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Visualization of Miscanthus × giganteus cell wall deconstruction subjected to dilute acid pretreatment for enhanced enzymatic digestibility.

Ji Z, Zhang X, Ling Z, Zhou X, Ramaswamy S, Xu F - Biotechnol Biofuels (2015)

Bottom Line: DAP of M. × giganteus resulted in solubilization of arabinoxylan and cross-linking hydroxycinnamic acids in a temperature-dependent manner.The optimized pretreatment (1% H2SO4, 170°C for 30 min) resulted in significant enhancement in the saccharification efficiency (51.20%) of treated samples in 72 h, which amounted to 4.4-fold increase in sugar yield over untreated samples (11.80%).The consequently occurred changes in inner cell wall structure including damaging, increase of porosity and loss of mechanical resistance were also found to enhance enzyme access to cellulose and further sugar yield.

View Article: PubMed Central - PubMed

Affiliation: Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing, 100083 China ; Ministry of Education Key Laboratory of Wooden Material Science and Application, Beijing Forestry University, Tsinghua East Road, Beijing, 100083 China.

ABSTRACT

Background: The natural recalcitrance of lignocellulosic plant cell walls resulting from complex arrangement and distribution of heterogeneous components impedes deconstruction of such cell walls. Dilute acid pretreatment (DAP) is an attractive method to overcome the recalcitrant barriers for rendering enzymatic conversion of polysaccharides. In this study, the internodes of Miscanthus × giganteus, a model bioenergy crop, were subjected to DAP to yield a range of samples with altered cell wall structure and chemistry. The consequent morphological and compositional changes and their possible impact on saccharification efficiency were comprehensively investigated. The use of a series of microscopic and microspectroscopic techniques including fluorescence microscopy (FM), transmission electron microscopy (TEM) and confocal Raman microscopy (CRM)) enabled correlative cell wall structural and chemical information to be obtained.

Results: DAP of M. × giganteus resulted in solubilization of arabinoxylan and cross-linking hydroxycinnamic acids in a temperature-dependent manner. The optimized pretreatment (1% H2SO4, 170°C for 30 min) resulted in significant enhancement in the saccharification efficiency (51.20%) of treated samples in 72 h, which amounted to 4.4-fold increase in sugar yield over untreated samples (11.80%). The remarkable improvement could be correlated to a sequence of changes occurring in plant cell walls due to their pretreatment-induced deconstruction, namely, loss in the matrix between neighboring cell walls, selective removal of hemicelluloses, redistribution of phenolic polymers and increased exposure of cellulose. The consequently occurred changes in inner cell wall structure including damaging, increase of porosity and loss of mechanical resistance were also found to enhance enzyme access to cellulose and further sugar yield.

Conclusions: DAP is a highly effective process for improving bioconversion of cellulose to glucose by breaking down the rigidity and resistance of cell walls. The combination of the most relevant microscopic and microanalytical techniques employed in this work provided information crucial for evaluating the influence of anatomical and compositional changes on enhanced enzymatic digestibility.

No MeSH data available.


Related in: MedlinePlus

Multiscale imaging of M. × giganteus cell wall architecture by FM and TEM. FM images showed intact clusters of cells in raw M. × giganteus tissues (a, b) with trace evidence of mechanical damage near Pxv from the cutting process (a, asterisk). The ultrastructure of Sf including compound cell corner (Ccml), compound middle lamella (Cml), secondary wall (Sw) and cell lumen (CL) were observed by TEM (c). In samples pretreated at 160°C, 0.5% H2SO4 for 15 min, individual cell walls were shown to be separated, particularly at the St and Com regions (d, white arrows) and at the Sf–Par boundaries (e, white arrows). A TEM scan of Sf presented lighter staining in the Ccml indicating lower density in these regions (f, white triangle). Samples pretreated at 170°C, 1% H2SO4 for 30 min exhibited many crushes of broken cells (g). Increased disjoining of Par walls from the Cml can be clearly distinguished at higher magnifications (h, white arrows). Additionally, intercellular spaces at the Ccml of Sf were gradually generated, resulting from the removal of hemicelluloses and lignin (i, white triangle). Sf sclerenchyma fibers, Par parenchyma, Pxv protoxylem vessel, Mxv metaxylem vessel, St sieve tube, Com companion cell.
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Fig2: Multiscale imaging of M. × giganteus cell wall architecture by FM and TEM. FM images showed intact clusters of cells in raw M. × giganteus tissues (a, b) with trace evidence of mechanical damage near Pxv from the cutting process (a, asterisk). The ultrastructure of Sf including compound cell corner (Ccml), compound middle lamella (Cml), secondary wall (Sw) and cell lumen (CL) were observed by TEM (c). In samples pretreated at 160°C, 0.5% H2SO4 for 15 min, individual cell walls were shown to be separated, particularly at the St and Com regions (d, white arrows) and at the Sf–Par boundaries (e, white arrows). A TEM scan of Sf presented lighter staining in the Ccml indicating lower density in these regions (f, white triangle). Samples pretreated at 170°C, 1% H2SO4 for 30 min exhibited many crushes of broken cells (g). Increased disjoining of Par walls from the Cml can be clearly distinguished at higher magnifications (h, white arrows). Additionally, intercellular spaces at the Ccml of Sf were gradually generated, resulting from the removal of hemicelluloses and lignin (i, white triangle). Sf sclerenchyma fibers, Par parenchyma, Pxv protoxylem vessel, Mxv metaxylem vessel, St sieve tube, Com companion cell.

Mentions: Figure 2 illustrates the morphology and layered structure of M. × giganteus cell walls as a function of acid treatment severity by multi-scale imaging strategies. FM was used to investigate the cell wall structure in vascular bundles. M. × giganteus culm tissues were well organized, consisting of several cell types, including sclerenchyma fiber (Sf) surrounding the vascular bundles, parenchyma (Par), protoxylem vessel (Pxv), metaxylem vessel (Mxv), sieve tube (St) and companion cell (Com). Prior to treatment, vascular bundle tissues appeared intact (Figure 2a, b) with trace evidence of mechanical damage near Pxv from the cutting process (asterisk in Figure 2a). After pretreatment under moderate condition (160°C, 0.5% H2SO4 for 15 min), the samples showed separation of cell walls in many locations (Figure 2d, e), particularly at St and Com regions (white arrows in Figure 2d) and at Sf–Par boundaries (white arrows in Figure 2e), which indicated loosening of the original structure. With increasing pretreatment severity, vascular bundles displayed more pronounced alterations to tissues, such as sucrose-storing Par; many cells were crushed and broken (Figure 2g). Cell separations among neighboring Par cells in the treated biomass were more clearly visible in higher magnification images (white arrows in Figure 2h), which may be attributable to effective depolymerization of hemicelluloses and removal of lignin and pectin from P and Cml regions. In alfalfa, the deposition and distribution of pectin conforms to the patterns of lignin in the Ccml, where much of the pectin in cell walls is located and lignification is initiated [31]. In addition, the pectic arabinogalactans are reported to be removed concurrently with lignin during delignification of lupin upon chemical treatments [32, 33]. A recent study by DeMartini et al. [34] employing a novel glycome profiling technique on Populus biomass during hydrothermal pretreatment demonstrates significant loss of pectic and arabinogalactan epitopes corresponding to the disintegration of lignin–polysaccharide linkages. Briefly, the disjoining of cell walls likely enhanced the exposure of cellulose microfibrils and availability of more active surface area, consequently increasing the cellulose digestibility of the treated biomass as shown earlier.Figure 2


Visualization of Miscanthus × giganteus cell wall deconstruction subjected to dilute acid pretreatment for enhanced enzymatic digestibility.

Ji Z, Zhang X, Ling Z, Zhou X, Ramaswamy S, Xu F - Biotechnol Biofuels (2015)

Multiscale imaging of M. × giganteus cell wall architecture by FM and TEM. FM images showed intact clusters of cells in raw M. × giganteus tissues (a, b) with trace evidence of mechanical damage near Pxv from the cutting process (a, asterisk). The ultrastructure of Sf including compound cell corner (Ccml), compound middle lamella (Cml), secondary wall (Sw) and cell lumen (CL) were observed by TEM (c). In samples pretreated at 160°C, 0.5% H2SO4 for 15 min, individual cell walls were shown to be separated, particularly at the St and Com regions (d, white arrows) and at the Sf–Par boundaries (e, white arrows). A TEM scan of Sf presented lighter staining in the Ccml indicating lower density in these regions (f, white triangle). Samples pretreated at 170°C, 1% H2SO4 for 30 min exhibited many crushes of broken cells (g). Increased disjoining of Par walls from the Cml can be clearly distinguished at higher magnifications (h, white arrows). Additionally, intercellular spaces at the Ccml of Sf were gradually generated, resulting from the removal of hemicelluloses and lignin (i, white triangle). Sf sclerenchyma fibers, Par parenchyma, Pxv protoxylem vessel, Mxv metaxylem vessel, St sieve tube, Com companion cell.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig2: Multiscale imaging of M. × giganteus cell wall architecture by FM and TEM. FM images showed intact clusters of cells in raw M. × giganteus tissues (a, b) with trace evidence of mechanical damage near Pxv from the cutting process (a, asterisk). The ultrastructure of Sf including compound cell corner (Ccml), compound middle lamella (Cml), secondary wall (Sw) and cell lumen (CL) were observed by TEM (c). In samples pretreated at 160°C, 0.5% H2SO4 for 15 min, individual cell walls were shown to be separated, particularly at the St and Com regions (d, white arrows) and at the Sf–Par boundaries (e, white arrows). A TEM scan of Sf presented lighter staining in the Ccml indicating lower density in these regions (f, white triangle). Samples pretreated at 170°C, 1% H2SO4 for 30 min exhibited many crushes of broken cells (g). Increased disjoining of Par walls from the Cml can be clearly distinguished at higher magnifications (h, white arrows). Additionally, intercellular spaces at the Ccml of Sf were gradually generated, resulting from the removal of hemicelluloses and lignin (i, white triangle). Sf sclerenchyma fibers, Par parenchyma, Pxv protoxylem vessel, Mxv metaxylem vessel, St sieve tube, Com companion cell.
Mentions: Figure 2 illustrates the morphology and layered structure of M. × giganteus cell walls as a function of acid treatment severity by multi-scale imaging strategies. FM was used to investigate the cell wall structure in vascular bundles. M. × giganteus culm tissues were well organized, consisting of several cell types, including sclerenchyma fiber (Sf) surrounding the vascular bundles, parenchyma (Par), protoxylem vessel (Pxv), metaxylem vessel (Mxv), sieve tube (St) and companion cell (Com). Prior to treatment, vascular bundle tissues appeared intact (Figure 2a, b) with trace evidence of mechanical damage near Pxv from the cutting process (asterisk in Figure 2a). After pretreatment under moderate condition (160°C, 0.5% H2SO4 for 15 min), the samples showed separation of cell walls in many locations (Figure 2d, e), particularly at St and Com regions (white arrows in Figure 2d) and at Sf–Par boundaries (white arrows in Figure 2e), which indicated loosening of the original structure. With increasing pretreatment severity, vascular bundles displayed more pronounced alterations to tissues, such as sucrose-storing Par; many cells were crushed and broken (Figure 2g). Cell separations among neighboring Par cells in the treated biomass were more clearly visible in higher magnification images (white arrows in Figure 2h), which may be attributable to effective depolymerization of hemicelluloses and removal of lignin and pectin from P and Cml regions. In alfalfa, the deposition and distribution of pectin conforms to the patterns of lignin in the Ccml, where much of the pectin in cell walls is located and lignification is initiated [31]. In addition, the pectic arabinogalactans are reported to be removed concurrently with lignin during delignification of lupin upon chemical treatments [32, 33]. A recent study by DeMartini et al. [34] employing a novel glycome profiling technique on Populus biomass during hydrothermal pretreatment demonstrates significant loss of pectic and arabinogalactan epitopes corresponding to the disintegration of lignin–polysaccharide linkages. Briefly, the disjoining of cell walls likely enhanced the exposure of cellulose microfibrils and availability of more active surface area, consequently increasing the cellulose digestibility of the treated biomass as shown earlier.Figure 2

Bottom Line: DAP of M. × giganteus resulted in solubilization of arabinoxylan and cross-linking hydroxycinnamic acids in a temperature-dependent manner.The optimized pretreatment (1% H2SO4, 170°C for 30 min) resulted in significant enhancement in the saccharification efficiency (51.20%) of treated samples in 72 h, which amounted to 4.4-fold increase in sugar yield over untreated samples (11.80%).The consequently occurred changes in inner cell wall structure including damaging, increase of porosity and loss of mechanical resistance were also found to enhance enzyme access to cellulose and further sugar yield.

View Article: PubMed Central - PubMed

Affiliation: Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing, 100083 China ; Ministry of Education Key Laboratory of Wooden Material Science and Application, Beijing Forestry University, Tsinghua East Road, Beijing, 100083 China.

ABSTRACT

Background: The natural recalcitrance of lignocellulosic plant cell walls resulting from complex arrangement and distribution of heterogeneous components impedes deconstruction of such cell walls. Dilute acid pretreatment (DAP) is an attractive method to overcome the recalcitrant barriers for rendering enzymatic conversion of polysaccharides. In this study, the internodes of Miscanthus × giganteus, a model bioenergy crop, were subjected to DAP to yield a range of samples with altered cell wall structure and chemistry. The consequent morphological and compositional changes and their possible impact on saccharification efficiency were comprehensively investigated. The use of a series of microscopic and microspectroscopic techniques including fluorescence microscopy (FM), transmission electron microscopy (TEM) and confocal Raman microscopy (CRM)) enabled correlative cell wall structural and chemical information to be obtained.

Results: DAP of M. × giganteus resulted in solubilization of arabinoxylan and cross-linking hydroxycinnamic acids in a temperature-dependent manner. The optimized pretreatment (1% H2SO4, 170°C for 30 min) resulted in significant enhancement in the saccharification efficiency (51.20%) of treated samples in 72 h, which amounted to 4.4-fold increase in sugar yield over untreated samples (11.80%). The remarkable improvement could be correlated to a sequence of changes occurring in plant cell walls due to their pretreatment-induced deconstruction, namely, loss in the matrix between neighboring cell walls, selective removal of hemicelluloses, redistribution of phenolic polymers and increased exposure of cellulose. The consequently occurred changes in inner cell wall structure including damaging, increase of porosity and loss of mechanical resistance were also found to enhance enzyme access to cellulose and further sugar yield.

Conclusions: DAP is a highly effective process for improving bioconversion of cellulose to glucose by breaking down the rigidity and resistance of cell walls. The combination of the most relevant microscopic and microanalytical techniques employed in this work provided information crucial for evaluating the influence of anatomical and compositional changes on enhanced enzymatic digestibility.

No MeSH data available.


Related in: MedlinePlus