Woody biomass is one of the most promising renewable alternatives to fossil resources. However, some physical and chemical treatment is required to convert their chemical components into biofuels and valuable chemicals because of their low degradative properties. Recently, there has been considerable interest in ionic liquid treatment for biorefinery, and many fundamental studies on the reactivity of wood with ionic liquids have been performed from a chemical and morphological point of view. This chapter highlights the findings regarding morphological and topochemical features of wood cell walls in the degradation process as a result of ionic liquid treatment. Bright-field microscopy and scanning electron microscopy have revealed the swelling behavior of cell walls and the detailed ultrastructural features of wood tissues treated with ionic liquid. Polarized light microscopy and confocal Raman microscopy have clarified the changes in cellulose crystallinity and distribution of chemical compositions such as polysaccharides and lignin during ionic liquid treatment at the cellular level.
- Cell wall
- Ionic liquid
The efficient use of lignocellulosics has been an important approach to prevent exhaustion of fossil resources and global warming caused by increasing emissions of greenhouse gases. Among the various types of lignocellulosics, woody biomass is regarded as a promising resource because it is carbon neutral, abundantly available in many regions, and does not compete with agricultural production. Wood cell walls decompose to form persistent organic complexes mainly composed of cellulose (40–50%), hemicellulose (25–35%), and lignin (18–35%) [1, 2]. To convert their chemical components into transportable biofuels and chemical feedstocks, it is necessary to develop the processing technology to break their rigid structure. To date, various conversion methods such as acid hydrolysis [3–5], enzymatic saccharification [6, 7], pyrolysis [8–10], and supercritical or sub-critical fluid treatment [11–13] have been investigated. However, practical methods have not yet been established.
Ionic liquids are defined as organic salts with low melting points, and have many advantages including negligible vapor pressures, chemical and thermal stability, non-flammability, low viscosity, and reusability [14–16]. In addition, they can dissolve a wide range of organic and inorganic substances . Ionic liquid treatment is attractive as a new conversion technology for woody biomass. Figure 1 shows the typical cations and anions found in ionic liquids. There are an infinite number of combinations of cations and anions, and their physical properties, such as melting point, viscosity and dissolving power, can be easily changed by altering the combination. This is why ionic liquids are called “designer solvents” .
Recent studies revealed that certain types of ionic liquids can liquefy cellulose [18–20], lignin , and even wood cell walls [22–26]. Using ionic liquids as the solvent to process woody biomass, many fundamental studies on the reaction behavior of wood in ionic liquids have been carried out focusing on the chemical processes [27–31]. However, woody biomass is a very inhomogeneous composite at the cell level. Wood comprises various types of tissues such as the tracheid, wood fibers, vessels, and parenchyma. In addition, wood cell walls consist of several layers: a compound middle lamella (middle lamella + primary wall; CML) and a secondary wall (S), which is generally composed of S1, S2, and S3 sublayers. The chemical components and distribution vary depending on the wood species, types of tissues and their layers . Therefore, to improve the chemical conversion process using ionic liquids, a better understanding of the effects of ionic liquid treatment of wood, such as the interaction of wood with ionic liquids at the cell level and the deconstruction behavior of various types of tissues in ionic liquids, are required.
In this chapter, we focus on morphological and topochemical studies on the liquefaction of wood in ionic liquids, especially 1-ethyl-3-methylimidazolium chloride ([C2mim][Cl]) and 1-ethylpyridinium bromide ([EtPy][Br]), using various microscopy techniques. [C2mim][Cl] and [EtPy][Br] are known as the ionic liquids which can preferentially liquefy cellulose  and lignin , respectively. Bright-field microscopy and polarized light microscopy were employed to determine the swelling and decomposition behaviors of wood cell walls and the state of cellulose crystallinity during ionic liquids treatment. Scanning electron microscopy (SEM) was used to observe the detailed ultrastructural changes in various wood tissues treated with ionic liquids. Confocal Raman microscopy was employed to examine the changes in chemical components including polysaccharides and lignin at the cellular level and to visualize their distribution on the cell walls during ionic liquids treatment.
2. Application of microscopy techniques to examine wood liquefaction
2.1. Light microscopy analysis
Morphological features of wood cell walls during the liquefaction process in ionic liquid were determined by light microscopy [33–38]. Figure 2 shows bright-field microscopy and polarized light microscopy images of
To study the detailed swelling behavior of tracheids arising from [C2mim][Cl] treatment, we performed time sequential measurements of the cell wall area, cell lumen area, and the total of cell lumen and cell wall areas, in earlywood and latewood, in transverse sections (Figure 3). The cell wall area in earlywood increased only slightly at an early stage of [C2mim][Cl] treatment, whereas that in latewood increased significantly. After the initial swelling, the cell wall area in earlywood showed no further changes, whereas that in latewood increased gradually with prolonged treatment time. After 72 h of treatment, the cell wall area in earlywood and latewood had increased by 1.5 and 4 times, respectively. These results indicate that the swelling behavior of the tracheids of
Changes in the cell wall area of fibrous cells of various Japanese hardwood species during [C2mim][Cl] treatment were also measured (Figure 4). At the initial stages of [C2mim][Cl] treatment, the cell wall areas of all species increased rapidly. Thereafter, the cell wall areas of
Figure 5 shows bright-field microscopy and polarized light microscopy images of
2.2. Scanning electron microscopy observations
Ultrastructural changes in wood cell walls due to ionic liquid treatment were observed by SEM [34–38]. Figure 6 shows SEM images of various tissues of
Figure 7 shows SEM images of various tissues of
Figure 8 shows SEM images of transverse sections of two hardwood species treated with [C2mim][Cl]. Although wood fibers (indicated by arrowheads) of both
2.3. Confocal Raman microscopy analysis
Raman spectra can reveal much information about functional groups, hydrogen and chemical bonds, and the surrounding environment. Raman spectroscopy is used to identify the chemical structure of a substance. Confocal Raman microscopy couples Raman spectroscopy with a confocal microscope to perform detailed analysis quickly. In recent years, confocal Raman microscopy has received attention as a new method of spectroscopic analysis for plant cell walls because of its characteristic advantages. It is non-destructive, has a high spatial resolution (approximately 0.3–2 μm), is not hindered by the presence of water [43, 44], and little-to-no sample pre-treatment is required. Several research groups have studied the chemical composition of native wood cell walls using this method [45–51]. In addition, it has been reported that confocal Raman microscopy is an effective tool to investigate topochemical changes in wood after pre-treatment for biorefinery [52–54].
We applied confocal Raman microscopy to determine the changes in chemical components and their distribution in wood at the cellular level during [C2mim][Cl] and [EtPy][Br] treatment [37, 38, 55, 56]. Figure 9 shows Raman spectra obtained from S2 of the wood fibers of
To study the changes in the distribution of chemical components in the wood cell walls over a wide range during ionic liquids treatment, Raman mapping analysis was applied on transverse sections. Raman mapping was performed on tracheids and wood fibers because these tissues are the main elements of
Figure 10 shows the results of time-sequential Raman mapping analysis of the distribution of lignin and polysaccharides of tracheids of
Raman mapping analysis was also performed on the wood samples treated with [EtPy][Br] (Figure 11). Although the lignin concentration in S2 and in CML decreased with prolonged treatment time, the lignin in CML was preserved at relatively high concentration after 72 h of treatment in both tracheids of
To gain insight into the reactivity of [C2mim][Cl] with various morphological regions, Raman spectra were acquired for the S2 of wood fibers, the cell corner of wood fibers, vessels, and axial parenchyma cells of
Using various microscopy techniques, the morphological and topochemical features of wood cell walls treated with ionic liquids were studied. During the processing of wood liquefaction in ionic liquids, the ultrastructure and chemical compositions of wood showed inhomogeneous changes at the cellular level. The interaction of ionic liquid with wood cell walls was quite different depending on the types of ionic liquids, wood species, tissues, and cell wall layers. These findings will serve to cultivate a better understanding of the liquefaction mechanism of woody biomass in ionic liquids and accelerate development of ionic liquid treatment for wood-based biorefinery.
For research in wood chemistry and anatomy, many microscopy techniques have been applied to investigate the characteristics of the cell walls. However, it is hardly possible to examine the chemical compositions and their distribution with nanoscale spatial resolution while at the same time observing the ultrastructure such as ultrathin layers. The development of sensitive analytical methods in the wood cell walls for chemical information with much higher spatial resolution will open a new field of wood science and technology.
This work was partly supported by the “Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry” (No. 26052A) from the Ministry of Agriculture, Forestry and Fisheries of Japan, a Grant-in-Aid for Scientific Research (C) (No. 25450246) from the Japan Society for the Promotion of Science (JSPS), and a Grant-in-Aid for JSPS Fellows (No. 15J05592).
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