Recalcitrance Of Lignocellulose Research Articles

Hemicellulose modification and cell wall genetic improvement in plants

Hemicellulose, as a primary component of plant cell walls, constitutes approximately one third of cell wall dry matter and ranks as the second abundant renewable biomass resource in the nature after cellulose. Hemicellulose is tightly cross-linked with cellulose, lignin and other components in the plant cell wall, leading to lignocellulose recalcitrance. However, precise genetic modifications of plant cell walls can significantly improve the saccharification efficiency of lignocellulose while ensuring normal plant growth and development. We comprehensively review the research progress in the structural distribution of hemicellulose in plant cell walls, the cross-linking between hemicellulose and other components of the cell wall, and the impact of hemicellulose modification on the saccharification efficiency of the cell wall, proving a reference for the genetic improvement of energy crops.

Multi enzyme production from mixed untreated agricultural residues applied in enzymatic saccharification of lignocellulosic biomass for biofuel

Lignocellulose recalcitrance essentially dictates high cost and low efficiency of its enzymatic saccharification. The complex lignocellulosic structure is a major obstacle to enzymatic saccharification and green ionic liquid pretreatment methods face difficulty of water washing prior enzymatic hydrolysis. Ionic liquid tolerant lignocellulolytic enzymes overcome the inhibition of enzymes during enzymatic saccharification and eliminated water washing step after pretreatment of lignocellulosic biomass. The cell wall composition cellulose, hemicellulose, and lignin, three main wall polymers of various agricultural residues and food waste lignocellulose substrates also affected the enzyme activities secreted by the B. subtilis strain. This study explores the potential of the halophilic, alkalophilic, and ionic liquid (IL)-tolerant strain Bacillus subtilis BC-001 for the simultaneous production of hydrolytic enzymes essential for lignocellulosic bio-refinery processes. BC-001 produced cellulase, amylase, xylanase, pectinase and protease are 70.41, 87.14, 65.50, 122.55 and 66.48 U/mL, respectively under optimized fermentation conditions. Cellulase produced by BC-001 retained more than 80% activity after 72 hours in 20% w/w of different ILs tested and enzymes retained more than 68% activities after 12 h in 50% w/w ILs. Employing such IL-stable cellulase, enzymatic saccharification is conducted without water washing on rice straw (RS) that has been pretreated with various ILs, including 1-ethyl-3-methylimidazolium chloride, 1-butyl-3-methylimidazolium chloride, and choline chloride. This research is novel as the study marks the first instance of multi-hydrolytic enzyme production from a novel alkalophilic, halophilic, thermophilic, and IL-tolerant bacterial strain using a mixture of untreated agricultural residues.

The bioaugmentation effect of microbial inoculants on humic acid formation during co-composting of bagasse and cow manure

The effective degradation of recalcitrant lignocellulose has emerged as a bottleneck for the humification of compost, and strategies are required to improve the efficiency of bagasse composting. Bioaugmentation is a promising method for promoting compost maturation and improving the quality of final compost. In this study, the bioaugmentation effects of microbial inoculants on humic acid (HA) formation during lignocellulosic composting were explored. In the inoculated group, the maximum temperature was increased to 72.5 °C, and the phenol-protein condensation and Maillard humification pathways were enhanced, thus increasing the HA content by 43.85%. After inoculation, the intensity of the microbial community interactions increased, particularly for fungi (1.4-fold). Macrogenomic analysis revealed that inoculation enriched thermophilic bacteria and lignocellulose-degrading fungi and increased the activity of carbohydrate-active enzymes and related metabolic functions, which effectively disrupted the recalcitrant structure of lignocellulose to achieve a high humification degree. Spearman correlation analysis indicated that Stappia of the Proteobacteria phylum, Ilumatobacter of the Actinomycetes phylum, and eleven genera of Ascomycota were the main HA producers. This study provides new ideas for bagasse treatment and recycling and realizing the comprehensive use of resources.

Upgrading pectin methylation for consistently enhanced biomass enzymatic saccharification and cadmium phytoremediation in rice Ospmes site-mutants

Crop straws provide enormous biomass residues applicable for biofuel production and trace metal phytoremediation. However, as lignocellulose recalcitrance determines a costly process with potential secondary waste liberation, genetic modification of plant cell walls is deemed as a promising solution. Although pectin methylation plays an important role for plant cell wall construction and integrity, little is known about its regulation roles on lignocellulose hydrolysis and trace metal elimination. In this study, we initially performed a typical CRISPR/Cas9 gene-editing for site mutations of OsPME31, OsPME34 and OsPME79 in rice, and then determined significantly upgraded pectin methylation degrees in the young seedlings of three distinct site-mutants compared to their wild type. We then examined distinctively improved lignocellulose recalcitrance in three mutants including reduced cellulose levels, crystallinity and polymerization or raised hemicellulose deposition and cellulose accessibility, which led to specifically enlarged biomass porosity either for consistently enhanced biomass enzymatic saccharification under mild alkali pretreatments or for cadmium (Cd) accumulation up to 2.4-fold. Therefore, this study proposed a novel model to elucidate how pectin methylation could play a unique enhancement role for both lignocellulose enzymatic hydrolysis and Cd phytoremediation, providing insights into precise pectin modification for effective biomass utilization and efficient trace metal exclusion.

Lignin reduction in sugarcane by performing CRISPR/Cas9 site-direct mutation of SoLIM transcription factor

Genetic engineering of plant cell walls is limited for reducing lignocellulose recalcitrance, so mild and/or green-like pretreatment is still required for sequential enzymatic saccharification. Here, we report a method to reduce lignin content in sugarcane stalks using the CRISPR/Cas 9 technique. Three target sequences of SoLIM were designed and fused to pRGEB32. The cassette constructs were introduced into sugarcane calli cv. KK3 through Agrobacterium-mediated transformation. We produced one base substitution and one insertion line for the 1st target site; two insertions, one deletion, and one base substitution for the 2nd target site; and one base substitution and insertion for the 3rd target site. qRT-PCR analysis of SoLIM, SoPAL, SoC4H, and SoCAD showeded that downregulation of SoLIM by single nucleotide insertions or deletions reduced the expression of SoPAL, SoC4H, and SoCAD. Consequently, the edited lines contained 9.74 to 51.46% less lignin content compared to that in the wild-type plants. The syringyl/guaiacyl (S/G) ratio of the edited lines ranged between 0.23 and 0.49, while the wild-type was 0.22. The histochemical evaluation and scanning electron microscopy of the cell walls supported this observation. A low lignin content sugarcane will provide a better feedstock for second-generation bioethanol production.

Microbial electrochemical composting: A sustainable strategy to enhance lignocellulose conversion into humus

Achieving satisfactory levels of humification during composting is recognised as a challenge due to the recalcitrance of lignocellulose and the uncertainty of current biofortification techniques. Therefore, a novel microbial electrochemical composting technology was developed from insights into microbiology and electrochemistry in this study to strengthen microorganisms’ function promoting the conversion from lignocellulose into humus. Firstly, a fungus (Aspergillus oryzae) was introduced which exhibited a higher propagation rate (29.9%) and secreted more lignocellulose-degrading enzymes (18.9–74.9%) under electric field assistance. Subsequently, an in-situ composting trial was conducted with supplementary electric fields simultaneously combined with Aspergillus oryzae inoculation. The abundance of Aspergillus oryzae increased 3.0–7.8-fold under the influence of electric field, which enabled the production of more extracellular enzymes (by 8.4–149.5%) to promote the conversion of lignocellulose to humus (increased by 7.0–20.2%). The results indicated electric field can act as an activator of Aspergillus oryzae and magnify its function. It provides an innovative and efficient technique for enhancing humification during composting that contributes to the conversion of organic waste into high-quality soil conditioner products.

Performance and mechanism of various microaerobic pretreatments on anaerobic digestion of tobacco straw

Tobacco straw is an abundant biomass in China's agricultural ecosystems, and has high potential for methane production. However, the anaerobic digestion (AD) efficiency is limited by the recalcitrant lignocellulose structure of the tobacco straw. In this study, three microaerobic pretreatments were performed for the AD of tobacco straw to increase methane production. Among them, microbial pretreatment with biogas slurry at an oxygen concentration of 4 mL/g VS resulted in the highest methane production of 349.1 mL/g VS, increasing by 19.8 % than that of untreated. During this pretreatment, the relative abundances of Enterococcus and Clostridium sensu stricto 12, which are closely related to acetic acid production and cellulose degradation, were high, and these bacteria might have an important contribution to substrate hydrolysis and the methanogenesis efficiency of the AD process. This study advances the understanding of microaerobic pretreatment processes and provides technological guidance for the efficient utilization of tobacco straw.

Sustainable structural polysaccharides conversion: How does DES pretreatment affect cellulase adsorption, thereby improving enzymatic digestion of lignocellulose?

Biomass conversion aims at degrading the structural polysaccharides of lignocellulose into reducing sugars. Pretreatment is necessary to overcome the recalcitrance of lignocellulose. The DES La/ChCl in this paper was selected based on our previous study. To examine cellulase adsorption of lignocellulose after DES pretreatment, sorghum straw was pretreated with DES under different condition. The adsorption improvement of cellulase on lignocellulose after DES pretreatment has positive impact on reducing sugar production of biomass. After DES pretreatment, 1. pore corrosion caused the upward trend of pore radius and the downward trend of SSA. 2. the hydrogen bounding force of pretreated sorghum straw and MCC decreased, the hydrogen bounding force of pretreated lignin increased. 3. although the unsaturation of pretreated lignin increased, DES pretreatment is helpful for the removal of lignin. 4. The decrease in the hydrophobicity of sorghum straw make it easier to disperse. 5. the Zeta potential of pretreated sorghum straw shifted towards the positively charged region, while pretreated lignin shifted towards the negatively charged region. 6. different adsorption behaviors were observed in specific components of cellulase mixtures (BGs, CBHs, EGs and xlylanase). These results revealing the mechanism of enzyme adsorption are conductive for understanding the role of pretreatment in biomass conversion.

Integrated multi-objective optimization of sodium bicarbonate pretreatment for the outer anatomical portion of corncob using central composite design, artificial neural networks, and metaheuristic algorithms

Effective pretreatment of raw biomass is crucial in establishing a successful biorefinery process, and response surface methodology (RSM), a statistical optimization method, is routinely employed for optimizing pretreatment methodologies. However, relying solely on statistical methods fails to capture the nonlinear relationships between pretreatment methodology and lignocellulose recalcitrance. Machine learning (ML) approaches excel at understanding these relationships. Therefore, combining statistical and ML optimization can establish better pretreatment parameters. This study presents a multi-stage approach to identify and optimize a chemical pretreatment for the outer anatomical portion of corncobs (CO). Various chemicals were screened using fixed factor screening and regular two-level factorial design (2FI), and among them NaHCO3 was chosen for further optimization, using central composite design (CCD). Moderate delignification (33.96%), minimum sugar loss (5.26 mg/g of CO), and a high enzymatic saccharification yield of up to 82% were achieved with the design. Three distinct metaheuristic algorithms, namely Teaching-Learning-Based Optimization (TLBO), Particle Swarm Optimization (PSO), and Genetic Algorithm (GA) were employed to optimize three specific hyperparameters of the artificial neural network (ANN), to create hybrid metaheuristic-optimized ANN models, which were subsequently used to validate the results obtained from the CCD. Among the different models, The TLBO-optimized ANN model provided the most accurate predictions than the CCD.

Natural lignin modulators improve bagasse saccharification of sugarcane and energy cane in field trials

AbstractThe burgeoning cellulosic ethanol industry necessitates advancements in enzymatic saccharification, effective pretreatments for lignin removal, and the cultivation of crops more amenable to saccharification. Studies have demonstrated that natural inhibitors of lignin biosynthesis can enhance the saccharification of lignocellulose, even in tissues generated several months post‐treatment. In this study, we applied daidzin (a competitive inhibitor of coniferaldehyde dehydrogenase), piperonylic acid (a quasi‐irreversible inhibitor of cinnamate 4‐hydroxylase), and methylenedioxy cinnamic acid (a competitive inhibitor of 4‐coenzyme A ligase) to 60‐day‐old crops of two conventional Brazilian sugarcane cultivars and two energy cane clones, bred specifically for enhanced biomass production. The resultant biomasses were evaluated for lignin content and enzymatic saccharification efficiency without additional lignin‐removal pretreatments. The treatments amplified the production of fermentable sugars in both the sugarcane cultivars and energy cane clones. The most successful results softened the most recalcitrant lignocellulose to the level of the least recalcitrant of the biomasses tested. Interestingly, the softest material became even more susceptible to saccharification.

Facilitating enzymatic hydrolysis efficiency of rape straw by pretreatment with a novel glycerol-based deep eutectic solvent

Deep eutectic solvent (DES) pretreatment plays an indispensable role in destroying the natural anti-degradation barrier structure of lignocellulose. Here, the cetyltrimethylammonium bromide and glycerol were used to synthesize novel DES and introduced to the pretreatment process to facilitate the enzymatic hydrolysis efficiency of rape straw (RS). The results showed that the delignification and xylan removal could extremely reach 46.1% and 52.7% under optimal conditions (molar ratio of 1:6, 200 °C, 90 min), respectively. It also resulted in 171.4 mg/g higher accessibility, 7.2% higher crystallinity, and 122.3 m2/g lower lignin surface area in RS concerning morphological changes of deconstructed and porous cell wall structure. Ultimately, the reducing sugar yield of RS was increased from 27.4% to 67.9% after pretreatment with this type of DES. This study exploited a novel DES for facilely reducing the recalcitrance of lignocellulose and a promising process for efficient saccharification using renewable biomass.

Biotechnological Potential of the Stress Response and Plant Cell Death Regulators Proteins in the Biofuel Industry.

Production of biofuel from lignocellulosic biomass is relatively low due to the limited knowledge about natural cell wall loosening and cellulolytic processes in plants. Industrial separation of cellulose fiber mass from lignin, its saccharification and alcoholic fermentation is still cost-ineffective and environmentally unfriendly. Assuming that the green transformation is inevitable and that new sources of raw materials for biofuels are needed, we decided to study cell death-a natural process occurring in plants in the context of reducing the recalcitrance of lignocellulose for the production of second-generation bioethanol. "Members of the enzyme families responsible for lysigenous aerenchyma formation were identified during the root hypoxia stress in Arabidopsis thaliana cell death mutants. The cell death regulatory genes, LESION SIMULATING DISEASE 1 (LSD1), PHYTOALEXIN DEFICIENT 4 (PAD4) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) conditionally regulate the cell wall when suppressed in transgenic aspen. During four years of growth in the field, the following effects were observed: lignin content was reduced, the cellulose fiber polymerization degree increased and the growth itself was unaffected. The wood of transgenic trees was more efficient as a substrate for saccharification, alcoholic fermentation and bioethanol production. The presented results may trigger the development of novel biotechnologies in the biofuel industry.

Improvement of GH10 xylanase activity based on channel hindrance elimination strategy for better synergistic cellulase to enhance green bio-energy production

Xylanase is widely used for the degradation lignocellulosic biomass as an important accessory enzyme. But productive hydrolysis of the recalcitrant lignocellulose also requires for applicable pretreatment with environmental friendliness. Meanwhile, the complicated industrial processes also demand for catalytically efficient enzymes with more resistance to both pH and heating conditions, which barely exist for the wild types. In this study, the dominant mutant H51K/Q118A from Gloeophyllum trabeum derived GtXyn10 (WT) was obtained by directed evolution. Compared to the WT, apart from the impressive pH resistance from pH 1.0–9.0, the dominant mutant H51K/Q118A also exhibited higher specific activity (2.5-fold; 2510 vs. 1010 U/mg), higher catalytic efficiency (2.4-fold; 460 vs. 190 mL/s‧mg), and better thermostability (with t1/2 at 80 °C extend by 10 min). When ‘Seawater + Feton’ pretreatment was applied, the lignin clearance rate reached 62.5%, 86.6% higher than that of feton pretreatment (33.5%). After pretreatment of ‘Seawater + Feton’, Compared with the cellulase-only treatment (159 μmol/g), synergistic hydrolysis with both H51K/Q118A and cellulase (236 μmol/g) increased the fermentable sugar yields from bagasse by 48.4%. In combination with the optimized pretreatment and synergistic hydrolysis of the modified xylanase and cellulase, the fermentable sugar production for green bio-energy was realized with efficiency.

Integrating genetic-engineered cellulose nanofibrils of rice straw with mild chemical treatments for enhanced bioethanol conversion and bioaerogels production

Cellulose in crop straws represents an abundant biopolymer convertible for biofuels and bioproducts, while the inherent recalcitrance of lignocellulose limits these applications. By selecting desirable rice mutant (cesa7) from site-mutation of the OsCESA7 isoform essential for cellulose biosynthesis, this study examined much improved lignocellulose recalcitrance such as reduced cellulose crystallinity, polymerization and nanofibril length for increased lignocellulose porosity and accessibility. Compared with its wild type, the cesa7 mutant showed a near-complete biomass saccharification with hexose yields of over 96% (% cellulose) for bioethanol production raised by 58%− 71% under three green-like pretreatments performed. Meanwhile, the cellulose substrate of cesa7 mutant was applied to produce aerogels with high porosity and large specific surface under less-intensity ultrasonic treatment, and its further mild chemical modification led to significantly raised oil adsorption capacity by 55%, mainly due to higher proportion of nanofibrils and the honeycomb-like fibers structure observed. It has thus provided an applicable approach for high-yield bioethanol and high-quality aerogels by integrating genetic-modified cellulose substrates with cost-effective process technology.

Integrated laccase delignification with improved lignocellulose recalcitrance for enhancing enzymatic saccharification of ensiled rice straw

The recalcitrant of straw biomass due to its severe lignification hinders its efficient use as a biofuel of feedstock. Therefore, this study was aimed to develop an integrated approach for treatment of rice straw to improve enzymatic saccharification and increase bioethanol production. We evaluated the effect of Lactobacillus plantarum (LP) and cellulases (CL), along with the ligninolytic enzyme laccase (Lac), on the quality of silage, enzymatic hydrolysis, and ethanol production from rice straw ensiled for 60 days. The rice straw was either ensiled without additive (control) or treated with five combinations of the additives, i.e., LP, CL, LP+CL, LP+Lac, or LP+CL+Lac (LPCL+Lac). The results indicated that adding laccase improved the fermentation and silage preservation quality of rice straw biomass by increasing lactic acid production and decreasing pH (<4) (P < 0.05). It also maintained high water-soluble carbohydrate and mono- and disaccharides. The LPCL+Lac treatment managed to reduce the lignin content of rice straw up to 3.82%. Fourier transform infrared spectroscopic analysis and scanning electron microscopic images confirmed that laccase degraded lignin and altered the physico-chemical structure of rice straw. The delignification and reducing the recalcitrance of lignocellulose of rice straw biomass with integrated laccase pretreatment (LPCL+Lac) resulted in release of high content of reducing sugar (52.42 mg/mL), and produced the highest quantity of ethanol (24.45 mg/mL). These results demonstrate that laccase effectively degraded lignin content of rice straw, and its integrated use with LP and CL provides a feasible approach for improving the preservation and bioconversion efficiency of highly lignified and recalcitrant straw biomass.

Highly effective fractionation chemistry to overcome the recalcitrance of softwood lignocellulose

The efficient fractionation and thus production of individual biomass components are pivotal processes in the biorefinery concept. However, the recalcitrant nature of lignocellulose biomass, especially in the case of softwood, is one of the main obstacles to the wider application of biomass-based chemicals and materials. In this study, the use of aqueous acidic systems in the presence of thiourea was studied for the fractionation of softwood in mild conditions. Despite relatively low temperature (100 °C) and treatment times (30–90 min), notable high lignin removal efficiency (approximately 90 %) was obtained. Chemical characterization and the isolation of minor fraction of cationic, water-soluble lignin indicated that the fractionation proceed via nucleophilic addition of thiourea to lignin, resulting in dissolution of lignin in acidic water in relatively mild conditions. Besides high fractionation efficiency, both fiber and lignin fractions were obtained with bright color, significantly elevating their usability in material applications.

Aquaculture Sludge as Co-Substrate for Sustainable Olive Mill Solid Waste Pre-Treatment by Anthracophyllum discolor

Olive mill solid waste (OMSW) is an agro-industrial waste that has a high content of recalcitrant lignocellulose, which can adversely affect the environment. This study aimed to evaluate the phenol and lignin removal and the enzyme activity involved in the biological pre-treatment of OMSW supplemented with aquaculture sludge (AS) as an external nitrogen source by Anthracophyllum discolor. The highest lignin removal and enzymatic activity performance was obtained in the mixture of OMSW and AS prepared at a C/N ratio 45. In these conditions, the pre-treatment could remove 66% of lignin and 68% of phenols in the solid phase and 56% of phenols in the liquid phase and the maximum activity of laccase, manganese peroxidase and manganese independent peroxidase were of 10, 289 and 75 U L−1 in 25, 30, and 15 days of pre-treatment, respectively. These results propose that the addition of AS as a co-substrate for adjusting the C/N ratio allows a 41 and 141% increase in lignin removal and manganese peroxidase activity respectively, enabling the treatment of both OMSW and AS wastes and the possible recovery of an enzymatic extract of biotechnological interest.

Efficient Fractionation of Green Bamboo Using an Integrated Hydrothermal–Deep Eutectic Solvent Pretreatment for Its Valorization

Adopting an integrated strategy to realize efficient fractionation of lignocellulose into well-defined components for its valorization is challenging. Combinatorial pretreatments in this study decomposed hemicellulose of green bamboo during hydrothermal pretreatment (HP), and the hydrothermally pretreated bamboo was subsequently subjected to delignification using deep eutectic solvent (DES) consisting of choline chloride and lactic acid, finally facilitating enzymatic hydrolysis of cellulose residue. Upon hydrothermal treatment at 180 °C for 35 min, hemicellulose removal of 88.6% was achieved with xylo-oligosaccharide yield and purity of 50.9% and 81.6%, respectively. After DES treatment at 140 °C for 2 h, lignin removal was determined to be 79.1%. Notably, the regenerated lignin with high purity of 96.8% displayed superior antioxidant activity, and the decrease in the ratio of syringyl units to guaiacyl units led to a slight decrease in radical scavenging activity of lignin after five recycling runs of DES. Moreover, the two-step treated residue had much higher enzymatic digestibility than that of single HP residue and untreated green bamboo. Results show that synergistic pretreatment is a promising strategy to tackle the recalcitrance of lignocellulose towards high value-added utilization.

Large-scale screening of diverse barely lignocelluloses for simultaneously upgrading biomass enzymatic saccharification and plant lodging resistance coupled with near-infrared spectroscopic assay

Barley is an important economical crop for food and beer industry with abundant lignocellulose residues convertible for biofuels and biochemicals. Although lignocellulose recalcitrance provides mechanic strength to maintain plant lodging resistance for biomass production, it also leads to a costly process for biofuel production. By collecting total 94 barely samples with diverse genetic backgrounds, this study examined large variations of lignocellulose levels, sugars yields and lodging index values, and further identified that the cellulose level was a crucial factor either positively accounting for biomass enzymatic saccharification or negatively affecting plant lodging resistance. Despite lignin level showed slight impact on lodging resistance, it could negatively affect biomass saccharification in barley samples examined. Regarding normal distributions of barley samples, this study generated optimal near-infrared spectroscopy (NIRS) equations applicable for rapid assessment of lignocellulose levels, sugars yields and lodging-related parameters with high determination coefficients for external validation (0.71–0.97), cross-validation (0.89–0.95) and calibration (0.89–0.95), and acceptable ratio performance deviation (2.85–4.35). Notably, the established NIRS models could be employed for selection of the desirable barley samples that are simultaneously of better lodging resistance and higher biomass saccharification. Therefore, this study has established a powerful approach applicable for both rapidly screening lignocellulose-related traits in crop breeding and precisely selecting the ideal lignocellulose substrates for green-like biomass process into cost-effective biofuels and value-added bioproducts.

Cell Wall Glycan Changes in Different Brachypodium Tissues Give Insights into Monocot Biomass

The annual temperate grass Brachypodium distachyon has become a model system for monocot biomass crops and for understanding lignocellulosic recalcitrance to employ better saccharification and fermentation approaches. It is a monocot plant used to study the grass cell walls that differ from the cell walls of dicot plants such as the eudicot model Arabidopsis. The B. distachyon cell wall is predominantly composed of cellulose, arabinoxylans, and mixed-linkage glucans, and it resembles the cell walls of other field grasses. It has a vascular bundle anatomy similar to C3 grasses. These features make Brachypodium an ideal model to study cell walls. Cell walls are composed of polymers with complex structures that vary between cell types and at different developmental stages. Antibodies that recognize specific cell wall components are currently one of the most effective and specific molecular probes to determine the location and distribution of polymers in plant cell walls in situ. Here, we investigated the glycan distribution in the cell walls of the root and leaf tissues of Brachypodium by employing cell-wall-directed antibodies against diverse glycan epitopes. There are distinct differences in the presence of the epitopes between the root and leaf tissues as well as in the cell type level, which gives insights into monocot biomass.