FTIR显微成像表征碱处理后玉米秸秆木质素含量及分布
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摘要
探究木质素原位表征方法,能为研究农作物秸秆去木质化过程中木质素变化规律提供帮助。该研究以玉米秸秆为原料,碱处理玉米秸秆横切片后进行傅里叶变换红外(Fourier transform infrared,FTIR)显微成像,并结合快速非负最小二乘(fast non-negativity-constrained least squares,fast NNLS)拟合算法计算组织中木质素的分布。结果显示:1)FTIR显微图像结合fastNNLS算法可对组织中的木质素进行定位及定量分析。如拟合计算得到,原样组织薄壁细胞中木质素质量分数为7.7%,碱处理5、30、60min后下降至6.0%、4.8%、3.5%;2)基于fastNNLS拟合计算的组织中木质素变化趋势同试验测定的木质素变化趋势一致。研究结果表明,基于FTIR显微成像的表征方法可用于原位分析碱处理过程中秸秆组织的木质素分布及含量变化,实现介观尺度研究秸秆木质素降解规律。
Lignin is one of the major components of plant cell wall, and it can provide sufficient strength and hardness to plant cells while avoiding biological damage and water erosion. In recent years, studies on the lignification process and delignification of plant tissues have received extensive attention from scholars. A large number of studies have been carried out to determine the removal of lignin after pretreatment by conventional wet chemical analysis. The lignin content and distribution of biomass resources such as crop straw directly affect its conversion and utilization efficiency. It's meaningful to develop multi-scale and in situ analysis to identify lignin distribution and quantification on delignified plant cell wall for bio-resource commercial utilization. In this paper, we studied an in situ analysis method to visualize changing of lignin distribution in the alkali pretreated corn straw internodal transverse section based on Fourier transform infrared(FTIR) microspectroscopic imaging, with a fast non-negativity-constrained least squares(fast NNLS) fitting. We collected the middle of the 6 th node of fresh jointing stage corn straw. Corn straws were pretreated for series of times(0, 5, 30, 60 min) by 2% NaOH solution at 100℃. A paraffin embedding method was used to produce 18 μm-thick transverse sections for each sample. Then, the sections were transferred onto ZnS windows for FTIR microspectroscopic imaging. We acquired FTIR spectra of major components of corn straw for fast NNLS fitting. We also milled whole straws to 0.069 mm and pretreated in the same condition as sections to determine structure carbohydrate. The data of FTIR microspectroscopic images and FTIR spectra were processed by Savizky-Golay smoothing within 5 points, SNV, and automatic Whittaker filter baseline to correct baseline and offset. K-means cluster algorithm was used to distinguish various tissues including epidermis, vascular bundles and parenchyma cells. Fast NNLS fitting was carried out to calculate the lignin concentrations in pixels. For comparison between different pretreated times, lignin concentration in pixels was calibrated according to the sum of content of all components based on laboratory analysis. The lignin content by laboratory analysis decreased with the increasing of the pretreated time, and the lignin degradation rate was larger at the initial stage of the reaction. The lignin content in the epidermis, vascular bundle and parenchyma cells of the samples showed a decreasing trend with the increasing of pretreatment time, and the trends of lignin changing in the three tissues were consistent with the trend of laboratory analysis. By comparing laboratory analysis with fast NNLS fitting results, the results show that the FTIR microspectroscopic imaging combined with fast NNLS could locate and quantify the distribution of lignin in various tissues, and the trend of lignin changing calculated by fast NNLS fitting is the same as the laboratory chemical analysis results. The study demonstrated that FTIR microspectroscopic imaging combined with fast NNLS fitting could be successfully applied to in situ visualize lignin distribution within corn straw pretreatment. The characterization method provides reference for the lignin research of corn stover pretreatment and the study of lignin degradation during straw pretreatment.
Lignin is one of the major components of plant cell wall, and it can provide sufficient strength and hardness to plant cells while avoiding biological damage and water erosion. In recent years, studies on the lignification process and delignification of plant tissues have received extensive attention from scholars. A large number of studies have been carried out to determine the removal of lignin after pretreatment by conventional wet chemical analysis. The lignin content and distribution of biomass resources such as crop straw directly affect its conversion and utilization efficiency. It's meaningful to develop multi-scale and in situ analysis to identify lignin distribution and quantification on delignified plant cell wall for bio-resource commercial utilization. In this paper, we studied an in situ analysis method to visualize changing of lignin distribution in the alkali pretreated corn straw internodal transverse section based on Fourier transform infrared(FTIR) microspectroscopic imaging, with a fast non-negativity-constrained least squares(fast NNLS) fitting. We collected the middle of the 6 th node of fresh jointing stage corn straw. Corn straws were pretreated for series of times(0, 5, 30, 60 min) by 2% NaOH solution at 100℃. A paraffin embedding method was used to produce 18 μm-thick transverse sections for each sample. Then, the sections were transferred onto ZnS windows for FTIR microspectroscopic imaging. We acquired FTIR spectra of major components of corn straw for fast NNLS fitting. We also milled whole straws to 0.069 mm and pretreated in the same condition as sections to determine structure carbohydrate. The data of FTIR microspectroscopic images and FTIR spectra were processed by Savizky-Golay smoothing within 5 points, SNV, and automatic Whittaker filter baseline to correct baseline and offset. K-means cluster algorithm was used to distinguish various tissues including epidermis, vascular bundles and parenchyma cells. Fast NNLS fitting was carried out to calculate the lignin concentrations in pixels. For comparison between different pretreated times, lignin concentration in pixels was calibrated according to the sum of content of all components based on laboratory analysis. The lignin content by laboratory analysis decreased with the increasing of the pretreated time, and the lignin degradation rate was larger at the initial stage of the reaction. The lignin content in the epidermis, vascular bundle and parenchyma cells of the samples showed a decreasing trend with the increasing of pretreatment time, and the trends of lignin changing in the three tissues were consistent with the trend of laboratory analysis. By comparing laboratory analysis with fast NNLS fitting results, the results show that the FTIR microspectroscopic imaging combined with fast NNLS could locate and quantify the distribution of lignin in various tissues, and the trend of lignin changing calculated by fast NNLS fitting is the same as the laboratory chemical analysis results. The study demonstrated that FTIR microspectroscopic imaging combined with fast NNLS fitting could be successfully applied to in situ visualize lignin distribution within corn straw pretreatment. The characterization method provides reference for the lignin research of corn stover pretreatment and the study of lignin degradation during straw pretreatment.
引文
[1]杨增玲,梅佳琪,曹聪,等.基于红外光谱的不同农作物秸秆磨木木质素差异表征[J].农业工程学报,2018,34(19):219-224.Yang Zengling,Mei Jiaqi,Cao Cong,et al.Traits of milled wood lignin isolated from different crop straw based on FT-IR[J].Transactions of the Chinese Society of Agricultural Engineering(Transactions of the CSAE),2018,34(19):219-224.(in Chinese with English abstract)
[2]李雅丽,高锦红,王丽.碱处理对农作物秸秆中木质素含量的影响[J].化学与生物工程,2017,34(10):58-60.Li Yali,Gao Jinhong,Wang Li.Effect of alkali treatment on lignin content in crop straw[J].Chemistry&Bioengineering,2017,34(10):58-60.(in Chinese with English abstract)
[3]Cesarino I,Araújo Pedro,Domingues Júnior Adilson Pereira,et al.An overview of lignin metabolism and its effect on biomass recalcitrance[J].Brazilian Journal of Botany,2012,35(4):303-311.
[4]路瑶,魏贤勇,宗志敏,等.木质素的结构研究与应用[J].化学进展,2013,25(5):838-858.Lu Yao,Wei Xianyong,Zong Zhimin,et al.Structural investigation and application of lignins[J].Progress in Chemistry,2013,25(5):838-858.(in Chinese with English abstract)
[5]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.Ma Jianfeng,Yang Shumin,Tian Genlin,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.(in Chinese with English abstract)
[6]Zhu Shengdong,Wu Yuanxin,Yu Ziniu,et al.Pretreatment by microwave/alkali of rice straw and its enzymic hydrolysis[J].Process Biochemistry,2005,40(9):3082-3086.
[7]Hu Zhenhu,Wen Zhiyou.Enhancing enzymatic digestibility of switchgrass by microwave-assisted alkali pretreatment[J].Biochemical Engineering Journal,2008,38(3):369-378.
[8]Li Junbao,Lu Mingsheng,Guo Xiaomiao,et al.Insights into the improvement of alkaline hydrogen peroxide(AHP)pretreatment on the enzymatic hydrolysis of corn stover:chemical and microstructural analyses[J].Bioresource Technology,2018,265:2-7.
[9]Micco V D,Aronne G.Combined histochemistry and autofluorescence for identifying lignin distribution in cell walls[J].Biotechnic&Histochemistry,2007,82(4/5):209-216.
[10]Liljegren S.Phloroglucinol stain for lignin[J].Cold Spring Harbor Protocols,2010(1):pdb.prot4954.Doi:10.1101/pdb.prot4954
[11]Coletta V C,Rezende C A,Da Concei??o F R,et al.Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy[J].Biotechnology for Biofuels,2013,6(1):43-43.
[12]Ji Zhe,Ma Jianfeng,Zhang Zhiheng,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.
[13]刘苍伟,苏明垒,周贤武,等.FTIR及CLSM对转基因杨木细胞壁木质素含量及微区分布研究[J].光谱学与光谱分析,2017(11):89-93.Liu Cangwei,Su Minglei,Zhou Xianwu,et al.Analysis of content and distribution of lignin in cell wall of transgenic poplar with Fourier infrared spectrun(FTIR)and confocal laser scanning microscopy(CLSM)[J].Spectroscopy and Spectral Analysis,2017(11):89-93.(in Chinese with English abstract)
[14]Miyafuji H,Komai K,Kanbayashi T.Development of quantification method for lignin content in woody biomass by Raman micro-spectroscopy[J].Vibrational Spectroscopy,2017,88:9-13.
[15]Singh A,Daniel G,Nilsson T.Ultrastructure of the S2 layer in relation to lignin distribution inpinus radiata tracheids[J].Journal of Wood Science,2002,48(2):95-98.
[16]Westermark U,Lidbrandt O,Eriksson I.Lignin distribution in spruce(Picea abies)determined by mercurization with SEM-EDXA technique[J].Wood Science&Technology,1988,22(3):243-250.
[17]Lan Sun,Li Chenlin,Xue Zhengjun,et al.Unveiling high-resolution,tissue specific dynamic changes in corn stover during ionic liquid pretreatment[J].RSC Advances,2013,3:2017-2027.
[18]Ma Jianfeng,Ji Zhe,Zhou Xia,et al.Transmission electron microscopy,fluorescence microscopy,and confocal raman microscopic analysis of ultrastructural and compositional heterogeneity of Cornus alba L.wood cell wall[J].Microscopy and Microanalysis,2013,19(1):243-253.
[19]Pelletier M J,Pelletier C C.Spectroscopic Theory for Chemical Imaging[M]//Raman,Infrared,and Near-Infrared Chemical Imaging.John Wiley&Sons Inc,2010.
[20]Türker-Kaya S,Huck C W.A review of mid-infrared and near-infrared imaging:Principles,concepts and applications in plant tissue analysis[J].Molecules,2017,22(1):168-187.
[21]Dokken K M,Davis L C.Infrared imaging of sunflower and maize root anatomy[J].Journal of Agricultural and Food Chemistry,2007,55(26):10517-10530.
[22]Gorzsás A,Stenlund H,Persson P,et al.Cell‐specific chemotyping and multivariate imaging by combined FT‐IRmicrospectroscopy and orthogonal projections to latent structures(OPLS)analysis reveals the chemical landscape of secondary xylem[J].The Plant Journal,2011,66(5):12.
[23]Yang Zengling,Mei Jiaqi,Liu Zhiqiang,et al.Visualization and semiquantitative study of the distribution of major components in wheat straw in mesoscopic scale using fourier transform infrared microspectroscopic Imaging[J].Analytical Chemistry,2018,90(12):7332-7340.
[24]Shih C J,Lupoi J S,Smith E A.Raman spectroscopy measurements of glucose and xylose in hydrolysate:Role of corn stover pretreatment and enzyme composition[J].Bioresource Technology,2011,102(8):5169-5176.
[25]Rossi S,Menardi R,Anfodillo T.Trephor:A new tool for sampling microcores from tree stems[J].IAWA Journal,2006,27(1):89-97.
[26]Sluiter A,Hames B,Ruiz R,et al.Determination of Structural Carbohydrates and Lignin in Biomass:NREL/TP-510-42618[S].National Renewable Energy Laboratory,Golden,Co,2011:1-15.
[27]Sun Xiaofeng,Sun Runcang,Fowler P,et al.Extraction and characterization of original lignin and hemicelluloses from wheat straw[J].Journal of Agricultural and Food Chemistry,2005,53(4):860-870.
[28]Fang J M,Sun R C,Tomkinson J.Isolation and characterization of hemicelluloses and cellulose from rye straw by alkaline peroxide extraction[J].Cellulose,2000,7(1):87-107.
[29]Bro R,Jong S D.A fast non-negativity-constrained least squares algorithm[J].Journal of Chemometrics,1997,11(5):393-401.
[30]杨增玲,梅佳琪,韩鲁佳,等.茎秆切片显微红外“组织-组分”同步分析软件V1.0[Z].证书号:软著登字第BI44059号.登记号:2018SRBJ0229.
[2]李雅丽,高锦红,王丽.碱处理对农作物秸秆中木质素含量的影响[J].化学与生物工程,2017,34(10):58-60.Li Yali,Gao Jinhong,Wang Li.Effect of alkali treatment on lignin content in crop straw[J].Chemistry&Bioengineering,2017,34(10):58-60.(in Chinese with English abstract)
[3]Cesarino I,Araújo Pedro,Domingues Júnior Adilson Pereira,et al.An overview of lignin metabolism and its effect on biomass recalcitrance[J].Brazilian Journal of Botany,2012,35(4):303-311.
[4]路瑶,魏贤勇,宗志敏,等.木质素的结构研究与应用[J].化学进展,2013,25(5):838-858.Lu Yao,Wei Xianyong,Zong Zhimin,et al.Structural investigation and application of lignins[J].Progress in Chemistry,2013,25(5):838-858.(in Chinese with English abstract)
[5]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.Ma Jianfeng,Yang Shumin,Tian Genlin,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.(in Chinese with English abstract)
[6]Zhu Shengdong,Wu Yuanxin,Yu Ziniu,et al.Pretreatment by microwave/alkali of rice straw and its enzymic hydrolysis[J].Process Biochemistry,2005,40(9):3082-3086.
[7]Hu Zhenhu,Wen Zhiyou.Enhancing enzymatic digestibility of switchgrass by microwave-assisted alkali pretreatment[J].Biochemical Engineering Journal,2008,38(3):369-378.
[8]Li Junbao,Lu Mingsheng,Guo Xiaomiao,et al.Insights into the improvement of alkaline hydrogen peroxide(AHP)pretreatment on the enzymatic hydrolysis of corn stover:chemical and microstructural analyses[J].Bioresource Technology,2018,265:2-7.
[9]Micco V D,Aronne G.Combined histochemistry and autofluorescence for identifying lignin distribution in cell walls[J].Biotechnic&Histochemistry,2007,82(4/5):209-216.
[10]Liljegren S.Phloroglucinol stain for lignin[J].Cold Spring Harbor Protocols,2010(1):pdb.prot4954.Doi:10.1101/pdb.prot4954
[11]Coletta V C,Rezende C A,Da Concei??o F R,et al.Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy[J].Biotechnology for Biofuels,2013,6(1):43-43.
[12]Ji Zhe,Ma Jianfeng,Zhang Zhiheng,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.
[13]刘苍伟,苏明垒,周贤武,等.FTIR及CLSM对转基因杨木细胞壁木质素含量及微区分布研究[J].光谱学与光谱分析,2017(11):89-93.Liu Cangwei,Su Minglei,Zhou Xianwu,et al.Analysis of content and distribution of lignin in cell wall of transgenic poplar with Fourier infrared spectrun(FTIR)and confocal laser scanning microscopy(CLSM)[J].Spectroscopy and Spectral Analysis,2017(11):89-93.(in Chinese with English abstract)
[14]Miyafuji H,Komai K,Kanbayashi T.Development of quantification method for lignin content in woody biomass by Raman micro-spectroscopy[J].Vibrational Spectroscopy,2017,88:9-13.
[15]Singh A,Daniel G,Nilsson T.Ultrastructure of the S2 layer in relation to lignin distribution inpinus radiata tracheids[J].Journal of Wood Science,2002,48(2):95-98.
[16]Westermark U,Lidbrandt O,Eriksson I.Lignin distribution in spruce(Picea abies)determined by mercurization with SEM-EDXA technique[J].Wood Science&Technology,1988,22(3):243-250.
[17]Lan Sun,Li Chenlin,Xue Zhengjun,et al.Unveiling high-resolution,tissue specific dynamic changes in corn stover during ionic liquid pretreatment[J].RSC Advances,2013,3:2017-2027.
[18]Ma Jianfeng,Ji Zhe,Zhou Xia,et al.Transmission electron microscopy,fluorescence microscopy,and confocal raman microscopic analysis of ultrastructural and compositional heterogeneity of Cornus alba L.wood cell wall[J].Microscopy and Microanalysis,2013,19(1):243-253.
[19]Pelletier M J,Pelletier C C.Spectroscopic Theory for Chemical Imaging[M]//Raman,Infrared,and Near-Infrared Chemical Imaging.John Wiley&Sons Inc,2010.
[20]Türker-Kaya S,Huck C W.A review of mid-infrared and near-infrared imaging:Principles,concepts and applications in plant tissue analysis[J].Molecules,2017,22(1):168-187.
[21]Dokken K M,Davis L C.Infrared imaging of sunflower and maize root anatomy[J].Journal of Agricultural and Food Chemistry,2007,55(26):10517-10530.
[22]Gorzsás A,Stenlund H,Persson P,et al.Cell‐specific chemotyping and multivariate imaging by combined FT‐IRmicrospectroscopy and orthogonal projections to latent structures(OPLS)analysis reveals the chemical landscape of secondary xylem[J].The Plant Journal,2011,66(5):12.
[23]Yang Zengling,Mei Jiaqi,Liu Zhiqiang,et al.Visualization and semiquantitative study of the distribution of major components in wheat straw in mesoscopic scale using fourier transform infrared microspectroscopic Imaging[J].Analytical Chemistry,2018,90(12):7332-7340.
[24]Shih C J,Lupoi J S,Smith E A.Raman spectroscopy measurements of glucose and xylose in hydrolysate:Role of corn stover pretreatment and enzyme composition[J].Bioresource Technology,2011,102(8):5169-5176.
[25]Rossi S,Menardi R,Anfodillo T.Trephor:A new tool for sampling microcores from tree stems[J].IAWA Journal,2006,27(1):89-97.
[26]Sluiter A,Hames B,Ruiz R,et al.Determination of Structural Carbohydrates and Lignin in Biomass:NREL/TP-510-42618[S].National Renewable Energy Laboratory,Golden,Co,2011:1-15.
[27]Sun Xiaofeng,Sun Runcang,Fowler P,et al.Extraction and characterization of original lignin and hemicelluloses from wheat straw[J].Journal of Agricultural and Food Chemistry,2005,53(4):860-870.
[28]Fang J M,Sun R C,Tomkinson J.Isolation and characterization of hemicelluloses and cellulose from rye straw by alkaline peroxide extraction[J].Cellulose,2000,7(1):87-107.
[29]Bro R,Jong S D.A fast non-negativity-constrained least squares algorithm[J].Journal of Chemometrics,1997,11(5):393-401.
[30]杨增玲,梅佳琪,韩鲁佳,等.茎秆切片显微红外“组织-组分”同步分析软件V1.0[Z].证书号:软著登字第BI44059号.登记号:2018SRBJ0229.