 h. The recovery of the solid residue was 91.1%, which was similar to 2% Ca(OH)2-water treated SCB. Maybe some reagents like glycerol should be added into the supernatant to improve its pretreatment efficiency. 4. Conclusions NaOH pretreatment was more suitable for treating SCB than Ca(OH) 2 pretreatment due to its advantage for more significant enzymatic hydrolysis efficiency and easier lignin recovery. Glycerol might be an optional promotor for Ca(OH) 2 pretreatment to improve the effective enzymatic saccharification of SCB. Acknowledgements The authors greatly appreciate the Guangdong Public Welfare Research and Capacity Building Project (2016A020221025), the National Natural Science Foundation of China (21506216), the Project Funded by Engineering Technology Center of Foshan
8
City (2014GA000355), the Engineering Project for Innovating and for Enhancing Universities of Guangdong Province (2016GCZX008), and the Key Platform Financing Programs from the Education Department of Guangdong Province (gg041002). References 1. Akhtar, N., Gupta, K., Goyal, D., Goyal, A. 2016. Recent advances in pretreatment technologies for efficient hydrolysis of lignocellulosic biomass. Environ. Prog. Sustain. Energy. 35, 489-511. 2. Chen, X. 2010. Studies on pretreamtent of wheat straw by glycerol. Dissertation, Jiangnan University. 3. Ge, J., Xu, M., Wang, S. 2003. Pretreatment of soda straw pulp black-liquor with calcium chloride. Industrial Water Treatment. 23, 37-39. 4. Ghose, T.K. 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257-268. 5. Ghose, T.K., Bisaria, V.S. 1987. Measurement of hemicellulase activities: I-xylanase. Pure Appl. Chem. 59, 1739-1752. 6. Jin, S., Zhang, G., Zhang, P., Li, F., Fan, S., Li, J. 2016. Thermo-chemical pretreatment and enzymatic hydrolysis for enhancing saccharification of catalpa sawdust. Bioresour. Technol. 205, 34-39. 7. Kim, J.S., Lee, Y.Y., Kim, T.H. 2016. A review on alkaline pretreatment technology for bioconversion of lignocellulosic biomass. Bioresour. Technol. 199, 42-48.
9
8. Li, Y., Qi, B., Luo, J., Wan, Y. 2015. Alkali recycling from rice straw hydrolyzate by ultrafiltration: fouling mechanism and pretreatment efficiency. Ind. Eng. Chem. Res. 54, 7925-7932. 9. Park, J.Y., Shiroma, R., Al-Haq, M.I., Zhang, Y., Ike, M., Arai-Sanoh, Y., Ida, A., Kondo, M., Tokuyasu, K. 2010. A novel lime pretreatment for subsequent bioethanol production from rice straw - Calcium capturing by carbonation (CaCCO) process. Bioresour. Technol. 101, 6805-6811. 10. Piazza, G.J., Lora, J.H., Garcia, R.A. 2017. Flocculation of wheat straw soda lignin by hemoglobin and chicken blood: effects of cationic polymer or calcium chloride. J. Chem. Technol. Biotechnol. 92, 793-800. 11. Rahikainen, J.L., Evans, J.D., Mikander, S., Kalliola, A., Puranen, T., Tamminen, T., Marjamaa, K., Kruus, K. 2013a. Cellulase-lignin interactions—The role of carbohydrate-binding module and pH in non-productive binding. Enzyme Microb. Technol. 53, 315-321. 12. Rahikainen, J.L., Martin-Sampedro, R., Heikkinen, H., Rovio, S., Marjamaa, K., Tamminen, T., Rojas, O.J., Kruus, K. 2013b. Inhibitory effect of lignin during cellulose bioconversion—The effect of lignin chemistry on non-productive enzyme adsorption. Bioresour. Technol. 133, 270-278. 13. Rodrigues, C.I.S., Jackson, J.J., Montross, M.D. 2016. A molar basis comparison of calcium hydroxide, sodium hydroxide, and potassium hydroxide on the pretreatment of switchgrass and miscanthus under high solids conditions. Ind.
10
Crop. Prod. 92, 165-173. 14. Sanderson, J.R., Smith, W.A., Marquis, E.T., Keating, K.P. 1987. Purification of propylene oxide by treatment with calcium hydroxide in glycerol or sugar water. US Patent 4691034. 15. Silveira, M.H.L., Morais, A.R.C., Lopes, A.M.D., Olekszyszen, D.N., Bogel-Lukasik, R., Andreaus, J., Ramos, L.P. 2015. Current pretreatment technologies for the development of cellulosic ethanol and biorefineries. ChemSusChem, 8, 3366-3390. 16. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D. 2008. Determination of structural carbohydrates and lignin in biomass. Technical Report NREL/TP-510-42618. 17. Wang, W., Chen, X., Tan, X., Wang, Q., Liu, Y., He, M., Yu, Q., Qi, W., Luo, Y., Zhuang, X., Yuan, Z. 2017. Feasibility of reusing the black liquor for enzymatic hydrolysis and ethanol fermentation. Bioresour. Technol. 228, 235-240. 18. Wang, W., Wang, Q., Tan, X., Qi, W., Yu, Q., Zhou, G., Zhuang, X., Yuan, Z. 2016. High conversion of sugarcane bagasse into monosaccharides based on sodium hydroxide pretreatment at low water consumption and wastewater generation. Bioresour. Technol. 218, 1230-1236. 19. Yan, Z., Li, J., Chang, S., Cui, T., Jiang, Y., Yu, M., Zhang, L., Zhao, G., Qi, P., Li, S. 2015. Lignin relocation contributed to the alkaline pretreatment efficiency of sweet sorghum bagasse. Fuel. 158, 152-158.
11
Table 1 Compositions of the raw and treated SCB. Fig. 1 Enzymatic hydrolysis of treated SCB at 50 ºC, 150 rpm for 72 h with cellulase loading of 20 FPU/g cellulose. Fig. 2 pH value changes and lignin recoveries from NaOH-water pretreatment liquor with CaCl2 loading increasing.
12
Table 1 SCB Raw 2% NaOH-water 2% Ca(OH)2-water 2% NaOH-glycerol 2% Ca(OH)2-glycerol 70% glycerol
Solid yield (%) 73.2 90.4 78.7 90.0 99.9
Glucan (%)
Xylan (%)
Araban (%)
Klason lignin (%)
47.5±0.83 63.5±0.46 51.4±0.37 57.7±0.40 52.5±1.93 46.5±0.05
27.2±0.38 29.0±0.81 27.3±0.00 30.5±0.82 27.4±0.06 26.4±0.10
5.1±0.04 5.4±0.31 5.3±0.01 5.4±0.04 5.2±0.00 5.0±0.09
23.4±0.37 9.7±0.10 18.7±0.20 11.0±0.00 18.3±0.05 22.6±0.12
13
Fig. 1 75
Cellobiose Arabiose
Glucose Xylose Glycan conversion
70 65
55 50
60
Sugar concentration (g/L)
60
45
55 50
40
45
35
40
30
35 25
30 25
20
20
15
15
10
10
5
5 0
NaOH-water Ca(OH) -water 2
NaOH-glycerol Ca(OH)2-glycerol
Treated SCB
14
Glycerol
0
Glycan conversion (%)
80
Fig. 2 24
Lignin recovery pH value
Lignin recovery (%) & pH value
22 20 18 16 14 12 10 8 6 4 2 0
0
1%
3%
5%
7%
CaCl2 loadings (g/mL)
15
9%
11%
13%
Highlights
NaOH solution was superior to Ca(OH)2 solution to pretreat sugarcane bagasse.
Glycerol could improve Ca(OH)2 pretreatment, but weaken NaOH pretreatment.
Lignin in NaOH pretreatment liquor could be mostly recovered by CaCl2 at mild condition.
CaCl2 could not recover lignin from Ca(OH)2 pretreatment liquor.
16