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 (Rpulp) 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 C1 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 (Rpulp) 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 Rpulp 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]. 13C 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 C1 groups of cellulose. The overlapping signals in the region of 70-80 ppm are assigned to C2, C3 and C5 of cellulose. In the spectra of cellulose, the amorphous carbons of C4 are represented by a fairly broad signal from 80-85 ppm, while crystalline carbons of C4 generate a sharper resonance from 85-92 ppm. Two phases of C6 cellulose are found at about 63 and 69 ppm, respectively. The peaks around 21 and 172 ppm in the spectrum of Rpulp 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 C4 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 C4 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 (Mw) and number average molecular-weights (Mn) and polydispersity values (Mw/Mn) of six alkaline hemicelluloses from ryegrass. The Mw 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 H0.15% 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 13C 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 13C 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 13C NMR spectra. The signals at 102.2, 76.1, 74.6, 73.6 and 63.3 ppm correspondes to C1, C4, C3, C2, and C5 of β-(1-4)-linked-D-Xylp units, respectively. The signals for C1~C5 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 C6, C4, C5 and methoxyl group of 4-O-methyl-D-glucuronic acid, respectively. However, the C6 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 (C3) 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].