CAOSA-extracted lignin improves enzymatic hydrolysis of cellulose

Abstract

The conversion of biomass into sugar platform compounds is very important for the biorefinery industry. Pretreatment is essential to the biomass of the sugar platform, however, the lignin obtained by pretreatment, as a key part of lignocellulose, generally has a passive effect on the enzymatic hydrolysis of cellulose into sugars. In this study, p-TsOH (p-toluenesulfonic acid), DES (Deep eutectic solvent) and CAOSA (cooking with active oxygen and solid alkali) pretreatment ways were used to fraction lignin from bamboo biomass. After CAOSA treatment, the hydrolysis efficiency of the pulp was 95.57%. Moreover, the effect of different treatment methods on lignin properties was studied and the promotion effect of lignin was investigated by adding it to the cellulose enzymatic hydrolysis system. In this work, the results showed that CAOSA-extracted lignin with lower Đ (1.31-1.25) had a better adsorption effect on the enzyme protein. p-TsOH-extracted lignin with a larger S/G ratio enhanced the inhibition of enzymatic hydrolysis. In addition, the presence of -COOHs in lignin could reduce its inhibitory effect on cellulose saccharification.

Headnote

Received 23 February 2023; revised 8 May 2023; accepted 29 May 2023

Available online 31 May 2023

Abstract

The conversion of biomass into sugar platform compounds is very important for the biorefinery industry. Pretreatment is essential to the biomass of the sugar platform, however, the lignin obtained by pretreatment, as a key part of lignocellulose, generally has a passive effect on the enzymatic hydrolysis of cellulose into sugars. In this study, p-TsOH (p-toluenesulfonic acid), DES (Deep eutectic solvent) and CAOSA (cooking with active oxygen and solid alkali) pretreatment ways were used to fraction lignin from bamboo biomass. After CAOSA treatment, the hydrolysis efficiency of the pulp was 95.57%. Moreover, the effect of different treatment methods on lignin properties was studied and the promotion effect of lignin was investigated by adding it to the cellulose enzymatic hydrolysis system. In this work, the results showed that CAOSA-extracted lignin with lower Đ (1.31-1.25) had a better adsorption effect on the enzyme protein. p-TsOH-extracted lignin with a larger S/G ratio enhanced the inhibition of enzymatic hydrolysis. In addition, the presence of -COOHs in lignin could reduce its inhibitory effect on cellulose saccharification.

© 2023 Institute of Process Engineering, Chinese Academy of Sciences. Publishing services by Elsevier B.V. on behalf of Ke Ai Communications Co., Ltd. This is an open access article under the CC BY license (http://creativecommons.Org/licenses/by/4.0/).

Keywords: Biomass pretreatment; CAOSA; Cellulose hydrolysis; Lignin; Enzyme

(ProQuest: ... denotes formula omitted.)

1. Introduction

Lignocellulosic biomass is obviously a new clean, renewable and sustainable energy source that can be converted into fuels and chemicals on a large scale [1]. The conversion of lignocellulosic biomass to sugar platform compounds is a key step for the bio-refining industry. Agricultural waste, a type of lignocellulosic biomass, increases with the increasing demand for agricultural production. The generation of this agricultural waste is becoming a problem that must be addressed. In China, about 900 million tons of agricultural waste are produced every year [2]. Simple disposal of this agricultural waste has negative environmental impacts. These agricultural residues are being considered as alternatives to producing energy. Pretreatment, enzymatic hydrolysis and sugar fermentation to biofuels are the main three steps in the conversion of lignocellulose to ethanol [3]. These three components are closely related and complex in structure, especially the rigid combination of cellulose and lignin, which leads to the low accessibility of cellulose [4]. The reticular polyphenolic structure of lignin involves various functional groups, among which aliphatic hydroxyl groups, phenolic hydroxyl groups and methoxy groups (-OCH3) are dominant moieties [5-7]. Hydroxyl and aromatic structures are the most critical functional groups that influence the properties of lignin [8]. However, different ratios of -OCH3 may lead to diverse lignin [9]. In addition, lignin is formed mainly by the connection of syringy 1 monomers, guaiac monomers and p-hydroxy-phenol monomers, which lack regularity and order [10]. This leads to difficulties in the conversion of cellulose to sugar.

The ^-toluenesulfonic acid (^>-TsOH) can dissolve lignin rapidly at atmospheric pressure and below boiling point, resulting in a low degree of condensation of lignin, which helps to stabilize the valorization of lignin through further depolymerization. The rapid, nearly complete delignification reaction simplifies the separation process and efficient recovery of chemicals is also available by recrystallization, which could reduce energy input [11]. In addition, deep eutectic solvent (DES) has been proven to easily cleave the rich ethers in lignin macromolecules, thus showing the potential to depolymerize lignin under mild conditions [12]. Lehmann et al. reported that DESs usually do not inactivate enzymes, which makes them of great value in biorefinery processes [13]. Furthermore, they are simple, environmentally friendly and economically feasible, and the loss of holocellulose and the generation of harmful by-products are avoided [14-16]. Our research group developed a new biomass pretreatment technology based on cooking with active oxygen and solid alkali, referred to as CAOSA. CAOSA process showed great potential in both lignocellulosic biomass fractionation and clean pulping. It used a lower liquid-solid ratio for pulping, avoiding subsequent processing of large amounts of the reaction liquid. Unlike traditional pulping methods, the CAOSA process did not produce sulfur or fetid compounds. Due to the presence of MgO, the CAOSA method produced pulp with better brightness and lower kappa value [17]. After CAOSA pretreatment, the pulp yield of corn stover was 50.2%, the delignification rate was 85.5% and the maximum delignification rate of bagasse was 95.4% [18,19]. In addition, it was undeniable that the solid base catalyst (MgO) was easy to recycle with no caustic soda or sintering required [20,21]. The study also proved that the cellulose pulp produced by CAOSA was very suitable for the production of fermentable sugar and other high-value chemicals [22].

In the saccharification process, the conversion of renewable biomass substrates into fuel and chemicals depends largely on the hydrophobic effect, adsorption and hydrogen bonding between cellulase and lignin [23]. The non-productive adsorption of lignin and cellulase is regarded as one of the main factors limiting hydrolysis efficiency [24]. As a potential raw material for biorefinery, lignin is a key block material in the hydrolysis of lignocellulose. Lignin generally has an adverse effect on cellulose hydrolysis. The resistance effect of lignin is thought to be related to its physical and chemical properties and functional groups [25]. This obstruction is associated with the structural complicacy of lignin and cell wall polysaccharide substrates. Lignin hinders and inhibits the entry of cellulase, while reducing the accessibility of cellulose [26]. Since the binding efficiency of cellulase and cellulose substrate was lowered by the adsorption of cellulase on lignin, the hydrolysis of cellulose would be restrained, which resulted in the increase of both enzyme loading and overall cost [27]. It is necessary to research the influence of some pretreatment processes and the corresponding lignin chemical properties during the enzymatic hydrolysis of cellulose. In addition, identifying lignin with weaker inhibitory and understanding its structural properties can help reveal the stimulating effect of lignin on the corresponding raw materials [28]. It is important to study the adsorption mechanism of lignin and cellulase, which can provide a reference for the biorefinery of lignocellulose. Therefore, it is of great significance to improve the enzymatic hydrolysis and biorefinery by using appropriate pretreatment and lignin separation methods.

This work compared the effects of these three pretreatment technologies and their severity on the property changes of lignin. The lignin has adsorption effect on cellulase. Hence, it is necessary to understand how these physicochemical properties of lignin affect the cellulose hydrolyzed, which can provide a feasible reference for producing glucose. Therefore, this work studies the structural changes of the lignin extracted by /7-TsOH, DES and CAOSA methods. FT-IR, TGA, GPC and NMR were used on the characterization of lignin.

2. Materials and methods

2.7. Raw materials

Bamboo powder (Cellulose = 44.89%, Hemic ellulose = 18.37%, Lignin = 26.95%) was smashed to a size of about 10 mesh and supplied by HYA Group Co. Ltd (Sanming, Fujian, China). Deuterated dimethyl sulfoxide (DMSO-d6) (99.9%), dimethylformamide (DMF), chromium (III) acetylacetonate and cyclohexanol (99%) were supplied by Energy Chemical, Shanghai; 2-chloro-tetramethyl-1,2,3dioxaphospholane and LiBr (99.9%) were provided by TCI Shanghai. The CellicCTec2 cellulase was supplied by Novozyme. Magnesium oxide was from Xiamen Lvyin Reagent Glass Instrument Co., Ltd. Sodium hydroxide and acetyl bromide (99%) were from Sigma-Aldrich. The microcrystalline cellulose, lactic acid, choline chloride and /?-TsOH were provided by Beijing Bailingwei Technology Co., LTD, Shanghai Yuanye Biotechnology Co., LTD and Beijing Galaxy Tianhong Chemical Co. LTD, respectively.

2.2. Methods

2.2.1. Pretreatments of biomass

DES pretreatment: bamboo powder (10 g), lactic acid (180 g) and choline chloride (20 g) were mixed in a flask. The reaction was run under 120-160 °C for 1 h [29,30]. /7-TsOH pretreatment: bamboo powder (10 g) was mixed with 75 wt% p-TsOH solution (200 g) and reacted at 70-110 °C for 30 min [31,32]. The filtrate was blended with 1 L water to precipitate the lignin for further testing. CAOSA pretreatment: bamboo powder (60 g) and MgO (9 g) were mixed in distilled water (600 mL). Then the mixture was heated to 140-180 °C and maintained for 3 h under 2 MPa O2 atmosphere [20]. The miniature reactor is purchased from Yantai Langchuan Experimental Equipment Co., LTD. Then the reacted mixture is filtered and diluted hydrochloric acid is added into the filtrates to precipitate lignin. The obtained sample was centrifuged, dried, washed, re-dried and saved for analysis.

The cellulose-rich residue was washed and dried to constant weight for components test. The constituent analysis was carried out by referring to NREL procedure [33]. The sugar content of hydrolysis solution was tested by HPIC (Dionex ICS 6000, Thermo Fisher, USA) equipped with an integral amperometric chemical detector. The elution solvent was 200 mmol L-1 NaOH solution. The flow rate was 1 mL min-1.

2.2.2. Fourier transform infrared spectroscopy (FTIR) analysis

The groups composition of lignin was identified by a Fisher's Nicolet 370 FT-IR equipped with a detector of 4 cm-1 resolution and 16 scans. The lignin powder was then thoroughly blended with KBr and pressed.

2.2.3. Thermal stability

Thermogravimetric analyses were performed by TGA/STA 449 F5, Netzsch, Germany. The thermal stability of bamboo lignin extracted by p-TsOH (BLP), DES (BLD) and CAOS A (BEC) were determined, respectively. The specimen was analyzed at a heating rate of 10 К min-1 from 30 °C to 800 °C under N2 environment.

2.2.4. Gel Permeation Chromatography (GPC)

The lignin (50 mg) and acetic acid (10 mL) were mixed and stirred for 15 min in a flask. And then acetyl bromide (1.25 mL) was dropped and stirred at 50 °C for 2 h. After acetylation, most of solvent was separated by rotary evaporator at 55 °C, and then ethanol (10 mL) was added to repeat rotary evaporation for 2-3 times to eliminate the remaining acetyl bromide. The acetylated samples were kept for GPC analysis. GPC is performed to analysis the number average molecular weight (Mn), weight average molecular weight (Mw) and dispersity (Đ) of lignin. The mobile phase of GPC is N, N Dimethylformamide (DMF) at a flow rate of 0.6 mL min-1 and 55 °C, using a column oven PLgel 5 pm MIED-C and a G7162A 1260 RID in 1260 Infinity II, Agilent. 0.1% LiBr was put into DMF to prevent oxidation of the mobile phase. Calibration was performed using standard polystyrene curves ranging from 580 to 3152000 g mol-1.

2.2.5. Nuclear magnetic resonance (NMR)

The 31P NMR analysis was employed to evaluate types and quantification of -OHs of substrate. The solution containing 20 mg of lignin was detected by Wuhan Zhongke Oxford WNMR-I 400 MHz. Its specific steps refer to previous research [34]. 2D ^-13€ HSQC-NMR was a common method to obtain the structural features of lignin. The sample (20 mg) was added to DMSO-d6 (0.55 mL) and spectra width of ·H and 13C was set as 0-15 ppm and 0-180 ppm, respectively. The number of scans and dummy scan was 8 and 16, respectively. For 13C and ^-dimension, the size of fid was set as 1024 and 256, respectively. The hsqcetgpsisp was used as pulse sequence. The accurate data were acquired on the Bruker AV 600 MHz NMR spectrometer (Switzerland).

2.2.6. X-Ray diffraction (XRD)

XRD pictures of different species were collected on Ultima IV Rigaku Japan diffractometer. The instrument operates at 30 kV and 40 mA. Cellulose-riched solids were scanned at the speed of 3° min-1 and angle was recorded from 26 of 10-40°. The CrI of cellulose is calculated following the equation (1) [35].

... (1)

/002 means the peak intensity around 20-22.5°, and Iam means the peak intensity of diffraction around 29-18.8°.

2.2.7. Enzyme hydrolysis of pretreated biomass

The pretreated biomass (2% w/v) was introduced into a flask, followed by adding citric acid buffer (50 mL, 50 mmol L-1) and 20 FPU cellulase/g cellulose. It was hydrolyzed in the flask at 50 °C (180 r min-1, 48 h). Tetracycline is used during enzymatic hydrolysis to avoid sugar consumption. The samples at 48 h were subjected to S-40D Biosensors Analyzer (SBA) for glucose analysis consistent with the reported method [36].

2.2.8. Enzymatic hydrolysis of microcrystalline cellulose and lignin

The microcrystalline cellulose (1 g) was introduced into a flask, to which citric acid buffer (50 mL, 50 mmol L-1) and 7 FPU cellulase/g cellulose were then added. The BLP, BLD and BLC (0.2 g) were added to the hydrolysis system respectively. Finally, the substrate was hydrolyzed in the flask at 50 °C (180 r min-1, 48 h). After the reaction, the glucose concentration of the hydrolysate was analyzed.

3. Results and discussion

3.1. Effect of different pretreatment ways on lignin structure properties

Fig. 1, Fig. S5a, Fig. S5b and Fig. S5c implied that the signal of ß-O-4 linkages persisting weak with increasing temperature in all three pretreatments. The intense pretreatment condition lead to the break of the ß-O-4 linkages, which is consistent with the report on p-TsOH [37]. Fig. S5a showed that Aa, A'Y, Aß(G), Aß(S), Ba, Bß, BY, Ca, Cß and X4 signal intensity (side-chain region) of BLP decreased rapidly. Fig. S5b showed that Aa, Aß (G), By and Cy signal intensity (side-chain region) of BLD also decreased rapidly, and Aß(S), Ca and X3 even disappeared. These results indicated that molecular structure was degraded by the intense temperature. As shown in Fig. S5c, the signals at Aa, Aß(G), AP(S), Ba and Ca, Cß, CY of BLC, corresponding to ß-O4 4 Cy-Hy in phenylcoumaran were weakened, respectively. Compared with the side chain region of milled wood lignin (MWL), DES pretreatment preserved the native lignin structure well. The BLP has Cß (Cß-Hß in phenylcoumaran) structure. For CAOS A, lignin fractions contains Cß, X2 and X3 structure and lost A'y (Cy-Hy in y-acylated ß-O-4) structure. The signals at X2, X3, X4 belong to ß-D-xylopyranoside become attenuate with increasing temperature. Interestingly, DES solvents and hydroxyl groups in lignin may combine to form ester/ether bonds. The /7-TsOH (at milder temperatures) and DES-extracted lignin both had A'Y and Bß (Cß-Hß in ß-ß (resinol)). Fig. S6 also implied that FTIR of samples are similar, and the functional group is maintained. However, the shrinkage vibration strength of chemical bonds is different, which indicates that different pretreatment conditions have different effects on lignin.

3.2. Effect of different pretreatment ways on hydrolysis efficiency

The mass of bamboo biomass pretreated by p-TsOH, DES and CAOS A were 10 g, 10 g and 60 g, respectively. After enzymatic hydrolysis, for example, the obtained mass of glucose obtained by after CAOS A pretreatment at 160 °C (BC160) reached 23.69 g. The enzymatic hydrolysis efficiency of the control group (only microcrystalline cellulose) was 50.40%. Compositions of bamboo biomass after pretreatment and the yields of lignin recovered from pretreatment hydrolysates were listed in Fig. SI and Table SI, respectively. The crystallinity of biomass was shown in Fig. S2. All of the hydrolysis efficiency of pretreated bamboo biomass and microcrystalline cellulose were listed in Table 1.

Liu used DES for bamboo pretreatment at 120 °C for 3 h, which resulted in a 94.4% delignification ratio and an improvement in cellulose yield. The results demonstrated that temperature was the predominant factor of delignification efficiency [38]. Wang proved that 80.1% of lignin was removed by DES pretreatment of bamboo at 140 °C for 6 h, which showed a high cellulose ratio in the solid residue (83.4%) [39]. Ling pretreated bamboo with DES at 120 °C for 2 h. The results showed that the ratio of cellulose in the solid residue was 44.11% [40]. Wang used p-TsOH to pretreat bamboo at 140 °C for 30 min. They found that 95.16% bamboo hemicellulose was extracted [41]. Table 1 and Fig. SI showed that the cellulose content and lignin removal rate in solid residue increased to 64.73% and 92.44%, respectively. It implied that DES pretreatment used in this study efficiently separated bamboo biomass in a short time with high delignification efficiency. In this work, the hydrolysis rate of hemicellulose reached 96.99% and the delignification efficiency was 91.27% after p-TsOH pretreatment. It fractionated bamboo biomass at lower temperatures with higher removal rates of hemicellulose and lignin.

As is listed in Table 1, the hydrolysis efficiency of biomass pretreated by DES was 29.68%, and it reached 49.26% after pTsOH pretreatment. ^>-TsOH is a strong organic acid that can contribute a proton in the water to accelerate the degradation of the glycosidic and ether bonds [42]. p-TsOH has been added to the delignification process through the formation of aggregation. It promotes the degradation of lignin and hemicellulose under mild conditions because of its prominent acidcatalyzed properties and surface-active nature under high concentrations [32]. The degradation of lignin and hemicellulose destroys the dense structure of the cell wall and increases the accessibility of cellulose, thus improving the efficiency of enzymatic hydrolysis [43].

Also, it could be concluded from Table 1 that the enzyme hydrolysis results of samples pretreated by CAOS A were much better than those of /?-TsOH and DES. After the hydrolysis for 48 h, the glucose content of CAOSA pretreated substrate (160 °C) was 14.3 g L-1 and its hydrolysis efficiency was 95.57%. MgO showed high delignification activity in the presence of O2 [44]. During CAOSA, delignification was mainly driven by O2. The change of phenolic structure caused by oxygen makes the benzyl alcohol group oxidized to the carbonyl group, which easily destroyed the ring-conjugated structure and further increased the delignification rate. MgO provided an indispensable alkaline environment for initiating oxygen delignification. Lignin was difficult to react with oxygen to form water-soluble fragments until the phenol hydroxyl group in lignin reacts to form phenol ions under alkaline conditions. Delignification slowed rapidly once the pH drops below about 7. A key role of a solid base was to neutralize various acidic substances formed during CAOSA [21]. The current study obtained high hemicellulose and lignin removal efficiency (88.04% and 98.84%, respectively), leading to 95.57% hydrolysis efficiency.

These results implied that CAOSA pretreatment actually destroyed the structure of biomass and increased the accessibility of cellulose for enzyme hydrolysis. With the further increase in temperature, the mass of solid residues gradually decreased, indicating that the composition of solid residues was still unstable and can be further decomposed. According to ANOVA shown in Tables S4-6, there was no obvious difference between temperature and the quality of pulp, glucose and lignin. It was shown in Table 1 that the hydrolysis efficiency of CAOSA pretreated biomass decreased steadily with increasing temperature (>160 °C). Actually, the reduction in enzymatic hydrolysis digestibility of PC 170 and PC 180 samples was due to the partial hydrolysis of cellulose and carbonization of cellulose at high temperatures. In addition, the higher temperature was proved to promote the fracture of the hydrogen bonds as well as the dehydration and carbonizing of cellulose [45].

In the hydrolysis process, ineffective adsorption of cellulase by residual lignin results in low enzymatic hydrolysis efficiency [46,47]. However, in this study, Table 1 showed that the recovered lignin obtained under mild pretreatment conditions (with samples BLD 120-140 (bamboo lignin extracted by DES at 120-140 °C) and BLC140-150) promoted the enzymatic hydrolysis of cellulose. The lignin obtained by higher the pretreatment intensity could inhibit the enzymatic saccharification efficiency, which may be caused by lignin enhancing the ineffective adsorption of enzymes [48]. Lignin could bind enzymes through hydrophobic interactions, hydrogen bonding and electrostatic interactions [49-51]. Among them, the hydrophobic interaction occurred between the benzene ring of lignin and the aromatic amino acids of the enzyme, and the two compounds with the same aromatic ring were bound together by the hydrophobic interaction. Hydrogen bonding mainly refers to the interaction between the amide group of the enzyme and the hydroxyl group of the lignin [52]. Cellulase contains many amino acid residues in its structure. The lignin fractions also have functional groups such as aliphatic, phenolic -OHs and -COOHs. The combination and separation of functional groups of lignin (-OHs and -COOHs) and cellulase (amino acid residues) in an aqueous environment result in potential electrostatic interactions between them [53].

3.3. CAO SA-extracted lignin improves the enzymatic hydrolysis

3.3.1. Lignin with higher Đ improves the enzymatic hydrolysis of cellulose

Mw, Mn and Đ of lignin from pretreatment by DES, pTsOH and CAOSA were listed in Table 2. The depolymerization of lignin mainly occurred at high temperatures, resulting in the gradual decrease of Mn and Mw. The same conclusions can be also drawn on BLC (1.49-1.25). As reported, the lignin with high Mn and Mw commonly had higher Đ [54].

Compared BLP and BLD, the Mn, Mw and Đ of BLC were slightly lower due to the high-temperature cooking of biomass. This indicates that the strong solubility of CAOSA pretreatment induces the dissolution of lignin components with different molecular weight, accompanied by polysaccharides [55,56]. BLD160 has a slight increase in molecular weight for BLD 150, possibly induced by the increase of polymerization of lignin under the increase in temperature. A large number of hydrogen bonds formed between the free radicals in the solution and the hydrophilic groups of lignin, resulting in the formation of lignin fractions with higher Mn and Mw, which is assumed as the factor affecting molecular weight [57]. According to GPC Table 2, the temperature has a certain influence on the Mn and Mw of sample. With the temperature increasing, the depolymerization of lignin was enhanced causing the improvement of its hydrophobicity, which was related to the unproductive binding of cellulase. Meanwhile, the Đ of extracted lignin decreased, indicating that the distribution of molecular weight for lignin was narrow. For DES and CAOSA, the results showed that lignin with lower Đ had a better adsorption effect on the enzyme protein [58].

3.3.2. Lignin with lower S/G improves the enzymatic hydrolysis of cellulose

As shown in Fig. 2, the S, H and G unit signals were found in the aromatic region, particularly the signals of C2 б-Н2 6 associate with oxidation unit (Sz) and S at óc/¿h 106.4/7.35 and 103.9/6.72, respectively [59]. Different bonds C2-H2 exhibited singáis at ôc/ÔH 111.0/6.99, C5 -H5 at Ôc/ôH 114.7/ 6.72 and C6-H6 at ôc/ôH 119.0/6.80. Besides, the signal at óc/ ôH 127.8/7.20 corresponding to the C2 б-Н2 6 linkage of the H unit [57]. Figs. S2a and b showed that the PCE and G6 signal intensity of BLP and BLD became weak as the temperature increased, which indicated that the pretreatment could destroy the aromatic region of lignin. For CAOSA, the PCE signal in sample attenuated as the temperature increasing, which suggested that the C2 6-H2 6, C3 5-H3 5 and C7-H7 linkages in the p-coumarate structure was decreased. Compared with the aromatic region of MWL, most of the BLP, BLD and BLC have FA2 (C2-H2 in ferulate) and Scond structure. It is worth noting that unlike lignin obtained by other pretreatments, the lignin extracted by CAOSA has G6' (the C6-H6 structure in oxidized (Ca = O) guaiacyl (G') units) units due to the presence of oxygen that oxidizes the Ca = O bond in lignin.

HSQC NMR spectra demonstrated that ether and ß-O-4 linkages were ruptured during the pre treatment process. The results implied that the lignin with high Mn and Mw had a higher ratio of ß-O-4 bonds [60]. Hydrophobic interaction between organic solvent molecules and depolymerization structure including lower ß-O-4 bonds contributes to the dissolution of lignin fragments. This was consistent with GPC test results. Although lignin extracted from 72-TsOH has a higher Ð, BLP has a strong inhibitory effect on enzymatic hydrolysis. This could be attributed to its S to G unit ratio. The S/G ratios of BLP70 and BLP110 are 0.52 mol/mol and 0.40 mol/mol, respectively, which are lower than those of BLP80, 90 and 100. For p-TsOH and DES pretreatment, Table S7 showed that with the increase in temperature, the proportion of S/G in lignin improved. Table 1 showed that the addition of lignin with a high proportion of S/G ratio resulted in a decrease in the enzymatic hydrolysis efficiency of microcrystalline cellulose. As reported, S unit could limit cellulase accessibility, as it usual is found in more cross-linked lignin structures, which formed a larger physical barrier against the access of cellulase [61]. Therefore, higher S/G ratio tended to decrease hydrolysis efficiency of cellulose.

3.3.3. Effect of thermal stability of lignin on enzymatic hydrolysis

TGA and DTG (derivative thermogravimetric analysis) were conducted to record the thermal stability of lignin with p-TsOH, DES and CAOSA extracted. The reagents used in these pretreatments had different properties, including polarity and the ability to form hydrogen bonds with lignin. The lignin recovered from each solvent was thoroughly characterized. Temperature strongly affected the properties of the lignin fraction. This was important when considering the selection of lignin fractions with customized properties for the preparation of specific materials, which provided a reference for the rational utilization of lignin. For example, 1) lignin with high molecular weight can be applied as a filler for composites; 2) low-molecular-weight lignin is usually used for plasticizers; 3) lignin with high phenolic -OH content is ideal for phenol-formaldehyde resin production, and 4) lignin with high thermal stability are widely used as the carbon-fiber precursor [62].

Moreover, it has been reported that the type and contents of lignin functional groups generally affect the thermal properties of lignin, and the thermal degradation behavior of lignin is mainly based on the cleavage of ß-O-4, C-C, ß-ß linkages [63,64]. As shown in Table S2, the major mass loss of lignin happened between 200 °C and 350 °C due to the cleavage of ß-O-4 linkages. Then most C-C linkages were oxidized at 350-400 °C. The ß-ß and ß-5 linkages were cleaved above 400 °C. The mass loss was minor after the temperature reached 600 °C compared with previous stages.

Table S2 shows molecules with a large number of ß-O-4 bond are available under relatively mild pretreatment conditions. From Table S2 and Fig. 3., it could be seen that the ß-O4 4 in lignin decreased with temperature increasing. Table S7 showed that the lignin extracted from CAOSA had larger S/ G, which had a negative effect on enzymatic hydrolysis. The lignin extracted under mild conditions (especially BLC140 and BLC150) had a large proportion of hydrophilic ß-O-4 linkage, which led to the low hydrophobicity of lignin and reduced the lignin adsorption of cellulase, eventually resulting in increased hydrolysis efficiency [65].

3.3.4. Lignin with higher -COOHs improves the enzymatic hydrolysis of cellulose

The -OHs in the lignin fractions extracted by /2-TsOH, DES and CAOSA were tested by 31P NMR. The domain of integration was shown in Table S3. Fig. S3 used MWL as an example to show the process of quantifying the primary and secondary OH content of lignin. The 31P NMR data of all the lignin fractions were shown in Fig. S4. 31P NMR indicated that more a-OH was kept after DES process, and lower a-OH content was detected in the lignin with p-TsOH and CAOSA. Table 3 indicated that a-OH was oxidized during the CAOSA process and modified in the pTsOH process due to the elimination reaction catalyzed by acid. The high content of the phenolic OH illustrates the fracture of ßO-4 bond, which was also found in ^-130 HSQC spectra [66]. In addition, the carboxyl content of lignin can also reveal the degradation and polymerization of lignin [67]. As shown in Table 3, the carboxyl content of lignin decreased with the increase of reaction severity, for CAOSA, where the carboxyl content was 1.18-0.95 mmol g-1. The main reason is deduced that in alkaline and high-temperature conditions, carboxyl is converted into CO2 in a decarboxylation reaction. For /э-TsOH and DES, the decrease of carboxyl group content is speculated to be due to the presence of acid reagent, resulting in esterification reaction. For ВЕС, the ß-O-4 content and molecular weight decreased along with depolymerization at high temperatures. The interaction between the solvent and the lower molecular weight contributed to the dissolution of the lignin. HSQC and GPC results proved that the poor solubility of BEC 140 and BEC 150 were due to higher ß-O-4 content and molecular weight [60].

Moreover, for p-TsOH-extracted lignin, Table 3 showed that increasing reaction severity can destroy more ß-O-4 bonds and decrease S and G phenolic-OHs. Notably, for CAOSA lignin, the break of ß-O-4 bond may promote the formation of aliphatic hydroxyl. For DES-extracted lignin, the content of S-phenol -OHs in BLD 160 is almost 2.5 times that in BLD 120 due to the increased treatment severity. By comprehensive comparison, it can be seen that BLP70, BLD 120 and BLC160 contain a large amount of phenolic OHs (2.40,2.07 and 0.84 mmol g-1), which shows great utilization value and can be used as raw materials for producing polyurethane, epoxy resin and phenolic resin [68]. In this study, with the improvement of pretreatment intensity, the content of -COOHs in lignin gradually reduced which led to a decrease in its hydrophilicity. The content of the -COOHs in lignin (BLD 120-140) is higher. The presence of -COOHs in lignin could reduce its inhibitory effect on cellulose saccharification [69].

4. Conclusions

Three pretreatments (p-TsOH, DES and CAOSA) have been used to prepare glucose from biomass. Biomass has a better cellulose enzymatic hydrolysis efficiency after CAOSA treatment. The existence of BLD120-140, BLC140 and BLC150 improved the enzymatic hydrolysis of microcrystalline cellulose. With increasing pretreatment severity, the number of ß-O-4 bonds and the -COOHs content of lignin fragment decreased. The presence of carboxyl groups in lignin reduces its inhibitory effect on cellulose saccharification. Lignin with lower Đ had a better adsorption effect on the enzyme protein and a larger S/G ratio causing the inhibition of saccharification. This work provides a feasible reference in the depolymerization of lignin and yield sugar products.

Declaration of competing interest

No conflict of interest exists in the submission of this manuscript, and the manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and is not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed. The author Xianhai Zeng is a Youth Editorial Board Member for Green Energy & Environment and was not involved in the editorial review or the decision to publish this article.

Acknowledgements

This work was financially supported by the National Key R&D Program of China (No. 2021YFC2101604), National Natural Science Foundation of China (No. 22278339, 21978248), Fujian Provincial Key Science and Technology Program of China (No. 2022YZ037013).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.Org/10.1016/j.gee.2023.05.009.

Footnote

* Corresponding authors.

E-mail addresses: [email protected] (J. Liu), [email protected] (X. Zeng).

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