Enzymatic Response of Ryegrass Cellulose and Hemicelluloses Valorization Introduced by Sequential Alkaline Extractions

Enzymatic Response of Ryegrass Cellulose and Hemicelluloses Valorization Introduced by Sequential Alkaline Extractions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Enzymatic Response of Ryegrass Cellulose and Hemicelluloses Valorization Introduced by Sequential Alkaline Extractions Shao-Fei Sun, Jing Yang, Da-Wei Wang, Hai-Yan Yang, Shao-Ni Sun, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-218658/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Mar, 2021 Read the published version in Biotechnology for Biofuels and Bioproducts → Version 1 posted 12 You are reading this latest preprint version Abstract Background In view of the natural resistance of hemicelluloses in lignocellulosic biomass on bioconversion of cellulose into fermentable sugars, alkali extraction is considered as an effective method for gradually fractionating hemicelluloses and enhancing the bioconversion efficiency of cellulose. In the present study, sequential alkaline extractions were performed on the delignified ryegrass material to achieve high bioconversion efficiency of cellulose and comprehensively investigated the structural feature of hemicellulosic fractions for further application. Results Sequential alkaline extractions removed hemicelluloses from cellulose-rich substrates and degraded part of amorphous cellulose, reducing yields of cellulose-rich substrates from 73.0 to 27.7% and increasing crystallinity indexes of which from 31.7 to 41.0%. Alkaline extraction enhanced bioconversion of cellulose by removal of hemicelluloses and swelling of cellulose, increasing of enzymatic hydrolysis from 72.3 to 95.3%. In addition, alkaline extraction gradually fractionated hemicelluloses into six fractions, containing arabinoxylans as the main polysaccharides and part of β -glucans. Simultaneously, increasing of alkaline concentration degraded hemicellulosic polysaccharides, which resulted in a decreasing their molecular weights from 67510 to 50720 g/mol. Conclusions The present study demonstrated that sequential alkaline extraction conditions had a significant effects on the enzymatic hydrolysis efficiency of cellulose and the investigation of the physicochemical properties of hemicellulose. Overall, the investigation the enzymatic hydrolysis efficiency of cellulose-rich substrates and the structural features of hemicelluloses from ryegrass will provide useful information for the efficient utilization of cellulose and hemicelluloses in biorefineries. Biotechnology and Bioengineering Ryegrass Cellulose Hemicelluloses structure Enzymatic hydrolysis Alkaline extraction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background The global energy and financial crisis promote the development of renewable energies [ 1 ]. Bioethanol derived from lignocellulosic biomass is considered as an alternative energy to fossil oil due to its environmentally attractive and technologically feasible. Among the lignocelluloses, short rotation grasses are targeted as potential energy crops due to their abundance, availability and high productivity [ 2 – 4 ]. In addition, grasses can be utilized as whole plants due to high percentage of total carbohydrates and comparatively less lignin content. Ryegrass is the most common bunch type of grass that widely used across the world as a forage and cover crop. The high abundance of ryegrass allows it to be a promising lignocellulose feedstock for bioethanol production [ 5 ]. However, bioethanol production from grass is impeded by the recalcitrant structure of cell wall. Thus, an efficient pretreatment process is required to disrupt the intact structure of biomass and release carbohydrate polymers for further fermentation [ 3 , 4 ]. Pretreatment of lignocelluloses is the process which removes lignin, preserves hemicelluloses, reduces cellulose crystallinity and increases accessibility of material for enzymes [ 6 , 7 ]. Among the pretreatment technologies, alkaline pretreatment is one of the major chemical pretreatments due to practical advantages such as low reaction temperature and pressure, no need for complicated reactors [ 8 ]. Up to now, alkaline pretreatment has been widely applied on lignocellulosic biomass with low lignin contents, such as agricultural wastes, herbaceous crops and hardwoods. During alkaline pretreatment, hydrogen and covalent bonds (such as ester and ether bonds) are broken, resulting in alteration of lignin structure and disruption of crosslinks between hemicelluloses and other components [ 9 ]. The cleavage of these linkages facilitates dissociation of entire cell wall of lignocelluloses and solubilization of hemicelluloses and lignin, improving accessibility of lignocellulose. In addition, more hemicelluloses are dissolved during the alkaline pretreatment as compared to lignin and cellulose [ 10 ]. The removal of hemicelluloses is often correlated well with the increase of enzymatic hydrolysis of lignocellulosic biomass. Although hemicelluloses can be hydrolyzed into its component sugars, which are fermentable to produce bioethanol. The pentoses such as xylose and arabinose in hemicelluloses are difficult to be fermented to ethanol because of the lack of the natural microorganisms that metabolize xylose or arabinose [ 11 ]. Thus, hemicelluloses can be recovered by alkaline treatment for further utilization. Hemicelluloses, the second most abundant structural polymers in lignocellulosic biomass, contains different types of sugars according to plant resources. Generally, glucouronoxylan (xylan) is found to be the principal constituent of the hemicelluloses in hardwood and agriculture residues, while galactoglucomannan is the principal component of softwood hemicelluloses [ 12 – 14 ]. Different from cellulose, hemicelluloses are branched heteropolysaccharides of several different neutral and acidic monosaccharides [ 15 ]. Apart from origin of hemicelluloses, extraction methods also affect the properties of hemicelluloses. Among different hemicelluloses extraction methods, alkaline and hot water extractions are the most popular processes. After extraction, the obtained hemicelluloses can used directly as natural polymers in industries or used as feedstock for producing platform chemicals [ 15 ]. In this study, cellulose-rich substrates and hemicellulosic fractions of ryegrass were gradually recovered by sequential alkaline extractions from delignified material. The effects of sequential alkaline treatments on chemical compositions and structural characteristics of the samples were analyzed by sugar analysis, Fourier transform infrared (FT-IR), and nuclear magnetic resonance (NMR) spectroscopy, respectively. In addition, the effect of sequential alkaline treatments on enzymatic hydrolysis of cellulose-rich substrate was also evaluated. Results And Discussion Yields and chemical compositions of cellulose - rich substrates Hemicellulosic compounds mutually adhered with cellulose microfibrils by hydrogen bonds and van der Waals forces, holding the stiff cellulose fibrils in place. However, hemicelluloses have been considered as major obstacle of physically penetrating and attacking the cellulose by cellulase in bioconversion process [16]. Aqueous alkaline treatment has been considered as an efficient process for hemicelluloses extraction. Yields and chemical compositions of cellulose-rich substrates obtained from sequential alkaline extractions are shown in Table 1. Cellulose-rich substrate (R pulp ) obtained by delignification contained 47.8% glucan as the major sugar. Hemicellulosic compounds, including xylan (19.7%), arabinan (7.7%), galactan (2.6%), mannan (0.1%), galacturonic acid (2.3%) and glucuronic acid (0.3%), totally accounted 32.7% of the substrate. The chemical compositions of hemicellulosic compounds in delignified ryegrass indicated that arabinoxylans was the main compound of hemicellulosic fractions. This result was consistent with the chemical compositions of hemicellulosic fractions obtained from sequential alkaline extractions. Besides, 3.2% Klason lignin and 0.7% acid-soluble lignin residued in the cellulose-rich substrate. After alkaline extraction, part of the hemicellulosic compounds and lignin were removed. As the alkaline concentration increased from 0.15 to 2.5%, the yields of solid cellulose-rich substrates also decreased from 73.0 to 27.7%. The contents of hemicellulosic compounds and lignin decreased from 30.3 to 19.2%, and from 2.3 to 0.7%, respectively. The residual hemicelluloses in substrates were xylans, which were the main compounds, and their contents decreased from 18.2 to 14.3%. The solubilization of hemicellulosic fractions was accompanied with increase of cellulose contents from 51.1 to 62.2%. Increasing of cellulose content is usually preferred for ethanol production due to the direct proportional relationship of ethanol yield and cellulose content of substrate [17]. These results were similar with the composition analysis of cellulosic samples obtained from sequential NaOH extractions of oat straw holocellulose [18]. However, a less amount of glucan in cellulose-rich substrate was observed after alkaline extraction in this study. It might be ascribed to the lower extraction temperature performed in this study. Besides, the dilute acid pretreatment of sugarcane bagasse before alkaline extraction also largely removes hemicellulosic fraction and releases higher content of cellulose in solid fraction than which in ryegrass cellulosic substrates [19]. FT - IR spectra Analysis of cellulose - rich substrates Under alkaline condition, the ester linkages in lignocellulose can be cleaved at relatively high temperature [20]. IR spectroscopy is a widely used to determine functional groups of complex polymers. FT-IR spectra of cellulose-rich substrates are shown in Fig. 2. The stretching vibration of -OH groups in substrates is observed at 3397 cm -1 . The bands at about 1319, 1245, and 1206 cm -1 are due to the in-plane bending of -OH. The bands at 2910 and 1379 cm -1 are assigned to C-H stretching and C-H bending along the chain, respectively. The intense absorption band at 1630 cm -1 corresponds to the bending mode of the absorbed water. The attributions of the main adsorptions are characteristic of glycosidic structures at 1171, 1110, 1060, and 1035 cm -1 for antisymmetric bridge C-O-C and C-O stretching, respectively. A small band at about 899 cm -1 in the spectra is characteristic of C 1 group of frequency/antisymmetric out-of-plane ring stretching due to β -glycosidic linkages. Although the spectral pattern of the samples was similar, the band (1725 cm -1 ) assigned for C=O stretching of acetyl groups in the spectrum of delignified ryegrass (R pulp ) disappeared in the spectra of samples after alkaline extraction. This result indicated the deacetylation of the substrates under alkaline conditions. Pretreatment of corn stalk with 0.5% KOH solution at 30 °C for 24 h also obtains 91.34% deacetylation [21]. The disappearance of ester bonds in FT-IR spectra is consisted with the results observed in solid NMR spectra of cellulose-rich substrates (Fig. 3). In addition, the signal at around 1539 cm -1 in spectrum of R pulp is assigned to the residual lignin (3.9%) in ryegrass holocellulose. Crystallinity analysis of cellulose - rich substrates Solid-state NMR methodologies particular useful for studying structural characteristics of lignocellulose and individual plant cell wall components due to the fact that they can provide much chemical information and ultrastructural details [22]. 13 C CP/MAS is one of the modern solid-state NMR methodologies, it can be used for a qualitative identification of the main chemical and structural changes taking place in the samples as a consequence of the pretreatments. CP/MAS spectra of cellulose-rich substrates obtained from sequential alkaline extractions are shown in Fig. 3. The signals between 60 and 110 ppm are singled to carbohydrates. The signal at about 105 ppm origins from C 1 groups of cellulose. The overlapping signals in the region of 70-80 ppm are assigned to C 2 , C 3 and C 5 of cellulose. In the spectra of cellulose, the amorphous carbons of C 4 are represented by a fairly broad signal from 80-85 ppm, while crystalline carbons of C 4 generate a sharper resonance from 85-92 ppm. Two phases of C 6 cellulose are found at about 63 and 69 ppm, respectively. The peaks around 21 and 172 ppm in the spectrum of R pulp origin from for methyl and carboxylic carbons of acetyl groups attached to the hemicellulosic fraction. After alkaline extraction, the disappearance of these peaks in the spectra of cellulose-rich substrates indicated that the cleavage of bonds between acetyl groups and backbone during alkaline extraction. Crystallinity index (CrI) is an important characteristic affecting the enzymatic hydrolysis of cellulose. The C 4 peak in the carbon spectrum of cellulose is the most commonly utilized peak used to extract ultrastructural information, such as crystalline domains [23]. During the alkaline treatment, alkali molecule can penetrate into the cellulose macromolecule and disrupt the hydrogen bonds between intro- and inter- molecule chains, thereby changing the ultrastructure of cellulose. The effect of sequential alkaline extractions on ordered structure of cellulose are shown as crystallinity index in CP/MAS spectra, which calculated as the peak area ratio of crystalline to total of C 4 signals. After alkaline extraction, the peak intensity for amorphous cellulose decrease, introducing an increase of cellulose-rich substrates crystallinity index (31.7, 33.8, 35.7, 39.1, and 41.0%). The increment of crystallinity index of cellulose was ascribed to the fact that alkaline treatments resulted in greater hydrolyzation of amorphous regions than crystalline regions and peeling reaction of the amorphous regions in cellulose [8]. In addition, an increase of crystalline index of the cellulose residue was also due to the removal of amorphous hemicelluloses from the pulp. Enzymatic hydrolysis of cellulose - rich substrates Hemicelluloses are considered as physical barriers for enzyme to attack cellulosic substrate. The effect of gradual fractionation of hemicellulosic compounds on enzymatic hydrolysis of cellulose-rich substrates are shown in Fig. 4. The delignified ryegrass achieved 59.0% cellulose conversion rate by enzymatic hydrolysis in first 3 h and 72.3% final glucose conversion in 48 h. The enzymatic conversion of cellulose was further enhanced by removal of hemicellulosic compounds. With the decrease content of hemicelluloses in substrates from 32.7 to 19.2%, the glucose yields of enzymatic hydrolysis increased gradually from 59.0 to 74.5% and 72.3 to 95.3%, respectively. The increase of initial enzymatic conversion was ascribed to the fact that sequential alkaline treatments removed hemicelluloses and increased accessibility of material [6]. NaOH pretreatment of Napier grass removes 84% lignin and achieves 94% glucan conversion rate by enzymatic hydrolysis [24]. Pretreatment with ryegrass and surfactant also improves the enzymatic conversion and achieves 87% reducing sugar yield as the maximum [25]. The high glucose yield in this study may be ascribed to the fact that sequential alkaline extraction not only removed hemicelluloses, but also swelled cellulose macromolecule. Swelling of biomass also occurs during alkaline pretreatment of rice husk with 2% NaOH [26]. However, the successively extracted poplar holocellulose also has yielded an increment of cellulose enzymatic conversion and achieved 61.9% cellulose conversion as the maximum [18]. This higher glucose conversion of ryegrass may be ascribed to the structure difference of these two materials. Yields, chemical compositions, and molecular weights of hemicellulosic fractions Hydroxyl ions can swell of cellulose, disrupt intermolecular hydrogen bonds between cellulose and hemicelluloses and dissolve hemicelluloses. Thus, alkaline extraction is one of the most efficient methods for isolation of hemicellulosic compounds [27]. Besides, alkaline extraction can gradually recover hemicellulosic polymers from lignocellulosic materials depending on components and molecular weights [28]. Yields, chemical compositions, and molecular weights of hemicellulosic fractions are shown in Tables 2 and 3. Sequential alkaline extractions of delignified ryegrass with 0.15, 0.3, 0.5, 1.0, 1.5, and 2.5% KOH solution recovered 7.3, 5.8, 33.9, 13.9, 11.3, and 8.7% hemicelluloses, respectively, equal to 80.9% of total hemicelluloses in holocellulose. It can be seen that the yields of hemicelluloses increased with increasing of alkaline concentration from 0.15 to 0.5%. This result suggested that most hemicelluloses were recovered in the early part of the alkaline extraction procedure. However, a continuous increase of alkali concentration to 2.5% declined yields to 8.7%. This result indicated the degradation of hemicellulosic fractions under alkaline condition, which was consisting with molecular weight of hemicellulosic fractions. The monosaccharide in hemicellulosic compounds is always determined by the neutral sugars and uronic acids released during acid hydrolysis of it. Hemicelluloses in ryegrass were fractionated into six parts by sequential alkaline extractions. It can be seen that xylose was the major neutral sugar of six hemicellulosic fractions followed by arabinose, glucose, galactose. Mannose, glucuronic acid and galacturonic acid were found to be minor amount components in hemicelluloses. As the increase of alkaline concentration, the contents of xylose increased from 45.1 to 62.5%, accompanying with decrease contents of arabinose and galactose from 29.4 to 18.3%, and from 9.4 to 3.9%, respectively. These phenomena suggested that xylan was the backbone of ryegrass hemicelluloses. Arabinose and minor quantity of uronic acids might substitute on the backbone of xylan as side chains. Besides, the ratio of arabinose to xylose decreased form 0.65 to 0.29 indicated that the linkages between side chains and backbone were cleaved under the alkaline concentration. In addition, glucose was found to be in the third large amount of neutral sugars and its content decreased from 10.3 to 3.7% as alkaline concentration increased from 0.15 to 1.0%. It revealed that β -glucans was one of polysaccharides in ryegrass hemicelluloses. However, a further increase of alkaline concentration leds to an increase of glucose concentration in hemicelluloses. This result might be ascribed the fact that cellulose was degraded during 1.5% and 2.5% KOH extractions. An increment of glucose content in hemicelluloses with increasing of alkaline concentration is also observed in the research of alkaline extraction of Caragana korshinskii Kom [29]. Molecular mass is an important parameter which affects physicochemical properties of hemicelluloses. Generally, the molecularly uniformed polysaccharides always have polymerization degrees in excess of 50 and polydispersities below 3 [30]. Table 3 shows the weight-average ( M w ) and number average molecular-weights ( M n ) and polydispersity values ( M w / M n ) of six alkaline hemicelluloses from ryegrass. The M w of hemicellulosic fractions gradually decreased from 67510 to 52120 g/mol as the alkaline concentration rose from 0.15 to 1.0%. It indicated that polysaccharides were degraded under the alkaline condition with the increase of the alkaline concentrations. The polydispersity indexes of hemicelluloses ranged from 1.66 to 2.01, implying a structural homogeneity of all hemicellulosic fractions. Further increase of KOH concentration to 1.5% and 2.5% degraded both hemicelluloses and amorphous cellulose. The co-participation of cellulose fragments and hemicellulosic polysaccharides introduced a slight increase of polydispersity indexes of hemicelluloses from 2.09 to 2.26. FT - IR spectra analysis of hemicellulosic fractions FT-IR spectra of hemicellulosic fractions are shown in Fig. 5. The spectra are dominant by signals at 3413 and 2935 cm -1 due to stretching vibration of -OH and C-H, respectively. The peaks for O-H in-plane bending occur at 1317, 1257, and 1215 cm -1 , while O-H out-of-plane bending is observed at 659 cm -1 . The signals origined from C-O stretching is distributed in the range of 1200-950 cm -1 , which are fingerprint region of hemicellulosic polysaccharides. The prominent band at 1049 cm -1 is attributed to the C-O, C-C stretching or C-OH bending typical of xylans. The shoulder band at 899 cm -1 is attributed to the β -linkages of hemicelluloses skeleton. All spectra of hemicelluloses showed similarities in this region, which was consistent with similar sugar components detected in hemicellulosic fractions (Table 2). The massive hydroxyl groups give hemicellulosic polysaccharides strong affinity for water. The band at 1637 cm -1 is identified the absorption of water on hemicelluloses. The signal at 1419 cm -1 is evidence for symmetric stretching of anion carboxylate, origining from salt state of the uronic acids side chain. Besides, the peak at 1552 cm -1 in spectrum of H 0.15% has a contribution from associated lignin. However, this absorbance disappeared in spectra of the hemicellulosic fractions obtained from further steps of the alkali extraction with the increasing its concentrations. This result is consistent with the signal for lignin observed in the spectra of cellulose-rich substrates. These phenomena were ascribed the fact that hemicelluloses associated with lignin through chemical bonds and form lignin-carbohydrate complexes (LCC) in plant cell wall [31]. Alkali can effectively cleave the linkages in LCC and promote the dissolution of it. The associated lignin was also determined in the alkali-soluble hemicelluloses from delignified peashrub [32]. NMR spectra analysis of hemicellulosic fractions NMR is an efficient technology to assay and identify the backbone and type of sidechain of polymers. The structural characteristics of hemicellulosic fractions were elucidated by 13 C and HSQC NMR, and are illustrated in Figs. 6 and 7, respectively. The assignment data of HSQC NMR spectra are given in Table 4. The signals for 13 C NMR were assigned on the basis of the HSQC spectra and previous literature [33]. The signals of different structural sugars are overlapped in the the 13 C NMR spectra. The signals at 102.2, 76.1, 74.6, 73.6 and 63.3 ppm correspondes to C 1 , C 4 , C 3 , C 2 , and C 5 of β -(1-4)-linked-D-Xylp units, respectively. The signals for C 1 ~C 5 of arabinose appeared at 109.4, 80.2, 78.5, 86.4 and 61.7 ppm, respectively. The signals observed at 173.3, 82.6, 72.3 and 59.7 ppm are originated from the C 6 , C 4 , C 5 and methoxyl group of 4- O -methyl-D-glucuronic acid, respectively. However, the C 6 of dissociative glucuronic acid was observed at 181.6 ppm. The present of β -glucans in hemicelluloses were identified by the signals at 80.3 ppm (C 3 ) and 60.6 ppm. The occurrence of galactose was observed as the signal at 69.0/3.88 ppm in HSQC of spectra. These results implied that the alkaline extract hemicelluloses from ryegrass presumably composed of galactoarabinoxylans, ʟ-arabino-(4- O -methyl-ᴅ-glucurono)xylans and β -glucans. The results is consisting with structural sugar components analysis and previous researches [34, 35]. Conclusions Cellulose-rich substrates and hemicellulosic fractions were recovered from ryegrass holocellulose by sequential KOH extractions, respectively. With the dissolution of hemicelluloses in alkaline aqueous, the hemicelluloses contents in cellulose-rich substrates decreased from 32.7 to 19.2%, accompanying decrease of cellulose-rich substrates yields from 100 to 27.7%. Alkaline extraction also removed amorphous cellulose, increasing crystallinity indexes of cellulose. The removal of hemicelluloses also reduced the physical barriers of substrates for enzyme, yielding 1.32 folds enhancement of enzymatic conversion of cellulose-rich substrates. In addition, the hemicellulosic fractions obtained from the sequential alkaline extractions contained arabinoxylans and parts of β -glucans. Materials And Methods Materials Ryegrass (35 days old) was harvested from the farm of Guangxi University. It was air dried and ground in a pulverizer. Next, the ryegrass powder was extracted with toluene-ethanol (2:1, v/v) for 5 h to remove wax and chlorophyll, and employed to delignification with NaClO 2 under acidic condition. The delignified residue was labeled as R pulp and submitted to alkaline extraction for cellulose-rich substrates and hemicellulosic fractions preparation. Sequential alkaline extractions and hemicellulosic fractions recovery Sequential alkaline extractions of delignified ryegrass were conducted at a solid-liquid ratio of 1:25 (w/v) with 0.15, 0.3, 0.5, 1.0, 1.5, and 2.5% (w/v) KOH aqueous at 50 o C for 3 h. After incubation, the solid fractions were filtered with a Brinell funnel, washed repeatedly with distilled water, and then oven dried at 55 °C for 16 h. The filtrates were regulated to pH 5.5-6.0 with acetic acid, and vaporized to 30 mL using a vacuum rotary evaporator. The soluble hemicellulosic fractions were obtained by the precipitation of the concentrated aqueous in 3 volumes of ethanol. Then, the precipitates were recovered by centrifugation and freeze-dried. All the cellulose-rich substrates and hemicellulosic fractions obtain by sequential alkaline extraction were labeled as R pulp , R 0.15% , R 0.3% , R 0.5% , R 1.0% , R 2.5% , and H 0.15% , H 0.3% , H 0.5% , H 1.0% , H 1.5% , and H 2.5% , respectively, according to the alkali concentration. The separation scheme of cellulose-rich and hemicellulosic fractions is illustrated in Fig. 1. All the extraction experiments were repeated at least in triplicate. The average yields of cellulose-rich and hemicellulosic fractions were given, and the standard deviation (SD) of the three determination was less than 3.3% (Tables 1 and 2). Physicochemical characterization of cellulose - rich substrates and hemicellulosic fractions Chemical components of cellulose-rich substrates and hemicellulosic fractions from the delignified ryegrass were analyzed according to the methods of US National Renewable Energy Laboratory (NREL) [36]. Particularly, the neutral sugars and uronic acids in the samples were analyzed by high-performance anion exchange chromatography (HPAEC), and the molecular weights of hemicellulosic fractions were determined by gel permeation chromatography (GPC) [27]. The analytical experiments were conducted with three parallel performs. The mean values of the chemical composition analysis from the samples were given in Tables 1 and 2, and the SD value of the three parallel performs was less than 2.1%. Meanwhile, the mean values of molecular weights of hemicellulosic fractions were given in Table 3, and the SD value of the three analysis results was less than 3038 (g/mol). FT-IR spectra of cellulose-rich substrates and hemicellulosic fractions were recorded on a Bruker Tesor 27 FT-IR spectrometer. 13 C and 2D-HSQC NMR spectra of hemicellulosic polymers were recorded on a Bruker AVIII 400 MHz spectrometer. Solid-state cross-polarization/magic angle spinning (CP/MAS) 13 C NMR spectra of cellulose-rich substrates were recorded on the spectrometer mentioned above. The procedures used for these spectral analyses were borrowed from the methods used in previous literature [28]. Enzymatic hydrolysis of cellulose - rich substrates Enzymatic hydrolysis was executed at 2% substrate (w/v) in 50 mM sodium acetate buffer (pH 4.8) with enzyme loading of 15 FPU/g substrates using a double-layer oscillating incubator at 170 rpm at 50 ºC for 48 h. Commercial cellulase (Cellic ® CTec2) was purchased from Novozymes (Beijing, China), which contained 100 FPU cellulase in 1 mL enzyme solution. During enzymatic hydrolysis, 0.2 mL hydrolyzates were sampled periodically and analyzed by a HPAEC system. All enzymatic hydrolysis experiments were carried out in triplicate. The average values of the three results were given in Fig. 4 and the SD value was less than 3.5%. Declarations Acknowledgements The authors are grateful to the funding support from the Key Laboratory for Forest Resources Conservation and Utilisation in the Southwest Mountains of China, Ministry of Education, Southwest Forestry University (KLESWFU-201807), Yunnan Provincal Department of Education (2020J0398), National Natural Science Foundation of China (No. 31760195, 31760194). Authors ' contributions SFS and HYY performed the major experiments, analyzed the data, and prepared the manuscript. JY and DWW helped with the overall pretreatment experiments and analyzed the data. SNS, and ZJS participated in proofreading and revising the manuscript critically. All authors read and approved the final manuscript. Funding Funding sources have been addressed in the Acknowlegements. Availability of data and materials All data generated or analyzed during this study are included in this published article. Ethics approval and consent to participate Not applicable. Consent for publication All authors consented on the publication of this work. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Cosentino SL, Scordia D, Testa G, Monti A, Alexopoulou E, Christou M. The importance of perennial grasses as a feedstock for bioenergy and bioproducts. Perennial Grasses for Bioenergy and Bioproducts: Elsevier; 2018. pp. 1-33. Mohapatra S, Mishra C, Behera SS, Thatoi H. Application of pretreatment, fermentation and molecular techniques for enhancing bioethanol production from grass biomass-a review. Renewable and Sustainable Energy Reviews. 2017; 78:1007-1032. Tye YY, Lee KT, Abdullah WNW, Leh CP. The world availability of non-wood lignocellulosic biomass for the production of cellulosic ethanol and potential pretreatments for the enhancement of enzymatic saccharification. Renewable and Sustainable Energy Reviews. 2016; 60:155-172. Wongwatanapaiboon J, Kangvansaichol K, Burapatana V, Inochanon R, Winayanuwattikun P, Yongvanich T, Chulalaksananukul W. The potential of cellulosic ethanol production from grasses in Thailand. Journal of Biomedicine and Biotechnology. 2012; 303748:1-10. Sharma H, Carmichael E, Muhamad M, Mccall D, Andrews F, Lyons G, Mcroberts WC, Hornsby PR. Biorefining of perennial ryegrass for the production of nanofibrillated cellulose. Rsc Adv. 2012; 2(16):6424-6437. Kim JS, Lee YY, Kim TH. A review on alkaline pretreatment technology for bioconversion of lignocellulosic biomass. Bioresource Technol. 2016; 199:42-48. Kumari D, Singh R. Pretreatment of lignocellulosic wastes for biofuel production: A critical review. Renewable and Sustainable Energy Reviews. 2018; 90:877-891. Hu F, Ragauskas A. Pretreatment and lignocellulosic chemistry. Bioenerg Res 5 (4): 1043–1066.; 2012. Zheng Y, Shi J, Tu M, Cheng Y. Principles and development of lignocellulosic biomass pretreatment for biofuels. Advances in Bioenergy, vol. 2: Elsevier; 2017. pp. 1-68. Yanfeng H, Yunzhi P, Yanping L, Xiujin L, Kuisheng W. Physicochemical characterization of rice straw pretreated with sodium hydroxide in the solid state for enchancing biogas production. Energy Fuels. 2008; 22:2775-2781. Kricka W, Fitzpatrick J, Bond U. Challenges for the production of bioethanol from biomass using recombinant yeasts. Advances in applied microbiology, vol. 92: Elsevier; 2015. pp. 89-125. Timell TE. Recent progress in the chemistry of wood hemicelluloses. Wood Sci Technol. 1967; 1(1):45-70. Willför S, Sundberg A, Hemming J, Holmbom B. Polysaccharides in some industrially important softwood species. Wood Sci Technol. 2005; 39(4):245-257. Willför S, Sundberg A, Pranovich A, Holmbom B. Polysaccharides in some industrially important hardwood species. Wood Sci Technol. 2005; 39(8):601-617. Li Z, Pan X. Strategies to modify physicochemical properties of hemicelluloses from biorefinery and paper industry for packaging material. Reviews in Environmental Science and Bio/Technology. 2018; 17(1):47-69. Qing Q, Wyman CE. Supplementation with xylanase and β-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover. Biotechnol Biofuels. 2011; 4(1):1-12. Bañuelos JA, Velázquez-Hernández I, Guerra-Balcázar M, Arjona N. Production, characterization and evaluation of the energetic capability of bioethanol from Salicornia Bigelovii as a renewable energy source. Renew Energ. 2018; 123:125-134. Yang H, Chen Q, Wang K, Sun R. Correlation between hemicelluloses-removal-induced hydrophilicity variation and the bioconversion efficiency of lignocelluloses. Bioresource Technol. 2013; 147:539-544. Rezende CA, de Lima MA, Maziero P, Deazevedo ER, Garcia W, Polikarpov I. Chemical and morphological characterization of sugarcane bagasse submitted to a delignification process for enhanced enzymatic digestibility. Biotechnol Biofuels. 2011; 4(1):54. Sun S, Sun S, Cao X, Sun R. The role of pretreatment in improving the enzymatic hydrolysis of lignocellulosic materials. Bioresource Technol. 2016; 199:49-58. Jiang W, Xu J. A novel stepwise pretreatment on corn stalk by alkali deacetylation and liquid hot water for enhancing enzymatic hydrolysis and energy utilization efficiency. Bioresource Technol. 2016; 209:115-124. Foston M. Advances in solid-state NMR of cellulose. Curr Opin Biotech. 2014; 27:176-184. Atalla RH, Vanderhart DL. The role of solid state 13 C NMR spectroscopy in studies of the nature of native celluloses. Solid State Nucl Mag. 1999; 15(1):1-19. Phitsuwan P, Sakka K, Ratanakhanokchai K. Structural changes and enzymatic response of Napier grass ( Pennisetum purpureum ) stem induced by alkaline pretreatment. Bioresource Technol. 2016; 218:247-256. Kataria R, Woods T, Casey W, Cerrone F, Davis R, O'Connor K, Ruhal R, Babu R. Surfactant-mediated hydrothermal pretreatment of ryegrass followed by enzymatic saccharification for polyhydroxyalkanoate production. Ind Crop Prod. 2018; 111:625-632. Castoldi R, Correa VG, de Morais GR, de Souza CG, Bracht A, Peralta RA, Moreira RFP, Peralta RM. Liquid nitrogen pretreatment of eucalyptus sawdust and rice hull for enhanced enzymatic saccharification. Bioresource Technol. 2017; 224:648-655. Peng F, Ren J, Xu F, Bian J, Peng P, Sun R. Comparative study of hemicelluloses obtained by graded ethanol precipitation from sugarcane bagasse. J Agr Food Chem. 2009; 57(14):6305-6317. Wen J, Sun Y, Xu F, Sun R. Fractional Isolation and Chemical Structure of Hemicellulosic Polymers Obtained from Bambusa rigida Species. J Agr Food Chem. 2010; 58(21):11372-11383. Yang H, Song X, Yuan T, Xu F, Sun R. Fractional characterization of hemicellulosic polymers isolated from Caragana korshinskii Kom. Ind Eng Chem Res. 2011; 50(11):6877-6885. Glasser WG, Kaar WE, Jain RK, Sealey JE. Isolation options for non-cellulosic heteropolysaccharides (HetPS). Cellulose. 2000; 7(3):299-317. Chesson A, Gordon AH, Lomax JA. Substituent groups linked by alkali-labile bonds to arabinose and xylose residues of legume, grass and cereal straw cell walls and their fate during digestion by rumen microorganisms. J Sci Food Agr. 1983; 34(12):1330-1340. Peng F, Bian J, Ren J, Peng P, Xu F, Sun R. Fractionation and characterization of alkali-extracted hemicelluloses from peashrub. Biomass and bioenergy. 2012; 39:20-30. Vignon MR, Gey C. Isolation, 1 H and 13 C NMR studies of (4- O -methyl-D-glucurono)-D-xylans from luffa fruit fibres, jute bast fibres and mucilage of quince tree seeds. Carbohyd Res. 1998; 307(1-2):107-111. Xu F, Geng ZC, Sun JX, Liu CF, Ren JL, Sun RC, Fowler P, Baird MS. Fractional and structural characterization of hemicelluloses from perennial ryegrass ( Lolium perenne ) and cocksfoot grass ( Dactylis glomerata ). Carbohyd Res. 2006; 341(12):2073-2082. Xu F, Sun J, Geng Z, Liu C, Ren J, Sun R, Fowler P, Baird M. Comparative study of water-soluble and alkali-soluble hemicelluloses from perennial ryegrass leaves ( Lolium peree ). Carbohyd Polym. 2007; 67(1):56-65. Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D. Determination of structural carbohydrates and lignin in biomass. Laboratory analytical procedure. 2008; 1617(1):1-16. Tables Table 1 Yields and chemical components of cellulose-rich substrates obtained from sequential alkaline extractions of delignified ryegrass . Samples Yield a,c (%) Substrates composition b,c (%) Ara Gal Glu Xyl Man GalA GluA KL ASL R pulp 100.0 ±0.6 7.7 ± 0.2 2.6 ± 0.0 47.8 ± 1.5 19.7 ± 0.6 0.1 ± 0.0 2.3 ± 0.0 0.3 ± 0.0 3.2 ± 0.1 0.7 ± 0.0 R 0.15% 73.0 ± 3.3 7.1 ± 0.2 1.8 ± 0.0 51.1 ± 1.7 18.2 ± 0.6 0.1 ± 0.0 2.7 ± 0.0 0.4 ± 0.0 1.8 ± 0.0 0.5 ± 0.0 R 0.3% 68.9 ± 3.2 6.6 ± 0.2 1.2 ± 0.0 52.7 ± 1.6 17.7 ± 0.5 0.1 ± 0.0 2.4 ± 0.0 0.3 ± 0.0 1.8 ± 0.0 0.4 ± 0.0 R 0.5% 63.9 ± 2.9 5.1 ± 0.1 1.2 ± 0.0 54.7 ± 1.8 17.0 ± 0.5 0.1 ± 0.0 2.1 ± 0.0 0.2 ± 0.0 1.7 ± 0.0 0.4 ± 0.0 R 1.0% 58.4 ± 2.6 4.3 ± 0.1 1.0 ± 0.0 57.9 ± 1.8 16.7 ± 0.5 0.2 ± 0.0 0.5 ± 0.0 0.2 ± 0.0 1.6 ± 0.0 0.3 ± 0.0 R 1.5% 50.8 ± 2.4 3.8 ± 0.1 1.0 ± 0.0 59.0 ± 1.8 16.1 ± 0.5 0.2 ± 0.0 0.4 ± 0.0 0.2 ± 0.0 0.9 ± 0.0 0.3 ± 0.0 R 2.5% 27.7 ± 1.3 3.3 ± 0.1 0.8 ± 0.0 62.2 ± 2.0 14.3 ± 0.4 0.2 ± 0.0 0.4 ± 0.0 0.2 ± 0.0 0.4 ± 0.0 0.3 ± 0.0 a Yields of cellulose-rich substrates obtained from the delignified ryegrass by sequential alkaline extractions, calculated as [(the weight of cellulose-rich substrate obtained after each alkaline extraction)/(the weight of the delignified ryegrass used for sequential alkaline extractions)]×100%; b Ara, arabinan; Gal, galactan; Glu, glucan; Xyl, xylan; Man, mannan; GalA, galacturonic acid; GluA, glucuronic acid; ASL, acid-soluble lignin; KL, Klason lignin. c The values are mean ± SD of three parallel determinations. Table 2 Yields and sugar compositions (relative %, w/w) of hemicellulosic fractions obtained from sequential alkaline extractions of delignified r yegrass . Samples Yield a,c (%) Sugar composition b,c (relative %) Ara Gal Glu Xyl Man GluA GalA H 0.15% 7.3 ± 0.3 29.4 ± 0.9 9.4 ± 0.3 10.3 ± 0.3 45.1 ± 1.5 1.1 ± 0.0 2.7 ± 0.0 2.0 ± 0.0 H 0.3% 5.8 ± 0.2 29.8 ± 1.0 10.2 ± 0.3 8.8 ± 0.2 46.2 ± 1.5 ND 2.0 ± 0.0 3.0 ± 0.1 H 0.5% 33.9 ± 1.6 28.6 ± 0.9 10.3 ± 0.3 7.8 ± 0.2 48.1 ± 1.5 1.0 ± 0.0 2.5 ± 0.0 1.6 ± 0.0 H 1.0% 13.9 ± 0.6 29.0 ± 0.9 8.4 ± 0.2 3.7 ± 0.1 52.8 ± 1.6 1.1 ± 0.0 1.8 ± 0.0 3.2 ± 0.1 H 1.5% 11.3 ± 0.5 22.7 ± 0.7 6.4 ± 0.2 12.8 ± 0.4 54.2 ± 1.7 ND 1.0 ± 0.0 2.9 ± 0.0 H 2.5% 8.7 ± 0.4 18.3 ± 0.6 3.9 ± 0.1 13.5 ± 0.4 62.5 ± 2.1 ND 0.6 ± 0.0 1.2 ± 0.0 a Represents the yields of the hemicelluloses, calculated as [(the weight of the hemicelluloses obtained in each alkaline extraction)/(the weight of the hemicelluloses in the delignified ryegrass)]×100%; b Ara, arabinose; Gal, galactose; Glu, glucose; Xyl, xylose; Man, mannose; GalA, galacturonic aicd; GluA, glucuronic aicd; ND, not detected. c The values are mean ± SD of three parallel determinations. Table 3 Weight-average molecular weights ( M w ) and number-average molecular weights ( M n ) (g/mol), and polydispersity ( M w /M n ) of hemicellulosic fractions isolated by sequential alkaline extractions of delignified ryegrass . Samples a M w b M n b M w /M n H 0.15% 67510 ± 3038 39670 ± 1786 1.70 H 0.3% 61480 ± 2829 37040 ± 1704 1.66 H 0.5% 55460 ± 2219 28730 ± 1150 1.93 H 1.0% 52120 ± 2242 25930 ± 1115 2.01 H 1.5% 51420 ± 2366 24660 ± 1135 2.09 H 2.5% 50720 ± 2283 22480 ± 1012 2.26 a Corresponding to the hemicellulosic fractions in Table 2. b Molecular weights values ( M w and M n ) are mean ± SD of three parallel determinations. Table 4 Assignments of 13 C - 1 H cross-signals in HSQC spectra of hemicellulosic fractions isolated by sequential alkaline extraction s from the delignified ryegrass . Glycosyl Assignments (ppm) 1 2 3 4 5eq d 5ax e OCH 3 X a 13 C 102.4 73.5 75.0 76.0 63.2 63.2 1 H 4.35 3.17 3.32 3.60 3.93 3.21 U b 13 C 71.2 73.8 59.6 1 H 3.40 3.61 3.31 A c 13 C 109.6 80.2 78.3 86.5 61.7 61.7 1 H 5.25 3.91 3.63 4.05 3.73 3.55 Gal 13 C 69.5 1 H 3.85 a X, (1→4)- β -ᴅ-Xyl p. b U, 4- O -methyl- α -ᴅ-Glc p A. c A, α -ʟ-Araf residues. d eq, equatorial. e ax, axial. Cite Share Download PDF Status: Published Journal Publication published 19 Mar, 2021 Read the published version in Biotechnology for Biofuels and Bioproducts → Version 1 posted Editorial decision: Minor revision 25 Feb, 2021 Review # 3 received at journal 13 Feb, 2021 Reviewer # 3 agreed at journal 12 Feb, 2021 Review # 1 received at journal 12 Feb, 2021 Reviewer # 2 agreed at journal 09 Feb, 2021 Reviewers invited by journal 08 Feb, 2021 Reviewer # 1 agreed at journal 08 Feb, 2021 Reviews received at journal 08 Feb, 2021 First submitted to journal 06 Feb, 2021 Editor assigned by journal 06 Feb, 2021 Submission checks completed at journal 06 Feb, 2021 Editor invited by journal 06 Feb, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-218658","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":10905444,"identity":"87190e3e-6eae-4ddf-88e8-8bd2d3055b17","order_by":0,"name":"Shao-Fei Sun","email":"","orcid":"","institution":"Southwest Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shao-Fei","middleName":"","lastName":"Sun","suffix":""},{"id":10905445,"identity":"87b68869-4ec8-47be-8df4-731ff0179673","order_by":1,"name":"Jing Yang","email":"","orcid":"","institution":"Southwest Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Yang","suffix":""},{"id":10905446,"identity":"87bba480-e5f3-4c7c-b5de-6839be06b989","order_by":2,"name":"Da-Wei Wang","email":"","orcid":"","institution":"Southwest Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Da-Wei","middleName":"","lastName":"Wang","suffix":""},{"id":10905447,"identity":"14f085cf-a2a4-4f38-ae50-30b3b02f9980","order_by":3,"name":"Hai-Yan Yang","email":"","orcid":"","institution":"Southwest Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hai-Yan","middleName":"","lastName":"Yang","suffix":""},{"id":10905448,"identity":"4df169f5-dd25-4632-82c5-d6a41e3e5b47","order_by":4,"name":"Shao-Ni Sun","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shao-Ni","middleName":"","lastName":"Sun","suffix":""},{"id":10905449,"identity":"8831102d-54f9-449a-8c0a-fdda5300f942","order_by":5,"name":"Zheng-Jun Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIie3QsUoDMRjA8YTAueScA4p5hZQbLa3giyQc9JaeON7gkCOQW/oAEcV3cHFOCDjdAxTsIAhOHSxdOpW2cIPL3XUsNL8lCXx/SAJAEJwiAiU6rFACYFfb4U10UdmjE+hMNEkucc17EgBQs0UeR168kTvWWdAXpdaPxWKkTG49xj7TBHCwKT5aE7ZwMjH1b1qaB+5eSZbrq9LCWf3VnhAh01j7FJops0t2m+tryxHU7Qk1Qvp42ySYoywinHUmYC5KFUs/OiQO23vem7C5UAh/eg5nS+ae5WSg95/sut5CTfazxk9+PKimyd9KDimtKve9KTou1hDv8t/J9s7vjekxU0EQBOdpB5JLYInBIeupAAAAAElFTkSuQmCC","orcid":"","institution":"Southwest Forestry University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Zheng-Jun","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2021-02-07 08:02:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-218658/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-218658/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13068-021-01921-1","type":"published","date":"2021-03-19T15:00:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":5908694,"identity":"a7d29bce-3f6b-4727-8fc3-6a018fc365bc","added_by":"auto","created_at":"2021-02-12 17:35:14","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":168703,"visible":true,"origin":"","legend":"Scheme for preparation of cellulose-rich and hemicellulosic substrates from delignified ryegrass. ","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-218658/v1/278fda188640bf81fb91ca93.jpeg"},{"id":5909295,"identity":"1fc20b72-0a4b-4a24-b0e0-2da0a2b57eae","added_by":"auto","created_at":"2021-02-12 17:41:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":545162,"visible":true,"origin":"","legend":"FT-IR spectra of the delignified ryegrass (Rpulp) and the cellulose-rich substrates (R0.15%, R0.3%, R0.5%, R1.0%, R1.5%, and R2.5%) obtained by sequential alkaline treatments of the delignified ryegrass. ","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-218658/v1/12881428c5051b6172ccd6bf.png"},{"id":5908919,"identity":"3431af41-4a55-48d0-a8d1-86ed2c59a85f","added_by":"auto","created_at":"2021-02-12 17:38:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":87346,"visible":true,"origin":"","legend":"CP/MAS 13C-NMR spectra of the delignified ryegrass (Rpulp) and the cellulose-rich substrates (R0.15%, R0.5%, R1.5%, and R2.5%) obtained by sequential alkaline treatments of the delignified ryegrass. ","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-218658/v1/e93f50519edd1ff4736dce9c.png"},{"id":5908700,"identity":"6dd65c6f-2d97-4d69-bcbb-6af2081492a1","added_by":"auto","created_at":"2021-02-12 17:35:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":326553,"visible":true,"origin":"","legend":"Glucose yields of enzymatic hydrolysis of the delignified ryegrass (Rpulp) and the cellulose-rich substrates (R0.15%, R0.3%, R0.5%, R1.0%, R1.5%, and R2.5%) obtained by sequential alkaline treatments of the delignified ryegrass.","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-218658/v1/b5d2fa93dad5d7582f72142e.png"},{"id":5908917,"identity":"fd18e4d3-fa2c-4552-a085-e1062753e3cf","added_by":"auto","created_at":"2021-02-12 17:38:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":471235,"visible":true,"origin":"","legend":"FT-IR spectra of hemicellulosic fractions (H0.15%, H0.3%, H1.0%, H1.5%, and H2.5%) isolated by sequential alkaline treatments of the delignified ryegrass. ","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-218658/v1/bef74f29d42e7e6c511aefb4.png"},{"id":5909297,"identity":"958472c5-3ecc-4d87-a9a6-5928badb95db","added_by":"auto","created_at":"2021-02-12 17:41:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":303024,"visible":true,"origin":"","legend":"13C NMR spectra of hemicellulosic fractions (H0.15%, H0.5%, and H2.5%) isolated by sequential alkaline treatments of the delignified ryegrass.","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-218658/v1/70935ae49fa8907ec6dd9e3b.png"},{"id":5909552,"identity":"dc6af3ee-aaca-4156-9a92-5a8acc149b31","added_by":"auto","created_at":"2021-02-12 17:44:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":284671,"visible":true,"origin":"","legend":"2D-HSQC NMR spectra of hemicellulosic fractions (H0.15%, H0.3%, H0.5%, H1.0%, H1.5%, and H2.5%) isolated by sequential alkaline treatments of the delignified ryegrass.","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-218658/v1/10442e1d7ee7173f77842509.png"},{"id":13659566,"identity":"46d0823e-f41b-4f11-a380-e73cb9c08fce","added_by":"auto","created_at":"2021-09-17 10:20:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2408276,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-218658/v1/667de738-bdad-44ae-98da-c72d46d96ada.pdf"}],"financialInterests":"","formattedTitle":"Enzymatic Response of Ryegrass Cellulose and Hemicelluloses Valorization Introduced by Sequential Alkaline Extractions","fulltext":[{"header":"Background","content":" \u003cp\u003eThe global energy and financial crisis promote the development of renewable energies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Bioethanol derived from lignocellulosic biomass is considered as an alternative energy to fossil oil due to its environmentally attractive and technologically feasible. Among the lignocelluloses, short rotation grasses are targeted as potential energy crops due to their abundance, availability and high productivity [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition, grasses can be utilized as whole plants due to high percentage of total carbohydrates and comparatively less lignin content. Ryegrass is the most common bunch type of grass that widely used across the world as a forage and cover crop. The high abundance of ryegrass allows it to be a promising lignocellulose feedstock for bioethanol production [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, bioethanol production from grass is impeded by the recalcitrant structure of cell wall. Thus, an efficient pretreatment process is required to disrupt the intact structure of biomass and release carbohydrate polymers for further fermentation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePretreatment of lignocelluloses is the process which removes lignin, preserves hemicelluloses, reduces cellulose crystallinity and increases accessibility of material for enzymes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Among the pretreatment technologies, alkaline pretreatment is one of the major chemical pretreatments due to practical advantages such as low reaction temperature and pressure, no need for complicated reactors [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Up to now, alkaline pretreatment has been widely applied on lignocellulosic biomass with low lignin contents, such as agricultural wastes, herbaceous crops and hardwoods. During alkaline pretreatment, hydrogen and covalent bonds (such as ester and ether bonds) are broken, resulting in alteration of lignin structure and disruption of crosslinks between hemicelluloses and other components [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The cleavage of these linkages facilitates dissociation of entire cell wall of lignocelluloses and solubilization of hemicelluloses and lignin, improving accessibility of lignocellulose. In addition, more hemicelluloses are dissolved during the alkaline pretreatment as compared to lignin and cellulose [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The removal of hemicelluloses is often correlated well with the increase of enzymatic hydrolysis of lignocellulosic biomass. Although hemicelluloses can be hydrolyzed into its component sugars, which are fermentable to produce bioethanol. The pentoses such as xylose and arabinose in hemicelluloses are difficult to be fermented to ethanol because of the lack of the natural microorganisms that metabolize xylose or arabinose [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Thus, hemicelluloses can be recovered by alkaline treatment for further utilization.\u003c/p\u003e \u003cp\u003eHemicelluloses, the second most abundant structural polymers in lignocellulosic biomass, contains different types of sugars according to plant resources. Generally, glucouronoxylan (xylan) is found to be the principal constituent of the hemicelluloses in hardwood and agriculture residues, while galactoglucomannan is the principal component of softwood hemicelluloses [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Different from cellulose, hemicelluloses are branched heteropolysaccharides of several different neutral and acidic monosaccharides [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Apart from origin of hemicelluloses, extraction methods also affect the properties of hemicelluloses. Among different hemicelluloses extraction methods, alkaline and hot water extractions are the most popular processes. After extraction, the obtained hemicelluloses can used directly as natural polymers in industries or used as feedstock for producing platform chemicals [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, cellulose-rich substrates and hemicellulosic fractions of ryegrass were gradually recovered by sequential alkaline extractions from delignified material. The effects of sequential alkaline treatments on chemical compositions and structural characteristics of the samples were analyzed by sugar analysis, Fourier transform infrared (FT-IR), and nuclear magnetic resonance (NMR) spectroscopy, respectively. In addition, the effect of sequential alkaline treatments on enzymatic hydrolysis of cellulose-rich substrate was also evaluated.\u003c/p\u003e "},{"header":"Results And Discussion","content":"\u003cp\u003e\u003cstrong\u003eYields and chemical compositions of cellulose\u003c/strong\u003e-\u003cstrong\u003erich substrates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHemicellulosic compounds mutually adhered with cellulose microfibrils by hydrogen bonds and van der Waals forces, holding the stiff cellulose fibrils in place. However, hemicelluloses have been considered as major obstacle of physically penetrating and attacking the cellulose by cellulase in bioconversion process [16]. Aqueous alkaline treatment has been considered as an efficient process for hemicelluloses extraction. Yields and chemical compositions of cellulose-rich substrates obtained from sequential alkaline extractions are shown in Table 1. Cellulose-rich substrate (R\u003csub\u003epulp\u003c/sub\u003e) obtained by delignification contained 47.8% glucan as the major sugar. Hemicellulosic compounds, including xylan (19.7%), arabinan (7.7%), galactan (2.6%), mannan (0.1%), galacturonic acid (2.3%) and glucuronic acid (0.3%), totally accounted 32.7% of the substrate. The chemical compositions of hemicellulosic compounds in delignified ryegrass indicated that arabinoxylans was the main compound of hemicellulosic fractions. This result was consistent with the chemical compositions of hemicellulosic fractions obtained from sequential alkaline extractions. Besides, 3.2% Klason lignin and 0.7% acid-soluble lignin residued in the cellulose-rich substrate. After alkaline extraction, part of the hemicellulosic compounds and lignin were removed. As the alkaline concentration increased from 0.15 to 2.5%, the yields of solid cellulose-rich substrates also decreased from 73.0 to 27.7%. The contents of hemicellulosic compounds and lignin decreased from 30.3 to 19.2%, and from 2.3 to 0.7%, respectively. The residual hemicelluloses in substrates were xylans, which were the main compounds, and their contents decreased from 18.2 to 14.3%. The solubilization of hemicellulosic fractions was accompanied with increase of cellulose contents from 51.1 to 62.2%. Increasing of cellulose content is usually preferred for ethanol production due to the direct proportional relationship of ethanol yield and cellulose content of substrate [17]. These results were similar with the composition analysis of cellulosic samples obtained from sequential NaOH extractions of oat straw holocellulose [18]. However, a less amount of glucan in cellulose-rich substrate was observed after alkaline extraction in this study. It might be ascribed to the lower extraction temperature performed in this study. Besides, the dilute acid pretreatment of sugarcane bagasse before alkaline extraction also largely removes hemicellulosic fraction and releases higher content of cellulose in solid fraction than which in ryegrass cellulosic substrates [19].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFT\u003c/strong\u003e-\u003cstrong\u003eIR spectra Analysis of cellulose\u003c/strong\u003e-\u003cstrong\u003erich substrates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder alkaline condition, the ester linkages in lignocellulose can be cleaved at relatively high temperature [20]. IR spectroscopy is a widely used to determine functional groups of complex polymers. FT-IR spectra of cellulose-rich substrates are shown in Fig. 2. The stretching vibration of -OH groups in substrates is observed at 3397 cm\u003csup\u003e-1\u003c/sup\u003e. The bands at about 1319, 1245, and 1206 cm\u003csup\u003e-1\u003c/sup\u003e are due to the in-plane bending of -OH. The bands at 2910 and 1379 cm\u003csup\u003e-1\u003c/sup\u003e are assigned to C-H stretching and C-H bending along the chain, respectively. The intense absorption band at 1630 cm\u003csup\u003e-1\u003c/sup\u003e corresponds to the bending mode of the absorbed water. The attributions of the main adsorptions are characteristic of glycosidic structures at 1171, 1110, 1060, and 1035 cm\u003csup\u003e-1\u003c/sup\u003e for antisymmetric bridge C-O-C and C-O stretching, respectively.\u003c/p\u003e\n\u003cp\u003eA small band at about 899 cm\u003csup\u003e-1\u003c/sup\u003e in the spectra is characteristic of C\u003csub\u003e1\u003c/sub\u003e group of frequency/antisymmetric out-of-plane ring stretching due to \u003cem\u003e\u0026beta;\u003c/em\u003e-glycosidic linkages. Although the spectral pattern of the samples was similar, the band (1725 cm\u003csup\u003e-1\u003c/sup\u003e) assigned for C=O stretching of acetyl groups in the spectrum of delignified ryegrass (R\u003csub\u003epulp\u003c/sub\u003e) disappeared in the spectra of samples after alkaline extraction. This result indicated the deacetylation of the substrates under alkaline conditions. Pretreatment of corn stalk with 0.5% KOH solution at 30 \u0026deg;C for 24 h also obtains 91.34% deacetylation [21]. The disappearance of ester bonds in FT-IR spectra is consisted with the results observed in solid NMR spectra of cellulose-rich substrates (Fig. 3). In addition, the signal at around 1539 cm\u003csup\u003e-1\u003c/sup\u003e in spectrum of R\u003csub\u003epulp\u003c/sub\u003e is assigned to the residual lignin (3.9%) in ryegrass holocellulose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCrystallinity analysis of cellulose\u003c/strong\u003e-\u003cstrong\u003erich substrates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSolid-state NMR methodologies particular useful for studying structural characteristics of lignocellulose and individual plant cell wall components due to the fact that they can provide much chemical information and ultrastructural details [22]. \u003csup\u003e13\u003c/sup\u003eC CP/MAS is one of the modern solid-state NMR methodologies, it can be used for a qualitative identification of the main chemical and structural changes taking place in the samples as a consequence of the pretreatments. CP/MAS spectra of cellulose-rich substrates obtained from sequential alkaline extractions are shown in Fig. 3. The signals between 60 and 110 ppm are singled to carbohydrates. The signal at about 105 ppm origins from C\u003csub\u003e1\u003c/sub\u003e groups of cellulose. The overlapping signals in the region of 70-80 ppm are assigned to C\u003csub\u003e2\u003c/sub\u003e, C\u003csub\u003e3\u003c/sub\u003e and C\u003csub\u003e5\u003c/sub\u003e of cellulose. In the spectra of cellulose, the amorphous carbons of C\u003csub\u003e4\u003c/sub\u003e are represented by a fairly broad signal from 80-85 ppm, while crystalline carbons of C\u003csub\u003e4\u003c/sub\u003e generate a sharper resonance from 85-92 ppm. Two phases of C\u003csub\u003e6\u003c/sub\u003e cellulose are found at about 63 and 69 ppm, respectively. The peaks around 21 and 172 ppm in the spectrum of R\u003csub\u003epulp\u003c/sub\u003e origin from for methyl and carboxylic carbons of acetyl groups attached to the hemicellulosic fraction. After alkaline extraction, the disappearance of these peaks in the spectra of cellulose-rich substrates indicated that the cleavage of bonds between acetyl groups and backbone during alkaline extraction.\u003c/p\u003e\n\u003cp\u003eCrystallinity index (CrI) is an important characteristic affecting the enzymatic hydrolysis of cellulose. The C\u003csub\u003e4\u003c/sub\u003e peak in the carbon spectrum of cellulose is the most commonly utilized peak used to extract ultrastructural information, such as crystalline domains [23]. During the alkaline treatment, alkali molecule can penetrate into the cellulose macromolecule and disrupt the hydrogen bonds between intro- and inter- molecule chains, thereby changing the ultrastructure of cellulose. The effect of sequential alkaline extractions on ordered structure of cellulose are shown as crystallinity index in CP/MAS spectra, which calculated as the peak area ratio of crystalline to total of C\u003csub\u003e4\u003c/sub\u003e signals. After alkaline extraction, the peak intensity for amorphous cellulose decrease, introducing an increase of cellulose-rich substrates crystallinity index (31.7, 33.8, 35.7, 39.1, and 41.0%). The increment of crystallinity index of cellulose was ascribed to the fact that alkaline treatments resulted in greater hydrolyzation of amorphous regions than crystalline regions and peeling reaction of the amorphous regions in cellulose [8]. In addition, an increase of crystalline index of the cellulose residue was also due to the removal of amorphous hemicelluloses from the pulp.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzymatic hydrolysis of cellulose\u003c/strong\u003e-\u003cstrong\u003erich substrates \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHemicelluloses are considered as physical barriers for enzyme to attack cellulosic substrate. The effect of gradual fractionation of hemicellulosic compounds on enzymatic hydrolysis of cellulose-rich substrates are shown in Fig. 4. The delignified ryegrass achieved 59.0% cellulose conversion rate by enzymatic hydrolysis in first 3 h and 72.3% final glucose conversion in 48 h. The enzymatic conversion of cellulose was further enhanced by removal of hemicellulosic compounds. With the decrease content of hemicelluloses in substrates from 32.7 to 19.2%, the glucose yields of enzymatic hydrolysis increased gradually from 59.0 to 74.5% and 72.3 to 95.3%, respectively. The increase of initial enzymatic conversion was ascribed to the fact that sequential alkaline treatments removed hemicelluloses and increased accessibility of material [6]. NaOH pretreatment of Napier grass removes 84% lignin and achieves 94% glucan conversion rate by enzymatic hydrolysis [24]. Pretreatment with ryegrass and surfactant also improves the enzymatic conversion and achieves 87% reducing sugar yield as the maximum [25]. The high glucose yield in this study may be ascribed to the fact that sequential alkaline extraction not only removed hemicelluloses, but also swelled cellulose macromolecule. Swelling of biomass also occurs during alkaline pretreatment of rice husk with 2% NaOH [26]. However, the successively extracted poplar holocellulose also has yielded an increment of cellulose enzymatic conversion and achieved 61.9% cellulose conversion as the maximum [18]. This higher glucose conversion of ryegrass may be ascribed to the structure difference of these two materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYields, chemical compositions, and molecular weights of hemicellulosic fractions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHydroxyl ions can swell of cellulose, disrupt intermolecular hydrogen bonds between cellulose and hemicelluloses and dissolve hemicelluloses. Thus, alkaline extraction is one of the most efficient methods for isolation of hemicellulosic compounds [27]. Besides, alkaline extraction can gradually recover hemicellulosic polymers from lignocellulosic materials depending on components and molecular weights [28]. Yields, chemical compositions, and molecular weights of hemicellulosic fractions are shown in Tables 2 and 3. Sequential alkaline extractions of delignified ryegrass with 0.15, 0.3, 0.5, 1.0, 1.5, and 2.5% KOH solution recovered 7.3, 5.8, 33.9, 13.9, 11.3, and 8.7% hemicelluloses, respectively, equal to 80.9% of total hemicelluloses in holocellulose. It can be seen that the yields of hemicelluloses increased with increasing of alkaline concentration from 0.15 to 0.5%. This result suggested that most hemicelluloses were recovered in the early part of the alkaline extraction procedure. However, a continuous increase of alkali concentration to 2.5% declined yields to 8.7%. This result indicated the degradation of hemicellulosic fractions under alkaline condition, which was consisting with molecular weight of hemicellulosic fractions.\u003c/p\u003e\n\u003cp\u003eThe monosaccharide in hemicellulosic compounds is always determined by the neutral sugars and uronic acids released during acid hydrolysis of it. Hemicelluloses in ryegrass were fractionated into six parts by sequential alkaline extractions. It can be seen that xylose was the major neutral sugar of six hemicellulosic fractions followed by arabinose, glucose, galactose. Mannose, glucuronic acid and galacturonic acid were found to be minor amount components in hemicelluloses. As the increase of alkaline concentration, the contents of xylose increased from 45.1 to 62.5%, accompanying with decrease contents of arabinose and galactose from 29.4 to 18.3%, and from 9.4 to 3.9%, respectively. These phenomena suggested that xylan was the backbone of ryegrass hemicelluloses. Arabinose and minor quantity of uronic acids might substitute on the backbone of xylan as side chains. Besides, the ratio of arabinose to xylose decreased form 0.65 to 0.29 indicated that the linkages between side chains and backbone were cleaved under the alkaline concentration. In addition, glucose was found to be in the third large amount of neutral sugars and its content decreased from 10.3 to 3.7% as alkaline concentration increased from 0.15 to 1.0%. It revealed that \u003cem\u003e\u0026beta;\u003c/em\u003e-glucans was one of polysaccharides in ryegrass hemicelluloses. However, a further increase of alkaline concentration leds to an increase of glucose concentration in hemicelluloses. This result might be ascribed the fact that cellulose was degraded during 1.5% and 2.5% KOH extractions. An increment of glucose content in hemicelluloses with increasing of alkaline concentration is also observed in the research of alkaline extraction of \u003cem\u003eCaragana korshinskii\u003c/em\u003e Kom [29].\u003c/p\u003e\n\u003cp\u003eMolecular mass is an important parameter which affects physicochemical properties of hemicelluloses. Generally, the molecularly uniformed polysaccharides always have polymerization degrees in excess of 50 and polydispersities below 3 [30]. Table 3 shows the weight-average (\u003cem\u003eM\u003csub\u003ew\u003c/sub\u003e\u003c/em\u003e) and number average molecular-weights (\u003cem\u003eM\u003csub\u003en\u003c/sub\u003e\u003c/em\u003e) and polydispersity values (\u003cem\u003eM\u003csub\u003ew\u003c/sub\u003e\u003c/em\u003e/\u003cem\u003eM\u003csub\u003en\u003c/sub\u003e\u003c/em\u003e) of six alkaline hemicelluloses from ryegrass. The \u003cem\u003eM\u003csub\u003ew\u003c/sub\u003e\u003c/em\u003e of hemicellulosic fractions gradually decreased from 67510 to 52120 g/mol as the alkaline concentration rose from 0.15 to 1.0%. It indicated that polysaccharides were degraded under the alkaline condition with the increase of the alkaline concentrations. The polydispersity indexes of hemicelluloses ranged from 1.66 to 2.01, implying a structural homogeneity of all hemicellulosic fractions. Further increase of KOH concentration to 1.5% and 2.5% degraded both hemicelluloses and amorphous cellulose. The co-participation of cellulose fragments and hemicellulosic polysaccharides introduced a slight increase of polydispersity indexes of hemicelluloses from 2.09 to 2.26.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFT\u003c/strong\u003e-\u003cstrong\u003eIR spectra analysis of hemicellulosic fractions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFT-IR spectra of hemicellulosic fractions are shown in Fig. 5. The spectra are dominant by signals at 3413 and 2935 cm\u003csup\u003e-1\u003c/sup\u003e due to stretching vibration of -OH and C-H, respectively. The peaks for O-H in-plane bending occur at 1317, 1257, and 1215 cm\u003csup\u003e-1\u003c/sup\u003e, while O-H out-of-plane bending is observed at 659 cm\u003csup\u003e-1\u003c/sup\u003e. The signals origined from C-O stretching is distributed in the range of 1200-950 cm\u003csup\u003e-1\u003c/sup\u003e, which are fingerprint region of hemicellulosic polysaccharides. The prominent band at 1049 cm\u003csup\u003e-1\u003c/sup\u003e is attributed to the C-O, C-C stretching or C-OH bending typical of xylans. The shoulder band at 899 cm\u003csup\u003e-1\u003c/sup\u003e is attributed to the \u003cem\u003e\u0026beta;\u003c/em\u003e-linkages of hemicelluloses skeleton. All spectra of hemicelluloses showed similarities in this region, which was consistent with similar sugar components detected in hemicellulosic fractions (Table 2). The massive hydroxyl groups give hemicellulosic polysaccharides strong affinity for water. The band at 1637 cm\u003csup\u003e-1 \u003c/sup\u003eis identified the absorption of water on hemicelluloses. The signal at 1419 cm\u003csup\u003e-1\u003c/sup\u003e is evidence for symmetric stretching of anion carboxylate, origining from salt state of the uronic acids side chain. Besides, the peak at 1552 cm\u003csup\u003e-1\u003c/sup\u003e in spectrum of H\u003csub\u003e0.15%\u003c/sub\u003e has a contribution from associated lignin. However, this absorbance disappeared in spectra of the hemicellulosic fractions obtained from further steps of the alkali extraction with the increasing its concentrations. This result is consistent with the signal for lignin observed in the spectra of cellulose-rich substrates. These phenomena were ascribed the fact that hemicelluloses associated with lignin through chemical bonds and form lignin-carbohydrate complexes (LCC) in plant cell wall [31]. Alkali can effectively cleave the linkages in LCC and promote the dissolution of it. The associated lignin was also determined in the alkali-soluble hemicelluloses from delignified peashrub [32].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNMR spectra analysis of hemicellulosic fractions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNMR is an efficient technology to assay and identify the backbone and type of sidechain of polymers. The structural characteristics of hemicellulosic fractions were elucidated by \u003csup\u003e13\u003c/sup\u003eC and HSQC NMR, and are illustrated in Figs. 6 and 7, respectively. The assignment data of HSQC NMR spectra are given in Table 4. The signals for \u003csup\u003e13\u003c/sup\u003eC NMR were assigned on the basis of the HSQC spectra and previous literature [33]. The signals of different structural sugars are overlapped in the the \u003csup\u003e13\u003c/sup\u003eC NMR spectra. The signals at 102.2, 76.1, 74.6, 73.6 and 63.3 ppm correspondes to C\u003csub\u003e1\u003c/sub\u003e, C\u003csub\u003e4\u003c/sub\u003e, C\u003csub\u003e3\u003c/sub\u003e, C\u003csub\u003e2\u003c/sub\u003e, and C\u003csub\u003e5\u003c/sub\u003e of \u003cem\u003e\u0026beta;\u003c/em\u003e-(1-4)-linked-D-Xylp units, respectively. The signals for C\u003csub\u003e1\u003c/sub\u003e~C\u003csub\u003e5\u003c/sub\u003e of arabinose appeared at 109.4, 80.2, 78.5, 86.4 and 61.7 ppm, respectively. The signals observed at 173.3, 82.6, 72.3 and 59.7 ppm are originated from the C\u003csub\u003e6\u003c/sub\u003e, C\u003csub\u003e4\u003c/sub\u003e, C\u003csub\u003e5\u003c/sub\u003e and methoxyl group of 4-\u003cem\u003eO\u003c/em\u003e-methyl-D-glucuronic acid, respectively. However, the C\u003csub\u003e6\u003c/sub\u003e of dissociative glucuronic acid was observed at 181.6 ppm. The present of \u003cem\u003e\u0026beta;\u003c/em\u003e-glucans in hemicelluloses were identified by the signals at 80.3 ppm (C\u003csub\u003e3\u003c/sub\u003e) and 60.6 ppm. The occurrence of galactose was observed as the signal at 69.0/3.88 ppm in HSQC of spectra. These results implied that the alkaline extract hemicelluloses from ryegrass presumably composed of galactoarabinoxylans, ʟ-arabino-(4-\u003cem\u003eO\u003c/em\u003e-methyl-ᴅ-glucurono)xylans and \u003cem\u003e\u0026beta;\u003c/em\u003e-glucans. The results is consisting with structural sugar components analysis and previous researches [34, 35].\u003c/p\u003e"},{"header":"Conclusions","content":" \u003cp\u003eCellulose-rich substrates and hemicellulosic fractions were recovered from ryegrass holocellulose by sequential KOH extractions, respectively. With the dissolution of hemicelluloses in alkaline aqueous, the hemicelluloses contents in cellulose-rich substrates decreased from 32.7 to 19.2%, accompanying decrease of cellulose-rich substrates yields from 100 to 27.7%. Alkaline extraction also removed amorphous cellulose, increasing crystallinity indexes of cellulose. The removal of hemicelluloses also reduced the physical barriers of substrates for enzyme, yielding 1.32 folds enhancement of enzymatic conversion of cellulose-rich substrates. In addition, the hemicellulosic fractions obtained from the sequential alkaline extractions contained arabinoxylans and parts of \u003cem\u003eβ\u003c/em\u003e-glucans.\u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRyegrass (35 days old) was harvested from the farm of Guangxi University. It was air dried and ground in a pulverizer. Next, the ryegrass powder was extracted with toluene-ethanol (2:1, v/v) for 5 h to remove wax and chlorophyll, and employed to delignification with NaClO\u003csub\u003e2\u003c/sub\u003e under acidic condition. The delignified residue was labeled as R\u003csub\u003epulp\u003c/sub\u003e and submitted to alkaline extraction for cellulose-rich substrates and hemicellulosic fractions preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequential alkaline extractions and hemicellulosic fractions recovery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequential alkaline extractions of delignified ryegrass were conducted at a solid-liquid ratio of 1:25 (w/v) with 0.15, 0.3, 0.5, 1.0, 1.5, and 2.5% (w/v) KOH aqueous at 50 \u003csup\u003eo\u003c/sup\u003eC for 3 h. After incubation, the solid fractions were filtered with a Brinell funnel, washed repeatedly with distilled water, and then oven dried at 55 \u0026deg;C for 16 h. The filtrates were regulated to pH 5.5-6.0 with acetic acid, and vaporized to 30 mL using a vacuum rotary evaporator. The soluble hemicellulosic fractions were obtained by the precipitation of the concentrated aqueous in 3 volumes of ethanol. Then, the precipitates were recovered by centrifugation and freeze-dried. All the cellulose-rich substrates and hemicellulosic fractions obtain by sequential alkaline extraction were labeled as R\u003csub\u003epulp\u003c/sub\u003e, R\u003csub\u003e0.15%\u003c/sub\u003e, R\u003csub\u003e0.3%\u003c/sub\u003e, R\u003csub\u003e0.5%\u003c/sub\u003e, R\u003csub\u003e1.0%\u003c/sub\u003e, R\u003csub\u003e2.5%\u003c/sub\u003e, and H\u003csub\u003e0.15%\u003c/sub\u003e, H\u003csub\u003e0.3%\u003c/sub\u003e, H\u003csub\u003e0.5%\u003c/sub\u003e, H\u003csub\u003e1.0%\u003c/sub\u003e, H\u003csub\u003e1.5%\u003c/sub\u003e, and H\u003csub\u003e2.5%\u003c/sub\u003e, respectively, according to the alkali concentration. The separation scheme of cellulose-rich and hemicellulosic fractions is illustrated in Fig. 1. All the extraction experiments were repeated at least in triplicate. The average yields of cellulose-rich and hemicellulosic fractions were given, and the standard deviation (SD) of the three determination was less than 3.3% (Tables 1 and 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysicochemical characterization of cellulose\u003c/strong\u003e-\u003cstrong\u003erich substrates and hemicellulosic fractions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChemical components of cellulose-rich substrates and hemicellulosic fractions from the delignified ryegrass were analyzed according to the methods of US National Renewable Energy Laboratory (NREL) [36]. Particularly, the neutral sugars and uronic acids in the samples were analyzed by high-performance anion exchange chromatography (HPAEC), and the molecular weights of hemicellulosic fractions were determined by gel permeation chromatography (GPC) [27]. The analytical experiments were conducted with three parallel performs. The mean values of the chemical composition analysis from the samples were given in Tables 1 and 2, and the SD value of the three parallel performs was less than 2.1%. Meanwhile, the mean values of molecular weights of hemicellulosic fractions were given in Table 3, and the SD value of the three analysis results was less than 3038 (g/mol).\u003c/p\u003e\n\u003cp\u003eFT-IR spectra of cellulose-rich substrates and hemicellulosic fractions were recorded on a Bruker Tesor 27 FT-IR spectrometer. \u003csup\u003e13\u003c/sup\u003eC and 2D-HSQC NMR spectra of hemicellulosic polymers were recorded on a Bruker AVIII 400 MHz spectrometer. Solid-state cross-polarization/magic angle spinning (CP/MAS) \u003csup\u003e13\u003c/sup\u003eC NMR spectra of cellulose-rich substrates were recorded on the spectrometer mentioned above. The procedures used for these spectral analyses were borrowed from the methods used in previous literature [28].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzymatic hydrolysis of cellulose\u003c/strong\u003e-\u003cstrong\u003erich substrates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnzymatic hydrolysis was executed at 2% substrate (w/v) in 50 mM sodium acetate buffer (pH 4.8) with enzyme loading of 15 FPU/g substrates using a double-layer oscillating incubator at 170 rpm at 50 \u0026ordm;C for 48 h. Commercial cellulase (Cellic\u003csup\u003e\u0026reg;\u003c/sup\u003e CTec2) was purchased from Novozymes (Beijing, China), which contained 100 FPU cellulase in 1 mL enzyme solution. During enzymatic hydrolysis, 0.2 mL hydrolyzates were sampled periodically and analyzed by a HPAEC system. All enzymatic hydrolysis experiments were carried out in triplicate. The average values of the three results were given in Fig. 4 and the SD value was less than 3.5%.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to the funding support from the Key Laboratory for Forest Resources Conservation and Utilisation in the Southwest Mountains of China, Ministry of Education, Southwest Forestry University (KLESWFU-201807), Yunnan Provincal Department of Education (2020J0398), National Natural Science Foundation of China (No. 31760195, 31760194).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e'\u003c/strong\u003e\u003cstrong\u003e contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSFS and HYY performed the major experiments, analyzed the data, and prepared the manuscript. JY and DWW helped with the overall pretreatment experiments and analyzed the data. SNS, and ZJS participated in proofreading and revising the manuscript critically. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding sources have been addressed in the Acknowlegements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consented on the publication of this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCosentino SL, Scordia D, Testa G, Monti A, Alexopoulou E, Christou M. The importance of perennial grasses as a feedstock for bioenergy and bioproducts. Perennial Grasses for Bioenergy and Bioproducts: Elsevier; 2018. pp. 1-33.\u003c/li\u003e\n\u003cli\u003eMohapatra S, Mishra C, Behera SS, Thatoi H. Application of pretreatment, fermentation and molecular techniques for enhancing bioethanol production from grass biomass-a review. Renewable and Sustainable Energy Reviews. 2017; 78:1007-1032.\u003c/li\u003e\n\u003cli\u003eTye YY, Lee KT, Abdullah WNW, Leh CP. The world availability of non-wood lignocellulosic biomass for the production of cellulosic ethanol and potential pretreatments for the enhancement of enzymatic saccharification. Renewable and Sustainable Energy Reviews. 2016; 60:155-172.\u003c/li\u003e\n\u003cli\u003eWongwatanapaiboon J, Kangvansaichol K, Burapatana V, Inochanon R, Winayanuwattikun P, Yongvanich T, Chulalaksananukul W. The potential of cellulosic ethanol production from grasses in Thailand. Journal of Biomedicine and Biotechnology. 2012; 303748:1-10.\u003c/li\u003e\n\u003cli\u003eSharma H, Carmichael E, Muhamad M, Mccall D, Andrews F, Lyons G, Mcroberts WC, Hornsby PR. Biorefining of perennial ryegrass for the production of nanofibrillated cellulose. Rsc Adv. 2012; 2(16):6424-6437.\u003c/li\u003e\n\u003cli\u003eKim JS, Lee YY, Kim TH. A review on alkaline pretreatment technology for bioconversion of lignocellulosic biomass. Bioresource Technol. 2016; 199:42-48.\u003c/li\u003e\n\u003cli\u003eKumari D, Singh R. Pretreatment of lignocellulosic wastes for biofuel production: A critical review. Renewable and Sustainable Energy Reviews. 2018; 90:877-891.\u003c/li\u003e\n\u003cli\u003eHu F, Ragauskas A. Pretreatment and lignocellulosic chemistry. Bioenerg Res 5 (4): 1043\u0026ndash;1066.; 2012.\u003c/li\u003e\n\u003cli\u003eZheng Y, Shi J, Tu M, Cheng Y. Principles and development of lignocellulosic biomass pretreatment for biofuels. Advances in Bioenergy, vol. 2: Elsevier; 2017. pp. 1-68.\u003c/li\u003e\n\u003cli\u003eYanfeng H, Yunzhi P, Yanping L, Xiujin L, Kuisheng W. Physicochemical characterization of rice straw pretreated with sodium hydroxide in the solid state for enchancing biogas production. Energy Fuels. 2008; 22:2775-2781.\u003c/li\u003e\n\u003cli\u003eKricka W, Fitzpatrick J, Bond U. Challenges for the production of bioethanol from biomass using recombinant yeasts. Advances in applied microbiology, vol. 92: Elsevier; 2015. pp. 89-125.\u003c/li\u003e\n\u003cli\u003eTimell TE. Recent progress in the chemistry of wood hemicelluloses. Wood Sci Technol. 1967; 1(1):45-70.\u003c/li\u003e\n\u003cli\u003eWillf\u0026ouml;r S, Sundberg A, Hemming J, Holmbom B. Polysaccharides in some industrially important softwood species. Wood Sci Technol. 2005; 39(4):245-257.\u003c/li\u003e\n\u003cli\u003eWillf\u0026ouml;r S, Sundberg A, Pranovich A, Holmbom B. Polysaccharides in some industrially important hardwood species. Wood Sci Technol. 2005; 39(8):601-617.\u003c/li\u003e\n\u003cli\u003eLi Z, Pan X. Strategies to modify physicochemical properties of hemicelluloses from biorefinery and paper industry for packaging material. Reviews in Environmental Science and Bio/Technology. 2018; 17(1):47-69.\u003c/li\u003e\n\u003cli\u003eQing Q, Wyman CE. Supplementation with xylanase and \u0026beta;-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover. Biotechnol Biofuels. 2011; 4(1):1-12.\u003c/li\u003e\n\u003cli\u003eBa\u0026ntilde;uelos JA, Vel\u0026aacute;zquez-Hern\u0026aacute;ndez I, Guerra-Balc\u0026aacute;zar M, Arjona N. Production, characterization and evaluation of the energetic capability of bioethanol from \u003cem\u003eSalicornia Bigelovii\u003c/em\u003e as a renewable energy source. Renew Energ. 2018; 123:125-134.\u003c/li\u003e\n\u003cli\u003eYang H, Chen Q, Wang K, Sun R. Correlation between hemicelluloses-removal-induced hydrophilicity variation and the bioconversion efficiency of lignocelluloses. Bioresource Technol. 2013; 147:539-544.\u003c/li\u003e\n\u003cli\u003eRezende CA, de Lima MA, Maziero P, Deazevedo ER, Garcia W, Polikarpov I. Chemical and morphological characterization of sugarcane bagasse submitted to a delignification process for enhanced enzymatic digestibility. Biotechnol Biofuels. 2011; 4(1):54.\u003c/li\u003e\n\u003cli\u003eSun S, Sun S, Cao X, Sun R. The role of pretreatment in improving the enzymatic hydrolysis of lignocellulosic materials. Bioresource Technol. 2016; 199:49-58.\u003c/li\u003e\n\u003cli\u003eJiang W, Xu J. A novel stepwise pretreatment on corn stalk by alkali deacetylation and liquid hot water for enhancing enzymatic hydrolysis and energy utilization efficiency. Bioresource Technol. 2016; 209:115-124.\u003c/li\u003e\n\u003cli\u003eFoston M. Advances in solid-state NMR of cellulose. Curr Opin Biotech. 2014; 27:176-184.\u003c/li\u003e\n\u003cli\u003eAtalla RH, Vanderhart DL. The role of solid state \u003csup\u003e13\u003c/sup\u003eC NMR spectroscopy in studies of the nature of native celluloses. Solid State Nucl Mag. 1999; 15(1):1-19.\u003c/li\u003e\n\u003cli\u003ePhitsuwan P, Sakka K, Ratanakhanokchai K. Structural changes and enzymatic response of Napier grass (\u003cem\u003ePennisetum purpureum\u003c/em\u003e) stem induced by alkaline pretreatment. Bioresource Technol. 2016; 218:247-256.\u003c/li\u003e\n\u003cli\u003eKataria R, Woods T, Casey W, Cerrone F, Davis R, O'Connor K, Ruhal R, Babu R. Surfactant-mediated hydrothermal pretreatment of ryegrass followed by enzymatic saccharification for polyhydroxyalkanoate production. Ind Crop Prod. 2018; 111:625-632.\u003c/li\u003e\n\u003cli\u003eCastoldi R, Correa VG, de Morais GR, de Souza CG, Bracht A, Peralta RA, Moreira RFP, Peralta RM. Liquid nitrogen pretreatment of eucalyptus sawdust and rice hull for enhanced enzymatic saccharification. Bioresource Technol. 2017; 224:648-655.\u003c/li\u003e\n\u003cli\u003ePeng F, Ren J, Xu F, Bian J, Peng P, Sun R. Comparative study of hemicelluloses obtained by graded ethanol precipitation from sugarcane bagasse. J Agr Food Chem. 2009; 57(14):6305-6317.\u003c/li\u003e\n\u003cli\u003eWen J, Sun Y, Xu F, Sun R. Fractional Isolation and Chemical Structure of Hemicellulosic Polymers Obtained from\u003cem\u003e Bambusa rigida\u003c/em\u003e Species. J Agr Food Chem. 2010; 58(21):11372-11383.\u003c/li\u003e\n\u003cli\u003eYang H, Song X, Yuan T, Xu F, Sun R. Fractional characterization of hemicellulosic polymers isolated from \u003cem\u003eCaragana korshinskii\u003c/em\u003e Kom. Ind Eng Chem Res. 2011; 50(11):6877-6885.\u003c/li\u003e\n\u003cli\u003eGlasser WG, Kaar WE, Jain RK, Sealey JE. Isolation options for non-cellulosic heteropolysaccharides (HetPS). Cellulose. 2000; 7(3):299-317.\u003c/li\u003e\n\u003cli\u003eChesson A, Gordon AH, Lomax JA. Substituent groups linked by alkali-labile bonds to arabinose and xylose residues of legume, grass and cereal straw cell walls and their fate during digestion by rumen microorganisms. J Sci Food Agr. 1983; 34(12):1330-1340.\u003c/li\u003e\n\u003cli\u003ePeng F, Bian J, Ren J, Peng P, Xu F, Sun R. Fractionation and characterization of alkali-extracted hemicelluloses from peashrub. Biomass and bioenergy. 2012; 39:20-30.\u003c/li\u003e\n\u003cli\u003eVignon MR, Gey C. Isolation, \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR studies of (4-\u003cem\u003eO\u003c/em\u003e-methyl-D-glucurono)-D-xylans from luffa fruit fibres, jute bast fibres and mucilage of quince tree seeds. Carbohyd Res. 1998; 307(1-2):107-111.\u003c/li\u003e\n\u003cli\u003eXu F, Geng ZC, Sun JX, Liu CF, Ren JL, Sun RC, Fowler P, Baird MS. Fractional and structural characterization of hemicelluloses from perennial ryegrass (\u003cem\u003eLolium perenne\u003c/em\u003e) and cocksfoot grass (\u003cem\u003eDactylis glomerata\u003c/em\u003e). Carbohyd Res. 2006; 341(12):2073-2082.\u003c/li\u003e\n\u003cli\u003eXu F, Sun J, Geng Z, Liu C, Ren J, Sun R, Fowler P, Baird M. Comparative study of water-soluble and alkali-soluble hemicelluloses from perennial ryegrass leaves (\u003cem\u003eLolium peree\u003c/em\u003e). Carbohyd Polym. 2007; 67(1):56-65.\u003c/li\u003e\n\u003cli\u003eSluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D. Determination of structural carbohydrates and lignin in biomass. Laboratory analytical procedure. 2008; 1617(1):1-16.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e \u003cstrong\u003eYields and chemical components of cellulose-rich substrates obtained from sequential alkaline extractions of delignified ryegrass\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 38px;\"\u003e\n\u003ctd style=\"height: 73.5256px;\" rowspan=\"2\" width=\"62\"\u003e\n\u003cp\u003eSamples\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 73.5256px;\" rowspan=\"2\" width=\"78\"\u003e\n\u003cp\u003eYield\u003cem\u003e\u003csup\u003ea,c\u003c/sup\u003e\u003c/em\u003e (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 38px;\" colspan=\"9\" width=\"611\"\u003e\n\u003cp\u003eSubstrates composition\u003cem\u003e\u003csup\u003eb,c\u003c/sup\u003e\u003c/em\u003e (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35.5256px;\"\u003e\n\u003ctd style=\"height: 35.5256px;\" width=\"65\"\u003e\n\u003cp\u003eAra\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35.5256px;\" width=\"65\"\u003e\n\u003cp\u003eGal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35.5256px;\" width=\"76\"\u003e\n\u003cp\u003eGlu\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35.5256px;\" width=\"74\"\u003e\n\u003cp\u003eXyl\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35.5256px;\" width=\"68\"\u003e\n\u003cp\u003eMan\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35.5256px;\" width=\"66\"\u003e\n\u003cp\u003eGalA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35.5256px;\" width=\"66\"\u003e\n\u003cp\u003eGluA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35.5256px;\" width=\"66\"\u003e\n\u003cp\u003eKL\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35.5256px;\" width=\"66\"\u003e\n\u003cp\u003eASL\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 37px;\"\u003e\n\u003ctd style=\"height: 37px;\" width=\"62\"\u003e\n\u003cp\u003eR\u003csub\u003epulp\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"78\"\u003e\n\u003cp\u003e100.0 \u0026plusmn;0.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"65\"\u003e\n\u003cp\u003e7.7 \u0026plusmn; 0.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"65\"\u003e\n\u003cp\u003e2.6 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"76\"\u003e\n\u003cp\u003e47.8 \u0026plusmn; 1.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"74\"\u003e\n\u003cp\u003e19.7 \u0026plusmn; 0.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"68\"\u003e\n\u003cp\u003e0.1 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e2.3 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.3 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e3.2 \u0026plusmn; 0.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.7 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 37px;\"\u003e\n\u003ctd style=\"height: 37px;\" width=\"62\"\u003e\n\u003cp\u003eR\u003csub\u003e0.15%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"78\"\u003e\n\u003cp\u003e73.0 \u0026plusmn; 3.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"65\"\u003e\n\u003cp\u003e7.1 \u0026plusmn; 0.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"65\"\u003e\n\u003cp\u003e1.8 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"76\"\u003e\n\u003cp\u003e51.1 \u0026plusmn; 1.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"74\"\u003e\n\u003cp\u003e18.2 \u0026plusmn; 0.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"68\"\u003e\n\u003cp\u003e0.1 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e2.7 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.4 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e1.8 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.5 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 37px;\"\u003e\n\u003ctd style=\"height: 37px;\" width=\"62\"\u003e\n\u003cp\u003eR\u003csub\u003e0.3%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"78\"\u003e\n\u003cp\u003e68.9 \u0026plusmn; 3.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"65\"\u003e\n\u003cp\u003e6.6 \u0026plusmn; 0.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"65\"\u003e\n\u003cp\u003e1.2 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"76\"\u003e\n\u003cp\u003e52.7 \u0026plusmn; 1.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"74\"\u003e\n\u003cp\u003e17.7 \u0026plusmn; 0.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"68\"\u003e\n\u003cp\u003e0.1 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e2.4 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.3 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e1.8 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.4 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 37px;\"\u003e\n\u003ctd style=\"height: 37px;\" width=\"62\"\u003e\n\u003cp\u003eR\u003csub\u003e0.5%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"78\"\u003e\n\u003cp\u003e63.9 \u0026plusmn; 2.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"65\"\u003e\n\u003cp\u003e5.1 \u0026plusmn; 0.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"65\"\u003e\n\u003cp\u003e1.2 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"76\"\u003e\n\u003cp\u003e54.7 \u0026plusmn; 1.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"74\"\u003e\n\u003cp\u003e17.0 \u0026plusmn; 0.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"68\"\u003e\n\u003cp\u003e0.1 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e2.1 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.2 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e1.7 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.4 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 37px;\"\u003e\n\u003ctd style=\"height: 37px;\" width=\"62\"\u003e\n\u003cp\u003eR\u003csub\u003e1.0%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"78\"\u003e\n\u003cp\u003e58.4 \u0026plusmn; 2.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"65\"\u003e\n\u003cp\u003e4.3 \u0026plusmn; 0.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"65\"\u003e\n\u003cp\u003e1.0 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"76\"\u003e\n\u003cp\u003e57.9 \u0026plusmn; 1.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"74\"\u003e\n\u003cp\u003e16.7 \u0026plusmn; 0.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"68\"\u003e\n\u003cp\u003e0.2 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.5 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.2 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e1.6 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.3 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 37px;\"\u003e\n\u003ctd style=\"height: 37px;\" width=\"62\"\u003e\n\u003cp\u003eR\u003csub\u003e1.5%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"78\"\u003e\n\u003cp\u003e50.8 \u0026plusmn; 2.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"65\"\u003e\n\u003cp\u003e3.8 \u0026plusmn; 0.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"65\"\u003e\n\u003cp\u003e1.0 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"76\"\u003e\n\u003cp\u003e59.0 \u0026plusmn; 1.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"74\"\u003e\n\u003cp\u003e16.1 \u0026plusmn; 0.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"68\"\u003e\n\u003cp\u003e0.2 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.4 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.2 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.9 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.3 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 37px;\"\u003e\n\u003ctd style=\"height: 37px;\" width=\"62\"\u003e\n\u003cp\u003eR\u003csub\u003e2.5%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"78\"\u003e\n\u003cp\u003e27.7 \u0026plusmn; 1.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"65\"\u003e\n\u003cp\u003e3.3 \u0026plusmn; 0.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"65\"\u003e\n\u003cp\u003e0.8 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"76\"\u003e\n\u003cp\u003e62.2 \u0026plusmn; 2.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"74\"\u003e\n\u003cp\u003e14.3 \u0026plusmn; 0.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"68\"\u003e\n\u003cp\u003e0.2 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.4 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.2 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.4 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 37px;\" width=\"66\"\u003e\n\u003cp\u003e0.3 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e Yields of cellulose-rich substrates obtained from the delignified ryegrass by sequential alkaline extractions, calculated as [(the weight of cellulose-rich substrate obtained after each alkaline extraction)/(the weight of the delignified ryegrass used for sequential alkaline extractions)]\u0026times;100%;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/em\u003e Ara, arabinan; Gal, galactan; Glu, glucan; Xyl, xylan; Man, mannan; GalA, galacturonic acid; GluA, glucuronic acid; ASL, acid-soluble lignin; KL, Klason lignin.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/em\u003e The values are mean \u0026plusmn; SD of three parallel determinations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2 Yields\u003c/strong\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cstrong\u003esugar compositions \u003c/strong\u003e\u003cstrong\u003e(relative %, w/w) \u003c/strong\u003e\u003cstrong\u003eof hemicellulosic fractions obtained from \u003c/strong\u003e\u003cstrong\u003esequential alkaline extractions of delignified r\u003c/strong\u003e\u003cstrong\u003eyegrass\u003c/strong\u003e\u003cstrong\u003e. \u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"66\"\u003e\n\u003cp\u003eSamples\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" width=\"76\"\u003e\n\u003cp\u003eYield\u003cem\u003e\u003csup\u003ea,c\u003c/sup\u003e\u003c/em\u003e (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"7\" width=\"495\"\u003e\n\u003cp\u003eSugar composition\u003cem\u003e\u003csup\u003eb,c\u003c/sup\u003e\u003c/em\u003e (relative %)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003eAra\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003eGal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eGlu\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eXyl\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eMan\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eGluA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eGalA\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eH\u003csub\u003e0.15%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e7.3 \u0026plusmn; 0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e29.4 \u0026plusmn; 0.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e9.4 \u0026plusmn; 0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e10.3 \u0026plusmn; 0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e45.1 \u0026plusmn; 1.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1.1 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e2.7 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e2.0 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eH\u003csub\u003e0.3%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e5.8 \u0026plusmn; 0.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e29.8 \u0026plusmn; 1.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e10.2 \u0026plusmn; 0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e8.8 \u0026plusmn; 0.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e46.2 \u0026plusmn; 1.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e2.0 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e3.0 \u0026plusmn; 0.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eH\u003csub\u003e0.5%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e33.9 \u0026plusmn; 1.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e28.6 \u0026plusmn; 0.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e10.3 \u0026plusmn; 0.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e7.8 \u0026plusmn; 0.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e48.1 \u0026plusmn; 1.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1.0 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e2.5 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1.6 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eH\u003csub\u003e1.0%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e13.9 \u0026plusmn; 0.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e29.0 \u0026plusmn; 0.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e8.4 \u0026plusmn; 0.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e3.7 \u0026plusmn; 0.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e52.8 \u0026plusmn; 1.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1.1 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1.8 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e3.2 \u0026plusmn; 0.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eH\u003csub\u003e1.5%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e11.3 \u0026plusmn; 0.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e22.7 \u0026plusmn; 0.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e6.4 \u0026plusmn; 0.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e12.8 \u0026plusmn; 0.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e54.2 \u0026plusmn; 1.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1.0 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e2.9 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eH\u003csub\u003e2.5%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e8.7 \u0026plusmn; 0.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e18.3 \u0026plusmn; 0.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"72\"\u003e\n\u003cp\u003e3.9 \u0026plusmn; 0.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e13.5 \u0026plusmn; 0.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003e62.5 \u0026plusmn; 2.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003eND\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e0.6 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"66\"\u003e\n\u003cp\u003e1.2 \u0026plusmn; 0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e Represents the yields of the hemicelluloses, calculated as [(the weight of the hemicelluloses obtained in each alkaline extraction)/(the weight of the hemicelluloses in the delignified ryegrass)]\u0026times;100%;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/em\u003e Ara, arabinose; Gal, galactose; Glu, glucose; Xyl, xylose; Man, mannose; GalA, galacturonic aicd; GluA, glucuronic aicd; ND, not detected.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/em\u003e The values are mean \u0026plusmn; SD of three parallel determinations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3 Weight-average molecular weights (\u003cem\u003eM\u003csub\u003ew\u003c/sub\u003e\u003c/em\u003e) and number-average molecular weights (\u003cem\u003eM\u003csub\u003en\u003c/sub\u003e\u003c/em\u003e) (g/mol), and polydispersity (\u003cem\u003eM\u003csub\u003ew\u003c/sub\u003e/M\u003csub\u003en\u003c/sub\u003e\u003c/em\u003e) of hemicellulosic fractions isolated by \u003c/strong\u003e\u003cstrong\u003esequential alkaline extractions of \u003c/strong\u003e\u003cstrong\u003edelignified ryegrass\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" width=\"100%\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"20%\"\u003e\n\u003cp\u003eSamples\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"30%\"\u003e\n\u003cp\u003e\u003cem\u003eM\u003csub\u003ew\u003c/sub\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"25%\"\u003e\n\u003cp\u003e\u003cem\u003eM\u003csub\u003en\u003c/sub\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"23%\"\u003e\n\u003cp\u003e\u003cem\u003eM\u003csub\u003ew\u003c/sub\u003e/M\u003csub\u003en\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"20%\"\u003e\n\u003cp\u003eH\u003csub\u003e0.15%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"30%\"\u003e\n\u003cp\u003e67510 \u0026plusmn; 3038\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"25%\"\u003e\n\u003cp\u003e39670 \u0026plusmn; 1786\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"23%\"\u003e\n\u003cp\u003e1.70\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"20%\"\u003e\n\u003cp\u003eH\u003csub\u003e0.3%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"30%\"\u003e\n\u003cp\u003e61480 \u0026plusmn; 2829\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"25%\"\u003e\n\u003cp\u003e37040 \u0026plusmn; 1704\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"23%\"\u003e\n\u003cp\u003e1.66\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"20%\"\u003e\n\u003cp\u003eH\u003csub\u003e0.5%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"30%\"\u003e\n\u003cp\u003e55460 \u0026plusmn; 2219\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"25%\"\u003e\n\u003cp\u003e28730 \u0026plusmn; 1150\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"23%\"\u003e\n\u003cp\u003e1.93\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"20%\"\u003e\n\u003cp\u003eH\u003csub\u003e1.0%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"30%\"\u003e\n\u003cp\u003e52120 \u0026plusmn; 2242\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"25%\"\u003e\n\u003cp\u003e25930 \u0026plusmn; 1115\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"23%\"\u003e\n\u003cp\u003e2.01\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"20%\"\u003e\n\u003cp\u003eH\u003csub\u003e1.5%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"30%\"\u003e\n\u003cp\u003e51420 \u0026plusmn; 2366\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"25%\"\u003e\n\u003cp\u003e24660 \u0026plusmn; 1135\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"23%\"\u003e\n\u003cp\u003e2.09\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"20%\"\u003e\n\u003cp\u003eH\u003csub\u003e2.5%\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"30%\"\u003e\n\u003cp\u003e50720 \u0026plusmn; 2283\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"25%\"\u003e\n\u003cp\u003e22480 \u0026plusmn; 1012\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"23%\"\u003e\n\u003cp\u003e2.26\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e Corresponding to the hemicellulosic fractions in Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/em\u003e Molecular weights values (\u003cem\u003eM\u003csub\u003ew\u003c/sub\u003e\u003c/em\u003e and \u003cem\u003eM\u003csub\u003en\u003c/sub\u003e\u003c/em\u003e) are mean \u0026plusmn; SD of three parallel determinations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable \u003c/strong\u003e\u003cstrong\u003e4 \u003c/strong\u003e\u003cstrong\u003eAssignments of \u003csup\u003e13\u003c/sup\u003eC\u003c/strong\u003e-\u003cstrong\u003e\u003csup\u003e1\u003c/sup\u003eH cross-signals in HSQC spectra of hemicellulosic fractions isolated by \u003c/strong\u003e\u003cstrong\u003esequential alkaline \u003c/strong\u003e\u003cstrong\u003eextraction\u003c/strong\u003e\u003cstrong\u003es from \u003c/strong\u003e\u003cstrong\u003ethe \u003c/strong\u003e\u003cstrong\u003edelignified \u003c/strong\u003e\u003cstrong\u003eryegrass\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" width=\"100%\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"12%\"\u003e\n\u003cp\u003eGlycosyl\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"8\" width=\"87%\"\u003e\n\u003cp\u003eAssignments (ppm)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"12%\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e5eq\u003cem\u003e\u003csup\u003ed\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e5ax\u003cem\u003e\u003csup\u003ee\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003eOCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"12%\"\u003e\n\u003cp\u003eX\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003e\u003csup\u003e13\u003c/sup\u003eC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"12%\"\u003e\n\u003cp\u003e102.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e73.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e75.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e76.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e63.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e63.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"12%\"\u003e\n\u003cp\u003e4.35\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e3.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e3.32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e3.60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e3.93\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e3.21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"12%\"\u003e\n\u003cp\u003eU\u003cem\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003e\u003csup\u003e13\u003c/sup\u003eC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"12%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e71.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e73.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003e59.6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"12%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e3.40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e3.61\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003e3.31\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"12%\"\u003e\n\u003cp\u003eA\u003cem\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003e\u003csup\u003e13\u003c/sup\u003eC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"12%\"\u003e\n\u003cp\u003e109.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e80.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e78.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e86.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e61.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e61.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"12%\"\u003e\n\u003cp\u003e5.25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e3.91\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e3.63\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e4.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e3.73\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e3.55\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"12%\"\u003e\n\u003cp\u003eGal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003e\u003csup\u003e13\u003c/sup\u003eC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"12%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e69.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"12%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e3.85\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e X, (1\u0026rarr;4)-\u003cem\u003e\u0026beta;\u003c/em\u003e-ᴅ-Xyl\u003cem\u003ep.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/em\u003e U, 4-\u003cem\u003eO\u003c/em\u003e-methyl-\u003cem\u003e\u0026alpha;\u003c/em\u003e-ᴅ-Glc\u003cem\u003ep\u003c/em\u003eA.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/em\u003e A, \u003cem\u003e\u0026alpha;\u003c/em\u003e-ʟ-Araf residues.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003ed\u003c/sup\u003e\u003c/em\u003e eq, equatorial.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003csup\u003ee\u003c/sup\u003e\u003c/em\u003e ax, axial.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ryegrass, Cellulose, Hemicelluloses structure, Enzymatic hydrolysis, Alkaline extraction ","lastPublishedDoi":"10.21203/rs.3.rs-218658/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-218658/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIn view of the natural resistance of hemicelluloses in lignocellulosic biomass on bioconversion of cellulose into fermentable sugars, alkali extraction is considered as an effective method for gradually fractionating hemicelluloses and enhancing the bioconversion efficiency of cellulose. In the present study, sequential alkaline extractions were performed on the delignified ryegrass material to achieve high bioconversion efficiency of cellulose and comprehensively investigated the structural feature of hemicellulosic fractions for further application.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSequential alkaline extractions removed hemicelluloses from cellulose-rich substrates and degraded part of amorphous cellulose, reducing yields of cellulose-rich substrates from 73.0 to 27.7% and increasing crystallinity indexes of which from 31.7 to 41.0%. Alkaline extraction enhanced bioconversion of cellulose by removal of hemicelluloses and swelling of cellulose, increasing of enzymatic hydrolysis from 72.3 to 95.3%. In addition, alkaline extraction gradually fractionated hemicelluloses into six fractions, containing arabinoxylans as the main polysaccharides and part of \u003cem\u003eβ\u003c/em\u003e-glucans. Simultaneously, increasing of alkaline concentration degraded hemicellulosic polysaccharides, which resulted in a decreasing their molecular weights from 67510 to 50720 g/mol.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe present study demonstrated that sequential alkaline extraction conditions had a significant effects on the enzymatic hydrolysis efficiency of cellulose and the investigation of the physicochemical properties of hemicellulose. Overall, the investigation the enzymatic hydrolysis efficiency of cellulose-rich substrates and the structural features of hemicelluloses from ryegrass will provide useful information for the efficient utilization of cellulose and hemicelluloses in biorefineries.\u003c/p\u003e","manuscriptTitle":"Enzymatic Response of Ryegrass Cellulose and Hemicelluloses Valorization Introduced by Sequential Alkaline Extractions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-12 17:35:12","doi":"10.21203/rs.3.rs-218658/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2021-02-25T11:37:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-02-14T00:00:00+00:00","index":3,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2021-02-13T00:00:00+00:00","index":3,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-02-13T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2021-02-10T00:00:00+00:00","index":2,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-02-09T00:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-02-09T00:00:00+00:00","index":1,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-02-09T00:00:00+00:00","index":0,"fulltext":""},{"type":"submitted","content":"Biotechnology for Biofuels","date":"2021-02-07T03:02:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-02-07T00:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-02-06T23:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-02-06T23:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-for-biofuels-and-bioproducts","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbio","sideBox":"Learn more about [Biotechnology for Biofuels](http://biotechnologyforbiofuels.biomedcentral.com/)","snPcode":"13068","submissionUrl":"https://submission.nature.com/new-submission/13068/3","title":"Biotechnology for Biofuels and Bioproducts","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"205f587d-358d-4d6a-8865-a25b707d2146","owner":[],"postedDate":"February 12th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":2340451,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2021-03-27T15:00:48+00:00","versionOfRecord":{"articleIdentity":"rs-218658","link":"https://doi.org/10.1186/s13068-021-01921-1","journal":{"identity":"biotechnology-for-biofuels-and-bioproducts","isVorOnly":false,"title":"Biotechnology for Biofuels and Bioproducts"},"publishedOn":"2021-03-19 15:00:30","publishedOnDateReadable":"March 19th, 2021"},"versionCreatedAt":"2021-02-12 17:35:12","video":"","vorDoi":"10.1186/s13068-021-01921-1","vorDoiUrl":"https://doi.org/10.1186/s13068-021-01921-1","workflowStages":[]},"version":"v1","identity":"rs-218658","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-218658","identity":"rs-218658","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}