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Spectroscopic/Microscopic Elucidation for Chemical Changes During Acid Pretreatment on Arundo donax

  • Corresponding author: ZHAI Shengcheng, zhais@njfu.edu.cn
  • Received Date: 2019-04-19
  • The Arundo donax is a typical fast-growing species from the family Gramineae, which is widely cultivated in China. With a huge yield of A. donax in China, this plant offers great potential for biofuels production. The different types of organization of cell and the tissue in the A. donax could influence the efficiency of enzymatic hydrolysis. In this study, A. donax was subjected to 0.5% (w/w) sulfuric acid (H2SO4) for pretreatment at 140℃ for 10 min, 20 min, 40 min, and 60 min, respectively. The changes in microstructure, chemical composition, topochemical properties were comprehensively analyzed. Using a series of spectroscopic and microscopic techniques including Fourier transform infrared spectroscopy (FT-IR), X-Ray diffraction (XRD), polarized light microscopy (PLM), and confocal Raman microscopy (CRM) to obtain the correlative structural and chemical information. Analysis results of chemical composition, FT-IR spectra and XRD indicated that with increasing reaction time, more hemicellulose and lignin would be removed. Correspondingly, there was an obvious increase of the cellulose relative crystallinity via extending reaction time. Results of the PLM observations showed that the birefringence gradually dimmed due to the diminishing of the cellulose component. Furthermore, the CRM mapping images showed the lignin component in compound middle lamellar (CML) was difficult to remove relatively as compared with that in secondary walls. These results indicated that the combination of spectroscopic and microscopic elucidation could give an insightful understanding of chemical changes in cellular level during pretreatment.
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Spectroscopic/Microscopic Elucidation for Chemical Changes During Acid Pretreatment on Arundo donax

    Corresponding author: ZHAI Shengcheng, zhais@njfu.edu.cn
  • College of Materials Science and Engineering, Nanjing Forestry University, Nanjing 210037

Abstract: The Arundo donax is a typical fast-growing species from the family Gramineae, which is widely cultivated in China. With a huge yield of A. donax in China, this plant offers great potential for biofuels production. The different types of organization of cell and the tissue in the A. donax could influence the efficiency of enzymatic hydrolysis. In this study, A. donax was subjected to 0.5% (w/w) sulfuric acid (H2SO4) for pretreatment at 140℃ for 10 min, 20 min, 40 min, and 60 min, respectively. The changes in microstructure, chemical composition, topochemical properties were comprehensively analyzed. Using a series of spectroscopic and microscopic techniques including Fourier transform infrared spectroscopy (FT-IR), X-Ray diffraction (XRD), polarized light microscopy (PLM), and confocal Raman microscopy (CRM) to obtain the correlative structural and chemical information. Analysis results of chemical composition, FT-IR spectra and XRD indicated that with increasing reaction time, more hemicellulose and lignin would be removed. Correspondingly, there was an obvious increase of the cellulose relative crystallinity via extending reaction time. Results of the PLM observations showed that the birefringence gradually dimmed due to the diminishing of the cellulose component. Furthermore, the CRM mapping images showed the lignin component in compound middle lamellar (CML) was difficult to remove relatively as compared with that in secondary walls. These results indicated that the combination of spectroscopic and microscopic elucidation could give an insightful understanding of chemical changes in cellular level during pretreatment.

1.   Introduction
  • Nowadays, utilizing sustainable and renewable lignocellulosic biomass for bioenergy and biofuels has gained great attention. The Arundo donax, a typical renewable biomass resource, has superior adaptability to humid condition and saline-alkali soils (You et al., 2016). According to the reports, the yield of the A. donax is 45–75 t/hm2 in China and the biomass income is 7500– 33 000 yuan(RMB)/hm2, offering attractive attention for biofuels production (Yu et al., 2009).

    Enzymatic hydrolysis of lignocellulosic materials has been considered as an environment-friendly process for bioconversion (Mou et al., 2013). However, the enzymatic efficiency is always limited by the intrinsic chemical, structural, and physicochemical properties according to different materials. In the cell walls of the lignocellulosic materials, the targeted cellulose micro- fibrils are embedded in amorphous components, such as lignin and hemicellulose, forming a complex matrix that prevents the chemical attack against cellulose cores (Himmel et al., 2007). Thus, an effective pretreatment is the key to break the tight structure of cell wall and improve enzymatic digestibility.

    Many pretreatment technologies have been studied including chemical, physical and biological methods. Acid pretreatment is a typical chemical pretreatment method and widely used (Singh et al., 2015; Loow et al., 2016). It has been reported that acid pretreatment is effective to hydrolyze hemicelluloses to soluble oligomers and monomers (Himmel et al., 2007; Yang et al., 2019). Hemicellulose, playing as a steric hindrance, physically limits the access of the cellulase to the surface of cellulose. Hence, the hydrolysis of hemicellulose helps to enhance the susceptibility of the cellulose to enzymatic hydrolysis (Zhu et al., 2008; Brienzo et al., 2016). Lignin inhibits enzyme attack on cellulose due to the non- productive absorption with the enzyme (Canilha et al., 2011). Although lignin was removed only to a limited extent in acidic media, the redistribution of lignin may open the tight structure matrix of the cell wall, resulting in improved porosity and cellulose digestibility (Donohoe et al., 2008).

    The effects of pretreatment on biomass composition and enzymatic hydrolysis efficiency have been analyzed deeply, while only a few works are focused on the microstructures of different cell types and topochemical changes in cell walls during pretreatment. Given the diversity of cell types and complexity of cell wall structures, the research at the hierarchical structure of the plant cell wall will contribute to indicating the micro-region chemical alteration during a gradient of thermochemical pretreatment. Polarized light microscopy (PLM), a technique in optical mineralogy and crystallography, has been used to determine the fibrillar orientation of various kinds of plant fibers for many years (Engels and Jung, 1998; Bergfjord et al., 2010; Zhai et al., 2014; Simonović et al., 2017). Due to the different orientation of the cellulose microfibril in cell walls, the multi-laminated cell walls display bright or dark images under polarized light, which gives a simple analytical approach to determine the structure of cell walls compared with the transmission electron microscope (TEM). Furthermore, confocal Raman microscopy (CRM) is garnering extensive interest in the study of dynamic chemical imaging in situ during lignocellulosic pretreatment and is proven to facilitate the understanding of topochemical changes in the cell wall during the pretreatment process (Ji et al., 2015).

    In this work, the internode sections of the A. donax were pretreated with 0.5% (w/w) sulfuric acid (H2SO4) at 140℃ with different reaction times. Microscopic and spectroscopic elucidations were used to get detailed understanding of changes occurring in the A. donax cell walls during dilute acid pretreatment. Analyses of the chemical composition, FT-IR spectra and XRD were used to discuss chemical and crystallinity alterations of the cell wall. Combining the PLM with CRM observation and then visualizing the chemical transferring during pretreatment in cellular level will explain how the pretreatment influences cell walls degradation.

2.   Materials and Methods
  • The A. donax was collected in December 2016, from Jiangsu Province, China, and air-dried at room temperature and kept in a desiccator at 10% R.H. The internodes of air-dried materials were then hand-cut into (1–2) cm × 0.5 cm (length × diameter) sticks. Sulfuric acid (H2SO4, ≥ 98%) was purchased from Sinopharm Chemical Reagent Co. Ltd. and diluted to 0.5% (w/w) with deionized water before using.

  • For the pretreatment (Fig. 1), 6 g (on a dry weight basis) A. donax was presoaked in 60 mL of 0.5% (w/w) H2SO4 aqueous solution at room temperature overnight. Then the mixture was transferred into a 100 mL Teflon-lined autoclave and heated in a hot oil bath. The initial temperature (90℃) was raised to the target temperature (140℃) at a heating rate of 2℃/min and the target temperature was maintained for 10 min, 20 min, 40 min, and 60 min, respectively. After the pretreatment, the containers were submerged in a cold water bath to terminate the reaction. The water-insoluble solid was washed by hot deionized water to neutral (pH=7.0) and saved under refrigeration (4℃).

    Figure 1.  Schematic illustration of pretreatment

  • The chemical compositions of the raw material and pretreated samples were determined according to the National Renewable Energy Laboratory (NREL) Analytical Procedure (Sluiter et al., 2012). All measurements were carried out in duplicate and average data were reported. The solid yield (Sy), recovery yields of cellulose or hemicellulose (Rce/he), and degree of delignification (DD) were calculated to evaluate the effectiveness of pretreatments, as expressed by equations (1)–(3) (Mou et al., 2014):

  • The chemical structure of the raw material and pretreated samples were characterized by FT-IR spectroscopy. The FT-IR spectra were obtained on an FT-IR spectrophotometer (Thermo Electron Nicolet-360, USA) from the sample (1%) in KBr pellets in the range of 500–4000 cm-1. And each spectrum of the thin films was acquired by the accumulation of 32 scans with a resolution of 4 cm-1.

  • The XRD was used to characterize the crystallinity of raw and treated samples. The samples were scanned and recorded by the Ultima IV diffractometer (Rigaku, Japan) with Cu Kα radiation (λ=0.15406 nm), operating at 40 kV and 30 mA. The scan was performed at 2θ from 5° to 40° with a step size of 0.05° and a rate of 2°/min. The crystallinity index (CrI) of the samples is calculated according to the following formula (Mou et al., 2014):

    where I(002) is the intensity for the crystalline portion of the sample at about 2θ=22.2°, and I(am) is the peak for the amorphous area at about 2θ=18.3°.

  • The raw material and pretreated samples were cut into 0.3 cm × 0.3 cm × 0.5 cm and treated with glutaraldehyde, followed by washing with 10×PBS (PBS, phosphate buffer saline). All small sticks were dehydrated with a graded ethanol series and then embedded in Epon812. To observe the changes of cell wall structure during pretreatment, semi-thin transverse sections (2 μm) were cut from embedded blocks, using an RM2265 microtome (Leica, Germany) with a glass knife. Part of the sections was stained with toluidine blue and photographed with a light microscope (Olympus BX51, Japan) equipped with a digital camera. The sections were observed directly between crossed nicols in a polarized light microscope (PLM) to analysis changes of birefringence.

  • The 5 μm thick cross sections were cut from the raw material and pretreated samples by sliding microtome (Yamato REM-710, Japan). For Raman microscopy, the cross sections were paved on a glass slide with a drop of D2O, covered by a coverslip and sealed with nail-polish to avoid evaporation. A LabRam Xplora confocal Raman microscope with ×100 objective lens and a laser in the visible wavelength (λ=532 nm) was used in this study. The wavenumber was set from 3200 cm–1 to 600 cm–1 and slit width at 100 μm. The spectra from each location were acquired by averaging 2 s cycles. For mapping, an integration time of 2 s was chosen and every pixel corresponded to one scan with a spectrum (scan) obtained by 1 μm steps.

3.   Results and Discussion
  • The purpose of pretreatment is to remove lignin or hemicelluloses and then to expose more cellulose cores for improving the enzymatic digestibility of biomass. In this work, the effects of treatments on the chemical components of the A. donax are shown in Table 1. The hemicellulose content in the raw material was about 21.88%, which decreased to 10.45% after 60 min of pretreatment. On the other hand, a relative increase in cellulose content was detected from 40.06% to 55.04%. For lignin content, it was changed from 23.25% to 27.31%.

    Reaction time Cellulose Hemicellulose Lignin Sy Rcellulose Rhemicellulose DD
    Raw material 40.06 (0.74) 21.88 (0.01) 23.25 (0.54)
    10 min 43.92 (0.17) 20.37 (0.18) 24.18 (0.20) 74.81 82.01 69.61 22.21
    20 min 47.05 (0.09) 17.61 (0.07) 25.53 (0.40) 72.81 80.84 55.38 24.42
    40 min 51.03 (0.68) 11.87 (1.09) 26.88 (0.15) 49.40 62.92 26.79 42.90
    60 min 55.04 (0.43) 10.45 (0.18) 27.31 (0.01) 43.15 59.28 20.61 49.32
    Notes: hemicellulose, sum of xylose and arabinose; lignin, sum of soluble and insoluble lignin; Sy, solid yield; DD, degree of delignification. The extractives content of raw A. donax was 5.32%.

    Table 1.  Chemical compositions of A. donax after pretreatment and effects of pretreatment (%)

    From Table 1, it could be found that the solid yield (Sy) of pretreated A. donax decreased from 74.81% to 43.15% with the reaction time increased from 10 min to 60 min, indicating that more chemical components were removed during the periods. For instance, the recovery ratios of cellulose (Rcellulose) and hemicellulose (Rhemicellulose) diminished from 82.01% and 69.61% to 59.28% and 20.61%, respectively. The results illustrated that increasing reaction time could facilitate the removal of carbohydrates. However, the Rcellulose was higher than Rhemicellulose at the same reaction time, indicating that cellulose has relative tough recalcitrance towards dilute acid hydrolysis. The glycosidic bonds in hemicellulose main chains are less stabilized than cellulose and rapidly depredated by thermochemical pretreatment due to its more amorphous, higher solubility and chemically reactivity than cellulose (Ibbett et al., 2011; Chen et al., 2016). In addition, the degree of delignification (DD) enhanced with time as well. About 22.21%–49.32% of the lignin was removed when time extended from 10 min to 60 min. The H+ protons (supplied by acids) could attack the ether bond in lignin and result in loss of lignin (Wu et al., 2018). Moreover, the acetyl groups in lignocellulose and monosaccharides in liquid could form acetic acid under high temperature and then the acidic decomposition effects upon native lignin structures were increased (Wu et al., 2018).

  • To characterize the structural changes of the chemical substrates, the FT-IR spectra of the raw material and pretreated samples are illustrated in Fig. 2. The main chemical bands are assigned and summarized in Table 2 as previous described (Tjeerdsma et al., 2005; Castillo et al., 2015; Karimi, 2016). The band at 1734 cm-1 is characteristic of C=O conjugates in hemicellulose (Oh et al., 2005), and it could be clearly found that the absorbance intensity decreased with increasing time for pretreatment, which meant the removal of hemicellulose (Fig. 2b). This finding was in agreement with the trend of hemicellulose content shown in Table 1. The absorbance band around 1605 cm–1 is associated with the aromatic skeletal stretching together with —COO— stretching in hemicellulose, which is related to the connection of lignin and hemicellulose (Mou et al., 2014). A clear disappearance in the peak at 1605 cm–1 represented the broken of linkages between lignin and carbohydrate (Fig. 2b). The intensity of absorbance band at 1505 cm–1 was ascribed with aromatic skeletal vibrations of lignin, which increased in comparison with that of the raw material. This phenomenon was consistent with the changes of lignin content in Table 1. Horikawa et al. (2019) found the band at 1508 cm–1 did not overlap those related to the polysaccharides and could be used for estimating the lignin content. The increase of relative content of lignin in pretreated samples illustrated that the acid conditions mainly destroyed the side chains of lignin, but could not break the aromatic rings of lignin significantly (Chen et al., 2018; Raj et al., 2018).

    Figure 2.  The FT-IR spectra of raw material and pretreated samples

    Functional group Wavenumber (cm–1)
    Hydrogrn bonded stretching (O—H) 3400
    Stretching in methyl and methylene groups 2909
    Unconjugated C=O in xylans (hemicellulose) 1734
    Conjugated C=O in lignin; aromatic skeletal in lignin 1605
    Aromatic skeletal in lignin 1505
    C—H bending vibration in cellulose and hemicellulose 1375
    C—H deformation in cellulose and C—O vibration in syringyl derivatives1330
    C—O—C vibration in cellulose and hemicellulose 1158
    C—H deformation in cellulose 897

    Table 2.  Summary of chemical band assignment by FT-IR

    The crystallinity of raw materials and pretreated samples was determined by the XRD and the results are shown in Fig. 3. Compared with the CrI of the raw A. donax (50.05%), all the CrI of pretreated samples increased, which might be ascribed to the removal of hemicellulose and lignin (Table 1). However, when the reaction time was prolonged to 60 min, there was a slight decrease of CrI (53.33%). The FT-IR spectrum is an approach to analyze the changes in relative cellulose crystallinity as well. The O—C in-plane bending vibrations at 1422 cm–1 and C—H bending vibration at 1376 cm–1 are very sensitive to the crystalline region of cellulose, while the C—O stretch at 897 cm–1 and C—H stretching vibration at 2900 cm–1 are assigned to the amorphous structure of cellulose (Karimi et al., 2016). According to Raj et al. (2018), two infrared ratios were calculated: (1) the lateral order index (LOI) was calculated from the ratio of peak areas of 1422/897 cm–1, which represents the overall degree of order of the cellulose chains; (2) the total crystallinity index (TCI) was calculated from the ratio of peak areas of 1375/2900 cm–1, which is known as proportional to the crystallinity index of cellulose. From Fig. 3, it could be found that with the increasing of pretreated time, the ratios of LOI and TCI tended to rise, then occurred a slight reduction, which were similar to the result performed by the XRD.

    Figure 3.  Crystallinity of A. donax by XRD and FT-IR after pretreatment

    The CrI value calculated from the XRD reflects the total sample crystallinity. It is sensitive to other amorphous components such as hemicellulose, lignin and disordered cellulose, which together contribute to the amorphous phase signal of the sample (Ling et al., 2017). Many studies have proposed that the acid pretreatment increased the CrI of biomass by eliminating hemicellulose, lignin and other amorphous components (Brienzo et al., 2016; Ji et al., 2016). Additionally, the increase of the CrI after dilute acid pretreatment may also result from the reconfiguration of amorphous domains into para-crystalline cellulose (Sun et al., 2014). However, the swelling of cellulose, removal, and redistribution of substances in pretreatment, or hydrolysis of crystalline zones under severe conditions may also lead to a decrease in crystallinity (Phitsuwan et al., 2016; Zhang et al., 2018). As shown in Fig. 3, the lower LOI values could indicate a more disordered form of cellulose. In addition, the severe condition might lead to the trace destruction of the crystal structure and superimposed the removal of hemicellulose and lignin, resulting in a decreasing tendency in the CrI of biomass when the time was prolonged from 20 min to 60 min.

  • The transverse sections of sample are shown in Fig. 4. The vascular bundle (VB) was distributed randomly and embedded in the soft parenchyma (Par) tissue. The VB comprised the sclerenchyma fibers (SF), metaxylem vessel (MV) and protoxylem vessel (PV). The structural complexity of the VBs formed the protective sheath, which contributed to the highest degree of recalcitrance in the inner part of grass culm (Evert, 2006).

    Figure 4.  Light micrographs and polarized light micrographs of transections from raw A. donax clum internode region (a–c) and sample pretreated under 140℃, 60 min (d–f)

    The results of Fig. 4a and Fig. 4b show that the structure of the untreated sample was complete, and all tissues presented the uniform chromaticity. However, after the pretreatment, as shown in Fig. 4d and Fig. 4e, the color in cell corner (CC), compound middle lamellar (CML) is relatively deepened, and the sclerenchyma fibers revealed the characteristically multilayered cell wall structure (indicated by black arrowheads). Toluidine blue is a common stain and generally, stains lignified cell walls into blue or blue-green color, which could be used to distinguish lignified tissues from non-lignified tissues (Abdul Khalil et al., 2008). Thus, the deep colored regions (CC and CML) presented higher lignin concentrations. This phenomenon was in agreement with a previous study that of the lignin aggregated in CC and CML after dilute acid pretreatment (Ji et al., 2015).

    The PLM clearly showed the double or more rings of birefringence in the cell wall of the A. donax, which indicated the cell wall of A. donax was typically multilayered structure (Fig. 4). Due to the variation of the helix structure of microfibrils in different layers, the cell wall will turn either slight blue or yellow, with a 530-nm full wave compensator inserted at 45° to the cross-polar (Haugan et al., 2013). The results of the PLM observations showed the brightness of birefringence weakened after pretreatment (Fig. 4c and Fig. 4f). Murata et al. (2015) have found the covariance between the cellulose component and the polarization was positive and the absolute cellulose content was more highly correlated with the brightness of birefringence than other compositions. The data in Table 1 showed the gradually reduce of cellulose recovery over time, and correspondingly, the cell wall dimmed with extended reaction time. Other studies (Thygesen et al., 2011; Gu et al., 2018) also found the brightness would decrease as the crystallization area broken down.

  • The CRM has been considered as a noninvasive way to provide chemical, quantitative and structural information in situ with a high spatial resolution (Richter et al., 2011). To get detailed insights into the molecular composition of different tissue types, the confocal Raman imaging technique was applied on untreated cross sections and a set of average Raman spectra extracted from secondary wall are shown in Fig. 5. The band at 1600 cm–1 is assigned to the aryl ring symmetric stretching. The bands located between 2900 cm–1and 3000 cm–1 are known for the C—H and C—H2 stretching modes, and 1050–1150 cm–1 corresponding mainly to C—C ring breathing and C—O—C stretching vibrations of carbohydrates (Richter et al., 2011; Gierlinger et al., 2012). As shown in Fig. 5, it is clear that the strongest Raman intensity of lignin is in the metaxylem vessel (MV), followed by the parenchyma far from the vascular bundle (P-far), the parenchyma near the vascular bundle (P-near). The lowest intensity of lignin is found in sclerenchyma fibers (SF). Compared with the lignin, the distribution of carbohydrates was the opposite.

    Figure 5.  Average Raman spectra acquired from secondary cell walls of different cell types in raw material

    To determine subtle dynamic changes of lignin distribution in different morphological regions for different pretreatment times, a set of mapping analyses for Raman images was carried out. As shown in Fig. 6, signals of Raman spectra are converted into digitized images showing lignin distribution by integrating over 1570–1680 cm–1 region which produced by the symmetric stretching of aromatic rings (Ji et al., 2015). The highest intensity is found in the CC in sclerenchyma fiber and the CML between sclerenchyma fiber and parenchyma which suggested accumulation of more lignin in these regions, in contrast to the secondary wall in sclerenchyma fiber. During pretreatment, the increase in lignin intensity was observed in the cell walls from samples pretreated for 10 min and 20 min (Fig. 6b and Fig. 6c), compared with the raw material. Acid pretreatment broke down the covalent bonds between lignin and polysaccharides, resulting in more signals of lignin being detected. In addition, lignin could become expanded and mobile within the cell wall matrix during thermochemical pretreatment then migrated to the CC and CML and coalesced in these regions (Donohoe et al., 2008). Migration and redistribution of lignin might be another reason that enhancing lignin signal intensity during the first 20 min period. When the duration was prolonged to 40 min, a remarkable delignification occurred in the CML regions and secondary walls. This finding was consistent with the aforementioned view that extending reaction time could facilitate lignin removal. As illustrated in Fig. 6, lignin was removed preferentially from the secondary walls compared with the CML. As previously reported, the solvent preferentially absorbed from the cell lumen to the CML. And the looser structure of the secondary wall, due to abundant syringly units and fewer cross-linkages structure, is easier to react during pretreatment (Ji et al., 2014).

    Figure 6.  Raman images showing lignin distribution within cell walls at various pretreatment conditions by integrating from 1570 cm–1 to 1680 cm–1

4.   Conclusions
  • In this work, the A. donax was subjected to 0.5% H2SO4 pretreatment at 140℃ with different reaction time. The results showed that increasing reaction time could improve the removal of carbohydrates and lignin, thus increase the CrI of cellulose. The PLM reflected the absolute cellulose content changes through the brightness of birefringence. Raman mapping images indicated that delignification was heterogeneous and lignin would transfer and redistribute in the CML. Both the PLM and CRM could support the visualization of gradually changes in the qualitative content of cellulose and lignin in different cell types during pretreatment. The combination of spectroscopic and microscopic measurements and chemical analysis is capable of providing intuitionistic and live vision information in cellular level during pretreatment.

Acknowledgments
  • This research was supported by National Natural Science Foundation of China (No. 31400496) and Natural Science Foundation of Jiangsu Province (No. BK20180774). The authors thank Prof. Junji Sugiyama for the technical assistance with confocal Raman microscopy and the Laboratory of Biomass Morphogenesis and Information, Research Institute for Sustainable Humanosphere, Kyoto University, Japan. The experiments in this research were mainly carried out at the advanced analysis and testing center of Nanjing Forestry University.

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