乙醇预处理对芦竹细胞壁的影响及荧光可视化分析
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摘要
为探究NaOH-乙醇预处理过程中NaOH浓度变化对芦竹纤维表面结构、细胞壁区域化学成分以及酶解效果的影响,采用NaOH质量浓度分别为2.5,5.0,10.0 g/L的NaOH-乙醇溶液和10.0 g/L的NaOH水溶液于90℃水浴条件下,分别对芦竹粉末和切片预处理2 h。结果表明:当碱质量浓度为10.0 g/L时,NaOH-乙醇预处理后木质素和木聚糖脱除率达到最大值,分别为47.11%和35.12%,芦竹酶解葡萄糖得率和木糖得率达到49.41%和77.61%,分别是未处理样品的6.2倍和7.4倍。场发射扫描电镜观察显示,NaOH-乙醇预处理后,芦竹纤维细胞壁表面微纤丝暴露。预处理过程的荧光显微镜跟踪观察表明,木质素的脱除均由薄壁细胞开始,逐渐向与之靠近的厚壁纤维过渡,最后到维管束内部的厚壁纤维,细胞角隅的木质素相对较难脱除,预处理后仍显示较明显的木质素信号; NaOH-乙醇溶液预处理后的切片整体木质素自发荧光现象减弱,细胞壁中木质素相对浓度下降,对酶解葡萄糖和木糖得率的提高都起到促进作用。
Using enzymes,such as cellulases,the hydrolysis of lignocellulosic biomass has been considered as one of the eco-friendly methods to produce bioethanol in bioconversion. However,for the enzymatic hydrolysis,the biomass materials should be pretreated to improve hydrolysis efficiency. This is because of the intrinsic recalcitrance of cell walls in the biomass materials,which is caused by the complexity of chemical composition,multi-laminated cell-wall structure,diversity of cell types and cell combinations. Thus,an efficient pretreatment is an essential step for increasing enzymatic digestibility of the biomass. Arundo donax,the typical monocot family Gramineae,is widely cultivated in China,which has the characteristics of the high biomass yield and fast-growing for cultivation and offers great potential for bioethanol production. In this study,A. donax was pretreated by NaOH-ethanol solution. Both sections and powder of A. donax were pretreated by 2.5 g/L,5.0 g/L and 10.0 g/L NaOH-ethanol solutions,and 10.0 g/L NaOH solution at a 90 ℃ water bath and kept for 2 h. After the pretreatment,using the field emission scanning electron microscope( FE-SEM),X-ray diffraction( XRD),fluorescence light microscope( FLM) and high-performance liquid chromatography( HPLC),the surface morphology of the fibers,the topochemical changes in the cell walls and the enzymatic hydrolysis efficiency were investigated. The 10.0 g/L NaOH-ethanol was demonstrated to be the optimal condition for the pretreatment and evidenced by the maximum yield of glucose and xylose,which was 6.2 times and7.4 times of the yields using the un-treated A. donax. The results of the wet-chemical analysis and XRD examination indicated that changes of crystallinity were affected by the removal of the amorphous cellulose,hemicellulose and lignin in the cell walls together with the swelling of cellulose fibrils. FE-SEM images showed that,after the 10.0 g/L NaOH-ethanol pretreatment,more separated microfibers were exposed on the surface of the cell walls,which could increase the accessibility of the enzyme. By tracing the pretreatment process,the autofluorescence images showed that the removal of lignin among different cell types began at the parenchyma,then the sclerenchyma fibers located at the outer regions of vascular bundles and followed by the inner parts. The fluorescence signal still occurred in the cell corner after the pretreatment,indicating that the lignin in this region was relatively difficult to remove. After the NaOHethanol pretreatment,the whole autofluorescence from the sections was reduced,which suggested that the effect of the NaOH-ethanol pretreatment was more homogeneous compared with the NaOH pretreatment. Thus,the NaOH-ethanol pretreatment could be an effective method to gain fermentable sugars from A. donax.
Using enzymes,such as cellulases,the hydrolysis of lignocellulosic biomass has been considered as one of the eco-friendly methods to produce bioethanol in bioconversion. However,for the enzymatic hydrolysis,the biomass materials should be pretreated to improve hydrolysis efficiency. This is because of the intrinsic recalcitrance of cell walls in the biomass materials,which is caused by the complexity of chemical composition,multi-laminated cell-wall structure,diversity of cell types and cell combinations. Thus,an efficient pretreatment is an essential step for increasing enzymatic digestibility of the biomass. Arundo donax,the typical monocot family Gramineae,is widely cultivated in China,which has the characteristics of the high biomass yield and fast-growing for cultivation and offers great potential for bioethanol production. In this study,A. donax was pretreated by NaOH-ethanol solution. Both sections and powder of A. donax were pretreated by 2.5 g/L,5.0 g/L and 10.0 g/L NaOH-ethanol solutions,and 10.0 g/L NaOH solution at a 90 ℃ water bath and kept for 2 h. After the pretreatment,using the field emission scanning electron microscope( FE-SEM),X-ray diffraction( XRD),fluorescence light microscope( FLM) and high-performance liquid chromatography( HPLC),the surface morphology of the fibers,the topochemical changes in the cell walls and the enzymatic hydrolysis efficiency were investigated. The 10.0 g/L NaOH-ethanol was demonstrated to be the optimal condition for the pretreatment and evidenced by the maximum yield of glucose and xylose,which was 6.2 times and7.4 times of the yields using the un-treated A. donax. The results of the wet-chemical analysis and XRD examination indicated that changes of crystallinity were affected by the removal of the amorphous cellulose,hemicellulose and lignin in the cell walls together with the swelling of cellulose fibrils. FE-SEM images showed that,after the 10.0 g/L NaOH-ethanol pretreatment,more separated microfibers were exposed on the surface of the cell walls,which could increase the accessibility of the enzyme. By tracing the pretreatment process,the autofluorescence images showed that the removal of lignin among different cell types began at the parenchyma,then the sclerenchyma fibers located at the outer regions of vascular bundles and followed by the inner parts. The fluorescence signal still occurred in the cell corner after the pretreatment,indicating that the lignin in this region was relatively difficult to remove. After the NaOHethanol pretreatment,the whole autofluorescence from the sections was reduced,which suggested that the effect of the NaOH-ethanol pretreatment was more homogeneous compared with the NaOH pretreatment. Thus,the NaOH-ethanol pretreatment could be an effective method to gain fermentable sugars from A. donax.
引文
[1]余醉,李建龙,李高扬.芦竹作为清洁生物质能源牧草开发的潜力分析[J].草业科学,2009,26(6):62-69.YU Z,LI J L,LI G Y. Analysis on the potential capacity of exploiting giant reed as an energy forage[J]. Pratacultural Science,2009,26(6):62-69.
[2]YOU T T,ZHANG L M,XU F. Progressive deconstruction of Arundo donax Linn. to fermentable sugars by acid catalyzed ionic liquid pretreatment[J]. Bioresource Technology,2016,199:271-274. DOI:10.1016/j.biortech.2015.08.152.
[3]许凤,张逊,周霞,等.农林生物质预处理过程中细胞壁主要组分溶解机理研究进展[J].林业工程学报,2016,1(4):1-9. DOI:10.13360/j.issn.2096-1359.2016.04.001.XU F,ZHANG S,ZHOU X,et al. An investigation of dissolution mechanism of major components in cell walls of agricultural and forest biomass[J]. Journal of Forestry Engineering,2016,1(4):1-9.
[4]ZHANG K,PEI Z J,WANG D H. Organic solvent pretreatment of lignocellulosic biomass for biofuels and biochemicals:a review[J]. Bioresource Technology,2016,199:21-33. DOI:10.1016/j.biortech.2015.08.102.
[5]元英进,秦磊,李炳志,等.乙醇预处理对水稻秸秆物质迁移和酶解的影响[J].天津大学学报,2012,45(9):757-762.DOI:10.3969/j.issn.0493-2137.2012.09.003.YUAN Y J,QIN L,LI B Z,et al. Effects of ethanol pretreatment on chemical conversion and enzymatic hydrolysis in rice straw[J].Journal of Tianjin University,2012,45(9):757-762.
[6]周静,沈葵忠,房桂干,等.响应面优化碱醇预处理麦草酶解效率及木质素组分分离[J].食品工业科技,2018,39(14):81-86. DOI:10.13386/j.issn.1002-0306.2018.14.016.ZHOU J,SHEN K Z,FANG G G,et al. Optimization of the alkali-ethanol pretreatment of wheat straw for increasing enzymatic hydrolysis efficiency with response surface methodology and separation of lignin component[J]. Science and Technology of Food Industry,2018,39(14):81-86.
[7]YUAN W,GONG Z W,WANG G H,et al. Alkaline organosolv pretreatment of corn stover for enhancing the enzymatic digestibility[J]. Bioresource Technology,2018,265:464-470.DOI:10.1016/j.biortech.2018.06.038.
[8] JI Z,MA J F,XU F. Multi-scale visualization of dynamic changes in poplar cell walls during alkali pretreatment[J]. Microscopy and Microanalysis,2014,20(2):566-576. DOI:10.1017/s1431927614000063.
[9]NI Y,HU Q. Alcelllignin solubility in ethanol-water mixtures[J]. Journal of Applied Polymer Science,1995,57(12):1441-1446. DOI:10.1002/app.1995.070571203.
[10]SLUITER A,HAMES B,RUIZ R,et al. Determination of structural carbohydrates and lignin in biomass[R]. National Renewable Energy Laboratory,2012:1-15.
[11]李新宇,张明辉.利用X射线衍射法探究木材含水率与结晶度的关系[J].东北林业大学学报,2014,42(2):96-99.DOI:10.13759/j.cnki.dlxb.2014.02.023.LI X Y,ZHANG M H. Relationship of wood moisture content and the degree of crystallinity by X-ray diffraction[J]. Journal of Northeast Forestry University,2014,42(2):96-99.
[12]ZHAO X B,CHENG K K,LIU D H. Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis[J]. Applied Microbiology and Biotechnology,2009,82(5):815-827. DOI:10.1007/s00253-009-1883-1.
[13]周静,沈葵忠,房桂干,等.不同预处理方法对麦草化学组分及其酶解性能的影响[J].林产化学与工业,2017,37(5):53-60. DOI:10.3969/j.issn.0253-2417.2017.05.007.ZHOU J,SHEN K Z,FANG G G,et al. Effects of different pretreatment methods on chemical composition and enzymatic hydrolysis of wheat straw[J]. Chemistry and Industry of Forest Products,2017,37(5):53-60.
[14]SELIG M J,ADNEY W S,HIMMEL M E,et al. The impact of cell wall acetylation on corn stover hydrolysis by cellulolytic and xylanolytic enzymes[J]. Cellulose,2009,16(4):711-722.DOI:10.1007/s10570-009-9322-0.
[15]马建锋,杨淑敏,田根林,等.植物细胞壁木质素区域化学紫外显微光谱研究进展[J].林产化学与工业,2016,36(1):147-154. DOI:10.3969/j.issn.0253-2417.2016.01.021.MA J F,YANG S M,TIAN G L,et al. Application of UV-microspectrophotometry on lignin topochemistry in plant cell wall[J]. Chemistry and Industry of Forest Products,2016,36(1):147-154.
[16]WU X X,HUANG C X,ZHAI S C,et al. Improving enzymatic hydrolysis efficiency of wheat straw through sequential autohydrolysis and alkaline post-extraction[J]. Bioresource Technology,2018,251:374-380. DOI:10.1016/j.biortech.2017.12.066.
[17]TOBA K,YAMAMOTO H,YOSHIDA M. Crystallization of cellulose microfibrils in wood cell wall by repeated dry-and-wet treatment,using X-ray diffraction technique[J]. Cellulose,2013,20(2):633-643. DOI:10.1007/s10570-012-9853-7.
[18]LING Z,CHEN S,ZHANG X,et al. Exploring crystalline-structural variations of cellulose during alkaline pretreatment for enhanced enzymatic hydrolysis[J]. Bioresource Technology,2017,224:611-617. DOI:10.1016/j.biortech.2016.10.064.
[19]ZENG Y N,ZHAO S,WEI H,et al. In situ micro-spectroscopic investigation of lignin in poplar cell walls pretreated by maleic acid[J]. Biotechnology for Biofuels,2015,8(1):126. DOI:10.1186/s13068-015-0312-1.
[20]邓建华,管梦灵,刘美芹.高粱茎杆中木质素分布的显微技术研究[J].植物学研究,2014,3:188-194. DOI:10.12677/br.2014.35024.DENG J H,GUAN M L,LIU M Q. Study on microscopy techniques for identifying lignin distribution in the stem of sorghum[J]. Botanical Research,2014,3:188-194.
[21]DONALDSON L A. Lignification and lignin topochemistry:an ultrastructural view[J]. Phytochemistry,2001,57(6):859-873.DOI:10.1016/s0031-9422(01)00049-8.
[22]JI Z,MA J F,ZHANG Z H,et al. Distribution of lignin and cellulose in compression wood tracheids of Pinus yunnanensis determined by fluorescence microscopy and confocal Raman microscopy[J]. Industrial Crops and Products,2013,47:212-217. DOI:10.1016/j.indcrop.2013.03.006.
[23]DONOHOE B S, DECKER S R, TUCKER M P, et al.Visualizing lignin coalescence and migration through maize cell walls following thermochemical pretreatment[J]. Biotechnology and Bioengineering,2008,101(5):913-925. DOI:10.1002/bit.21959.
[24]HARRIS P J,HARTLEY R D. Detection of bound ferulic acid in cell walls of the Gramineae by ultraviolet fluorescence microscopy[J]. Nature, 1976, 259(5543):508-510. DOI:10.1038/259508a0.
[25]SIQUEIRA G,MILAGRES A M,CARVALHO W,et al. Topochemical distribution of lignin and hydroxycinnamic acids in sugar-cane cell walls and its correlation with the enzymatic hydrolysis of polysaccharides[J]. Biotechnology for Biofuels,2011,4:7. DOI:10.1186/1754-6834-4-7.
[26]徐栋梁,任浩.木质素对纤维素酶水解抑制作用的研究进展与展望[J].中华纸业,2017,38(20):19-24.XU D L,REN H. The research progress and prospect on lignin inhibition in cellulose enzyme hydrolysis[J]. China Pulp&Paper Industry,2017,38(20):19-24.
[27]杨淑娟,游艳芝,张威伟,等. Na OH-乙醇预处理提高甘蔗渣酶法制备低聚木糖效率[J].北京林业大学学报,2018,40(2):114-120. DOI:10.13332/j.1000-1522.20170366.YANG S J,YOU Y Z,ZHANG W W,et al. Na OH-ethanol pretreatment increasing preparation efficiency of xylo-oligosaccharide from sugarcane bagasse with enzymatic hydrolysis[J]. Journal of Beijing Forestry University,2018,40(2):114-120.
[2]YOU T T,ZHANG L M,XU F. Progressive deconstruction of Arundo donax Linn. to fermentable sugars by acid catalyzed ionic liquid pretreatment[J]. Bioresource Technology,2016,199:271-274. DOI:10.1016/j.biortech.2015.08.152.
[3]许凤,张逊,周霞,等.农林生物质预处理过程中细胞壁主要组分溶解机理研究进展[J].林业工程学报,2016,1(4):1-9. DOI:10.13360/j.issn.2096-1359.2016.04.001.XU F,ZHANG S,ZHOU X,et al. An investigation of dissolution mechanism of major components in cell walls of agricultural and forest biomass[J]. Journal of Forestry Engineering,2016,1(4):1-9.
[4]ZHANG K,PEI Z J,WANG D H. Organic solvent pretreatment of lignocellulosic biomass for biofuels and biochemicals:a review[J]. Bioresource Technology,2016,199:21-33. DOI:10.1016/j.biortech.2015.08.102.
[5]元英进,秦磊,李炳志,等.乙醇预处理对水稻秸秆物质迁移和酶解的影响[J].天津大学学报,2012,45(9):757-762.DOI:10.3969/j.issn.0493-2137.2012.09.003.YUAN Y J,QIN L,LI B Z,et al. Effects of ethanol pretreatment on chemical conversion and enzymatic hydrolysis in rice straw[J].Journal of Tianjin University,2012,45(9):757-762.
[6]周静,沈葵忠,房桂干,等.响应面优化碱醇预处理麦草酶解效率及木质素组分分离[J].食品工业科技,2018,39(14):81-86. DOI:10.13386/j.issn.1002-0306.2018.14.016.ZHOU J,SHEN K Z,FANG G G,et al. Optimization of the alkali-ethanol pretreatment of wheat straw for increasing enzymatic hydrolysis efficiency with response surface methodology and separation of lignin component[J]. Science and Technology of Food Industry,2018,39(14):81-86.
[7]YUAN W,GONG Z W,WANG G H,et al. Alkaline organosolv pretreatment of corn stover for enhancing the enzymatic digestibility[J]. Bioresource Technology,2018,265:464-470.DOI:10.1016/j.biortech.2018.06.038.
[8] JI Z,MA J F,XU F. Multi-scale visualization of dynamic changes in poplar cell walls during alkali pretreatment[J]. Microscopy and Microanalysis,2014,20(2):566-576. DOI:10.1017/s1431927614000063.
[9]NI Y,HU Q. Alcelllignin solubility in ethanol-water mixtures[J]. Journal of Applied Polymer Science,1995,57(12):1441-1446. DOI:10.1002/app.1995.070571203.
[10]SLUITER A,HAMES B,RUIZ R,et al. Determination of structural carbohydrates and lignin in biomass[R]. National Renewable Energy Laboratory,2012:1-15.
[11]李新宇,张明辉.利用X射线衍射法探究木材含水率与结晶度的关系[J].东北林业大学学报,2014,42(2):96-99.DOI:10.13759/j.cnki.dlxb.2014.02.023.LI X Y,ZHANG M H. Relationship of wood moisture content and the degree of crystallinity by X-ray diffraction[J]. Journal of Northeast Forestry University,2014,42(2):96-99.
[12]ZHAO X B,CHENG K K,LIU D H. Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis[J]. Applied Microbiology and Biotechnology,2009,82(5):815-827. DOI:10.1007/s00253-009-1883-1.
[13]周静,沈葵忠,房桂干,等.不同预处理方法对麦草化学组分及其酶解性能的影响[J].林产化学与工业,2017,37(5):53-60. DOI:10.3969/j.issn.0253-2417.2017.05.007.ZHOU J,SHEN K Z,FANG G G,et al. Effects of different pretreatment methods on chemical composition and enzymatic hydrolysis of wheat straw[J]. Chemistry and Industry of Forest Products,2017,37(5):53-60.
[14]SELIG M J,ADNEY W S,HIMMEL M E,et al. The impact of cell wall acetylation on corn stover hydrolysis by cellulolytic and xylanolytic enzymes[J]. Cellulose,2009,16(4):711-722.DOI:10.1007/s10570-009-9322-0.
[15]马建锋,杨淑敏,田根林,等.植物细胞壁木质素区域化学紫外显微光谱研究进展[J].林产化学与工业,2016,36(1):147-154. DOI:10.3969/j.issn.0253-2417.2016.01.021.MA J F,YANG S M,TIAN G L,et al. Application of UV-microspectrophotometry on lignin topochemistry in plant cell wall[J]. Chemistry and Industry of Forest Products,2016,36(1):147-154.
[16]WU X X,HUANG C X,ZHAI S C,et al. Improving enzymatic hydrolysis efficiency of wheat straw through sequential autohydrolysis and alkaline post-extraction[J]. Bioresource Technology,2018,251:374-380. DOI:10.1016/j.biortech.2017.12.066.
[17]TOBA K,YAMAMOTO H,YOSHIDA M. Crystallization of cellulose microfibrils in wood cell wall by repeated dry-and-wet treatment,using X-ray diffraction technique[J]. Cellulose,2013,20(2):633-643. DOI:10.1007/s10570-012-9853-7.
[18]LING Z,CHEN S,ZHANG X,et al. Exploring crystalline-structural variations of cellulose during alkaline pretreatment for enhanced enzymatic hydrolysis[J]. Bioresource Technology,2017,224:611-617. DOI:10.1016/j.biortech.2016.10.064.
[19]ZENG Y N,ZHAO S,WEI H,et al. In situ micro-spectroscopic investigation of lignin in poplar cell walls pretreated by maleic acid[J]. Biotechnology for Biofuels,2015,8(1):126. DOI:10.1186/s13068-015-0312-1.
[20]邓建华,管梦灵,刘美芹.高粱茎杆中木质素分布的显微技术研究[J].植物学研究,2014,3:188-194. DOI:10.12677/br.2014.35024.DENG J H,GUAN M L,LIU M Q. Study on microscopy techniques for identifying lignin distribution in the stem of sorghum[J]. Botanical Research,2014,3:188-194.
[21]DONALDSON L A. Lignification and lignin topochemistry:an ultrastructural view[J]. Phytochemistry,2001,57(6):859-873.DOI:10.1016/s0031-9422(01)00049-8.
[22]JI Z,MA J F,ZHANG Z H,et al. Distribution of lignin and cellulose in compression wood tracheids of Pinus yunnanensis determined by fluorescence microscopy and confocal Raman microscopy[J]. Industrial Crops and Products,2013,47:212-217. DOI:10.1016/j.indcrop.2013.03.006.
[23]DONOHOE B S, DECKER S R, TUCKER M P, et al.Visualizing lignin coalescence and migration through maize cell walls following thermochemical pretreatment[J]. Biotechnology and Bioengineering,2008,101(5):913-925. DOI:10.1002/bit.21959.
[24]HARRIS P J,HARTLEY R D. Detection of bound ferulic acid in cell walls of the Gramineae by ultraviolet fluorescence microscopy[J]. Nature, 1976, 259(5543):508-510. DOI:10.1038/259508a0.
[25]SIQUEIRA G,MILAGRES A M,CARVALHO W,et al. Topochemical distribution of lignin and hydroxycinnamic acids in sugar-cane cell walls and its correlation with the enzymatic hydrolysis of polysaccharides[J]. Biotechnology for Biofuels,2011,4:7. DOI:10.1186/1754-6834-4-7.
[26]徐栋梁,任浩.木质素对纤维素酶水解抑制作用的研究进展与展望[J].中华纸业,2017,38(20):19-24.XU D L,REN H. The research progress and prospect on lignin inhibition in cellulose enzyme hydrolysis[J]. China Pulp&Paper Industry,2017,38(20):19-24.
[27]杨淑娟,游艳芝,张威伟,等. Na OH-乙醇预处理提高甘蔗渣酶法制备低聚木糖效率[J].北京林业大学学报,2018,40(2):114-120. DOI:10.13332/j.1000-1522.20170366.YANG S J,YOU Y Z,ZHANG W W,et al. Na OH-ethanol pretreatment increasing preparation efficiency of xylo-oligosaccharide from sugarcane bagasse with enzymatic hydrolysis[J]. Journal of Beijing Forestry University,2018,40(2):114-120.