Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation² and other products such as biocomposite materials⁶. Plant biomass remains one of the greatest untapped reserves on the planet⁴. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin⁵ and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation⁵. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant⁴. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose⁷. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I³.     

Biomass Enzymatic Saccharification Is Determined by the Non-KOH-Extractable Wall Polymer Features That Predominately Affect Cellulose Crystallinity in Corn

Corn is a major food crop with enormous biomass residues for biofuel production. Due to cell wall recalcitrance, it becomes essential to identify the key factors of lignocellulose on biomass saccharification. In this study, we examined total 40 corn accessions that displayed a diverse cell wall composition. Correlation analysis showed that cellulose and lignin levels negatively affected biomass digestibility after NaOH pretreatments at p<0.05 & 0.01, but hemicelluloses did not show any significant impact on hexoses yields. Comparative analysis of five standard pairs of corn samples indicated that cellulose and lignin should not be the major factors on biomass saccharification after pretreatments with NaOH and H₂SO₄ at three concentrations. Notably, despite that the non-KOH-extractable residues covered 12%–23% hemicelluloses and lignin of total biomass, their wall polymer features exhibited the predominant effects on biomass enzymatic hydrolysis including Ara substitution degree of xylan (reverse Xyl/Ara) and S/G ratio of lignin. Furthermore, the non-KOH-extractable polymer features could significantly affect lignocellulose crystallinity at p<0.05, leading to a high biomass digestibility. Hence, this study could suggest an optimal approach for genetic modification of plant cell walls in bioenergy corn.     

High‐level hemicellulosic arabinose predominately affects lignocellulose crystallinity for genetically enhancing both plant lodging resistance and biomass enzymatic digestibility in rice mutants

Rice is a major food crop with enormous biomass residue for biofuels. As plant cell wall recalcitrance basically decides a costly biomass process, genetic modification of plant cell walls has been regarded as a promising solution. However, due to structural complexity and functional diversity of plant cell walls, it becomes essential to identify the key factors of cell wall modifications that could not much alter plant growth, but cause an enhancement in biomass enzymatic digestibility. To address this issue, we performed systems biology analyses of a total of 36 distinct cell wall mutants of rice. As a result, cellulose crystallinity (CrI) was examined to be the key factor that negatively determines either the biomass enzymatic saccharification upon various chemical pretreatments or the plant lodging resistance, an integrated agronomic trait in plant growth and grain production. Notably, hemicellulosic arabinose (Ara) was detected to be the major factor that negatively affects cellulose CrI probably through its interlinking with β‐1,4‐glucans. In addition, lignin and G monomer also exhibited the positive impact on biomass digestion and lodging resistance. Further characterization of two elite mutants, Osfc17 and Osfc30, showing normal plant growth and high biomass enzymatic digestion in situ and in vitro, revealed the multiple GH9B candidate genes for reducing cellulose CrI and XAT genes for increasing hemicellulosic Ara level. Hence, the results have suggested the potential cell wall modifications for enhancing both biomass enzymatic digestibility and plant lodging resistance by synchronically overexpressing GH9B and XAT genes in rice.     

Lignocellulose conversion for biofuel: a new pretreatment greatly improves downstream biocatalytic hydrolysis of various lignocellulosic materials

Background

Lignocellulosic biomass is an attractive renewable resource for future liquid transport fuel. Efficient and cost-effective production of bioethanol from lignocellulosic biomass depends on the development of a suitable pretreatment system. The aim of this study is to investigate a new pretreatment method that is highly efficient and effective for downstream biocatalytic hydrolysis of various lignocellulosic biomass materials, which can accelerate bioethanol commercialization.

Results

The optimal conditions for the hydrogen peroxide–acetic acid (HPAC) pretreatment were 80 °C, 2 h, and an equal volume mixture of H₂O₂ and CH₃COOH. Compared to organo-solvent pretreatment under the same conditions, the HPAC pretreatment was more effective at increasing enzymatic digestibility. After HPAC treatment, the composition of the recovered solid was 74.0 % cellulose, 20.0 % hemicelluloses, and 0.9 % lignin. Notably, 97.2 % of the lignin was removed with HPAC pretreatment. Fermentation of the hydrolyzates by S. cerevisiae resulted in 412 mL ethanol kg^?1 of biomass after 24 h, which was equivalent to 85.0 % of the maximum theoretical yield (based on the amount of glucose in the raw material).

Conclusion

The newly developed HPAC pretreatment was highly effective for removing lignin from lignocellulosic cell walls, resulting in enhanced enzymatic accessibility of the substrate and more efficient cellulose hydrolysis. This pretreatment produced less amounts of fermentative inhibitory compounds. In addition, HPAC pretreatment enables year-round operations, maximizing utilization of lignocellulosic biomass from various plant sources.

     

Genetic modification of lignin biosynthesis for improved biofuel production

The energy in cellulosic biomass largely resides in plant cell walls. Cellulosic biomass is more difficult than starch to break down into sugars because of the presence of lignin and the complex structure of cell walls. Transgenic down-regulation of major lignin genes led to reduced lignin content, increased dry matter degradability, and improved accessibility of cellulases for cellulose degradation. This review provides background information on lignin biosynthesis and focuses on genetic manipulation of lignin genes in important monocot species as well as the dicot potential biofuel crop alfalfa. Reduction of lignin in biofuel crops by genetic engineering is likely one of the most effective ways of reducing costs associated with pretreatment and hydrolysis of cellulosic feedstocks, although some potential fitness issues should also be addressed.     

Unravelling the impact of lignin on cell wall mechanics: a comprehensive study on young poplar trees downregulated for CINNAMYL ALCOHOL DEHYDROGENASE (CAD)

Lignin engineering is a promising tool to reduce the energy input and the need of chemical pre‐treatments for the efficient conversion of plant biomass into fermentable sugars for downstream applications. At the same time, lignin engineering can offer new insight into the structure–function relationships of plant cell walls by combined mechanical, structural and chemical analyses. Here, this comprehensive approach was applied to poplar trees (Populus tremula × Populus alba) downregulated for CINNAMYL ALCOHOL DEHYDROGENASE (CAD) in order to gain insight into the impact of lignin reduction on mechanical properties. The downregulation of CAD resulted in a significant decrease in both elastic modulus and yield stress. As wood density and cellulose microfibril angle (MFA) did not show any significant differences between the wild type and the transgenic lines, these structural features could be excluded as influencing factors. Fourier transform infrared spectroscopy (FTIR) and Raman imaging were performed to elucidate changes in the chemical composition directly on the mechanically tested tissue sections. Lignin content was identified as a mechanically relevant factor, as a correlation with a coefficient of determination (r²) of 0.65 between lignin absorbance (as an indicator of lignin content) and tensile stiffness was found. A comparison of the present results with those of previous investigations shows that the mechanical impact of lignin alteration under tensile stress depends on certain structural conditions, such as a high cellulose MFA, which emphasizes the complex relationship between the chemistry and mechanical properties in plant cell walls.     

Visualization of biomass solubilization and cellulose regeneration during ionic liquid pretreatment of switchgrass

Auto‐fluorescent mapping of plant cell walls was used to visualize cellulose and lignin in pristine switchgrass (Panicum virgatum) stems to determine the mechanisms of biomass dissolution during ionic liquid pretreatment. The addition of ground switchgrass to the ionic liquid 1‐n‐ethyl‐3‐methylimidazolium acetate resulted in the disruption and solubilization of the plant cell wall at mild temperatures. Swelling of the plant cell wall, attributed to disruption of inter‐ and intramolecular hydrogen bonding between cellulose fibrils and lignin, followed by complete dissolution of biomass, was observed without using imaging techniques that require staining, embedding, and processing of biomass. Subsequent cellulose regeneration via the addition of an anti‐solvent, such as water, was observed in situ and provided direct evidence of significant rejection of lignin from the recovered polysaccharides. This observation was confirmed by chemical analysis of the regenerated cellulose. In comparison to untreated biomass, ionic liquid pretreated biomass produces cellulose that is efficiently hydrolyzed with commercial cellulase cocktail with high sugar yields over a relatively short time interval. Biotechnol. Bioeng. 2009; 104: 68–75 Published 2009 Wiley Periodicals, Inc.     

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels^1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals⁴. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation⁷. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor⁸ and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications^9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls^12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood¹⁵. Analyzing the lignin Raman bands^16,17 in the spectral region between 1,600 and 1,700 cm^-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-μm¹⁸. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.Download video file.^{(47M, mov)}     

Biomass digestibility is predominantly affected by three factors of wall polymer features distinctive in wheat accessions and rice mutants

Background

Wheat and rice are important food crops with enormous biomass residues for biofuels. However, lignocellulosic recalcitrance becomes a crucial factor on biomass process. Plant cell walls greatly determine biomass recalcitrance, thus it is essential to identify their key factors on lignocellulose saccharification. Despite it has been reported about cell wall factors on biomass digestions, little is known in wheat and rice. In this study, we analyzed nine typical pairs of wheat and rice samples that exhibited distinct cell wall compositions, and identified three major factors of wall polymer features that affected biomass digestibility.

Results

Based on cell wall compositions, ten wheat accessions and three rice mutants were classified into three distinct groups each with three typical pairs. In terms of group I that displayed single wall polymer alternations in wheat, we found that three wall polymer levels (cellulose, hemicelluloses and lignin) each had a negative effect on biomass digestibility at similar rates under pretreatments of NaOH and H₂SO₄ with three concentrations. However, analysis of six pairs of wheat and rice samples in groups II and III that each exhibited a similar cell wall composition, indicated that three wall polymer levels were not the major factors on biomass saccharification. Furthermore, in-depth detection of the wall polymer features distinctive in rice mutants, demonstrated that biomass digestibility was remarkably affected either negatively by cellulose crystallinity (CrI) of raw biomass materials, or positively by both Ara substitution degree of non-KOH-extractable hemicelluloses (reverse Xyl/Ara) and p-coumaryl alcohol relative proportion of KOH-extractable lignin (H/G). Correlation analysis indicated that Ara substitution degree and H/G ratio negatively affected cellulose crystallinity for high biomass enzymatic digestion. It was also suggested to determine whether Ara and H monomer have an interlinking with cellulose chains in the future.

Conclusions

Using nine typical pairs of wheat and rice samples having distinct cell wall compositions and wide biomass saccharification, Ara substitution degree and monolignin H proportion have been revealed to be the dominant factors positively determining biomass digestibility upon various chemical pretreatments. The results demonstrated the potential of genetic modification of plant cell walls for high biomass saccharification in bioenergy crops.

     

Understanding tissue specific compositions of bioenergy feedstocks through hyperspectral Raman imaging

Hyperspectral Raman imaging was used to study the tissue/cell type specific distribution of lignin and cellulose polymers within the plant cell walls. Distinct differences in cell wall compositions were identified between two potential bioenergy feedstocks: corn stover and Eucalyptus globulus. Characteristic bands of 627, 1,175, 1,206, and 1,428 cm⁻¹ were only observed for corn stover and 1,381 cm⁻¹ was only present in E. globulus. One‐dimensional and two‐dimensional chemical maps of lignin and cellulose were generated for the stem of corn stover, ranging from the epidermis to the pith area and revealed that lignin and cellulose abundance varies significantly among different cell types in the following order: sclerenchyma cells and tracheids (∼5 times) > epidermal cells (∼3 times) > bundle sheath cells > parenchyma cells. The Raman mapping methods developed on corn stover were also validated on E. globulus and clearly highlighted their difference in lignin composition. Biotechnol. Bioeng. 2011;108: 286–295. © 2010 Wiley Periodicals, Inc.     

Advanced biorefinery in lower termite-effect of combined pretreatment during the chewing process

Background

Currently the major barrier in biomass utilization is the lack of an effective pretreatment of plant cell wall so that the carbohydrates can subsequently be hydrolyzed into sugars for fermentation into fuel or chemical molecules. Termites are highly effective in degrading lignocellulosics and thus can be used as model biological systems for studying plant cell wall degradation.

Results

We discovered a combination of specific structural and compositional modification of the lignin framework and partial degradation of carbohydrates that occurs in softwood with physical chewing by the termite, Coptotermes formosanus, which are critical for efficient cell wall digestion. Comparative studies on the termite-chewed and native (control) softwood tissues at the same size were conducted with the aid of advanced analytical techniques such as pyrolysis gas chromatography mass spectrometry, attenuated total reflectance Fourier transform infrared spectroscopy and thermogravimetry. The results strongly suggest a significant increase in the softwood cellulose enzymatic digestibility after termite chewing, accompanied with utilization of holocellulosic counterparts and an increase in the hydrolysable capacity of lignin collectively. In other words, the termite mechanical chewing process combines with specific biological pretreatment on the lignin counterpart in the plant cell wall, resulting in increased enzymatic cellulose digestibility in vitro. The specific lignin unlocking mechanism at this chewing stage comprises mainly of the cleavage of specific bonds from the lignin network and the modification and redistribution of functional groups in the resulting chewed plant tissue, which better expose the carbohydrate within the plant cell wall. Moreover, cleavage of the bond between the holocellulosic network and lignin molecule during the chewing process results in much better exposure of the biomass carbohydrate.

Conclusion

Collectively, these data indicate the participation of lignin-related enzyme(s) or polypeptide(s) and/or esterase(s), along with involvement of cellulases and hemicellulases in the chewing process of C. formosanus, resulting in an efficient pretreatment of biomass through a combination of mechanical and enzymatic processes. This pretreatment could be mimicked for industrial biomass conversion.     

Imaging Lignin-Downregulated Alfalfa Using Coherent Anti-Stokes Raman Scattering Microscopy

Targeted lignin modification in bioenergy crops could potentially improve conversion efficiency of lignocellulosic biomass to biofuels. To better assess the impact of lignin modification on overall cell wall structure, wild-type and lignin-downregulated alfalfa lines were imaged using coherent anti-Stokes Raman scattering (CARS) microscopy. The 1,600-cm^?1 Raman mode was used in CARS imaging to specifically represent the lignin signal in the plant cell walls. The intensities of the CARS signal follow the general trend of lignin contents in cell walls from both wild-type and lignin-downregulated plants. In the downregulated lines, the overall reduction of lignin content agreed with the previously reported chemical composition. However, greater reduction of lignin content in cell corners was observed by CARS imaging, which could account for the enhanced susceptibility to chemical and enzymatic hydrolysis observed previously.     

The use of natural abundance stable isotopic ratios to indicate the presence of oxygen-containing chemical linkages between cellulose and lignin in plant cell walls

Qualitative and quantitative understanding of the chemical linkages between the three major biochemical components (cellulose, hemicellulose and lignin) of plant cell walls is crucial to the understanding of cell wall structure. Although there is convincing evidence for chemical bonds between hemicellulose and lignin and the absence of chemical bonds between hemicellulose and cellulose, there is no conclusive evidence for the presence of covalent bonds between cellulose and lignin. This is caused by the lack of selectivity of current GC/MS-, NMR- and IR-based methods for lignin characterisation as none of these techniques directly targets the possible ester and ether linkages between lignin and cellulose. We modified the widely-accepted “standard” three-step extraction method for isolating cellulose from plants by changing the order of the steps for hemicellulose and lignin removal (solubilisation with concentrated NaOH and oxidation with acetic acid-containing NaClO₂, respectively) so that cellulose and lignin could be isolated with the possible chemical bonds between them intact. These linkages were then cleaved with NaClO₂ reagent in aqueous media of contrasting ¹⁸O/¹⁶O ratios. We produced cellulose with higher purity (a lower level of residual hemicellulose and no detectable lignin) than that produced by the “standard” method. Oxidative artefacts may potentially be introduced at the lignin removal stage; but testing showed this to be minimal.Cellulose samples isolated from processing plant-derived cellulose–lignin mixtures in media of contrasting ¹⁸O/¹⁶O ratios were compared to provide the first quantitative evidence for the presence of oxygen-containing ester and ether bonds between cellulose and lignin in Zea mays leaves. However, no conclusive evidence for the presence or lack of similar bonds in Araucaria cunninghamii wood was obtained.     

Lignin Depletion Enhances the Digestibility of Cellulose in Cultured Xylem Cells

Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinnia elegans . Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.     

Identifying new lignin bioengineering targets: 1. Monolignol-substitute impacts on lignin formation and cell wall fermentability

Background  

Recent discoveries highlighting the metabolic malleability of plant lignification indicate that lignin can be engineered to dramatically alter its composition and properties. Current plant biotechnology efforts are primarily aimed at manipulating the biosynthesis of normal monolignols, but in the future apoplastic targeting of phenolics from other metabolic pathways may provide new approaches for designing lignins that are less inhibitory toward the enzymatic hydrolysis of structural polysaccharides, both with and without biomass pretreatment. To identify promising new avenues for lignin bioengineering, we artificially lignified cell walls from maize cell suspensions with various combinations of normal monolignols (coniferyl and sinapyl alcohols) plus a variety of phenolic monolignol substitutes. Cell walls were then incubated in vitro with anaerobic rumen microflora to assess the potential impact of lignin modifications on the enzymatic degradability of fibrous crops used for ruminant livestock or biofuel production.     

Modifying plants for biofuel and biomaterial production

The productivity of plants as biofuel or biomaterial crops is established by both the yield of plant biomass per unit area of land and the efficiency of conversion of the biomass to biofuel. Higher yielding biofuel crops with increased conversion efficiencies allow production on a smaller land footprint minimizing competition with agriculture for food production and biodiversity conservation. Plants have traditionally been domesticated for food, fibre and feed applications. However, utilization for biofuels may require the breeding of novel phenotypes, or new species entirely. Genomics approaches support genetic selection strategies to deliver significant genetic improvement of plants as sources of biomass for biofuel manufacture. Genetic modification of plants provides a further range of options for improving the composition of biomass and for plant modifications to assist the fabrication of biofuels. The relative carbohydrate and lignin content influences the deconstruction of plant cell walls to biofuels. Key options for facilitating the deconstruction leading to higher monomeric sugar release from plants include increasing cellulose content, reducing cellulose crystallinity, and/or altering the amount or composition of noncellulosic polysaccharides or lignin. Modification of chemical linkages within and between these biomass components may improve the ease of deconstruction. Expression of enzymes in the plant may provide a cost‐effective option for biochemical conversion to biofuel.     

Understanding biomass recalcitrance in grasses for their efficient utilization as biorefinery feedstock

One of the main challenges for the deployment of lignocellulosic biorefineries in future years is to find renewable and secured biomass sources in order to obtain bio-sourced products, as an alternative to petroleum-based commodities. Grass biomass, considering its characteristics (availability, composition, productivity, possibility of being harvested from both arable (post-harvest residues) and non-agricultural lands), can be considered as a biomass source for the future. Nevertheless, because of its complex structure and composition, which need deconstructive pre-treatments to render possible further biological conversions, grasses utilisation in biorefinery is today not widespread. Indeed, recalcitrance to polymers degradation in grasses concerns structural and compositional characteristics and can result in costly and complicated biorefinery processes. Grass recalcitrance is due to various natural factors strongly related and difficult to dissociate: rind and vascular structures; composition (lignin content is a key factor for cellulose hydrolysis acting like a physical barrier while hemicelluloses seem to play a more significant role in woody biomass than in grass plants); physical structures (crystalline nature and insoluble surface of cellulose, specific surface area, particle size), etc. Physico-chemical pretreatments are efficient solutions to overcome recalcitrance, while phenotypic selections are interesting but not efficient enough to obtain an optimal enzymatic hydrolysis. In some cases, the structural elements of grass biomass can be negatively affected by physico-chemical pretreatments, causing pre-treatment-induced recalcitrance, like cellulose hornification (irreversible alteration of cellulose microfibers), vascular structure collapsed and reduced cellulose bioaccesibility to enzymes due to cellulose covering by lignin, following lignin solubilisation.