Efficient biomass pretreatment
The release of reducing sugar
Generally, pretreatment is the primary step for the utilization of biomass to sugars, ethanol or other compounds [36]. The sugar yields after enzymatic digestibility is the major criteria for determining the efficiency of pretreatments. Enzymatic hydrolysis of CS under all pretreatment conditions was carried out and the corresponding reducing sugar yield was investigated (Fig. 1). All the reducing sugar yields produced from CS that pretreated by THF-H2O system under different conditions were higher than that (84.7 mg/g) of the untreated [24]. They are 301.23 mg/g, 588.45 mg/g, 619.62 mg/g of TH21, TH22, TH24 and 579.83 mg/g, 640.87 mg/g and 613.9 mg/g of TH51, TH52, TH54. The maximum yield of 640.87 mg/g was obtained after pretreatment under TH52, which was highly competitive compared with others in previous works [15, 37]. In addition, the yield of the reducing sugar increased from 301.23 mg/g (TH21) to 619.62 mg/g (TH24) with the prolongation of reaction time under 120 °C, which was attributed to the breakdown of the biomass recalcitrance structure. A large amount of lignin was dissolved by THF–H2O, and the internal cellulose was gradually exposed, which promoted the accessibility of the cellulase to the substrate [25]. In general, the reducing sugar yield of biomass pretreated by THF–H2O increases with the increase of the pretreated temperature and time [25]. However, it was surprised to observe that the reducing sugar yield of CS pretreated at 150 °C increased from 579.83 mg/g (TH51) to 640.87 mg/g (TH52) first, and then decreased to 613.9 mg/g (TH54) with the increase of the pretreated time. Although enzymatic digestion was significantly improved due to lignin removal from CS by THF–H2O under low temperature, there is still lignin remained in the pretreated CS, may play a physical adsorption role of nonproductive binding with cellulase in the enzymatic hydrolysis [38, 39]. This lignin residue can be further dissolved and removed from cell wall structure under severe condition [40], which resulted that the reducing sugar yields under 150 °C are higher than that under 120 °C with similar pretreated times. Although most of lignin has been removed from CS under TH54, the reducing sugar yield was not increased compared to that of TH52. In general, the more lignin removed under severe condition, the more internal cellulose exposed to the outside, which was benefit to the enzymatic hydrolysis of cellulose. For this phenomenon, we speculated some of the exposed cellulose might be attacked, which resulted in a decrease in the amount of degradable cellulose. The conceivable proof will be discussed and proved depending on the SEM analysis.
Morphology change of CS
The purpose of pretreatment is not only to remove lignin on the surface of biomass as much as possible but also to maximize the retention of cellulose to obtain a high yield of the reducing sugar. The efficiency of biomass pretreatment is closely related to the change of its structure. To deep understand the reducing sugar production from pretreated CS, the morphology changes of CS before and after pretreatment were analyzed. The surface of the untreated CS (Fig. 2a) is smooth and rigid, covered with a large amount of lignin which is the physical barrier for cellulase [41]. In contrast, the surface of CS pretreated under TH21 was changed (Fig. 2b). Some lignin droplets were formed and deposited on the surface of the pretreated residues,which was similar to the phenomenon that was found in dilute sulphuric acid pretreatment of lignocellulosic biomass [15]. The formation of lignin droplets was caused by the acidic environment created by adding sulfuric acid as a catalyst in THF–H2O system. However, the lignin droplets remained on the surface of CS hindered the enzymatic hydrolysis, resulting in the low yield of the reducing sugar under TH21. In contrast, the morphology of CS pretreated under the other five reaction conditions has undergone tremendous changes. For example, the CS pretreated by TH22 was split and the cellulose fiber was observed (Fig. 2c). The CS was seriously deformed after TH24 pretreatment (Fig. 2d), indicating that the structure of the CS was fragile and collapsed [42]; No matter TH22 or TH24, the integrated structure of CS has been destroyed, the most importantly, cellulose appeared. It means that the enzymatic hydrolysis was easier than the CS covering with rigid lignin and lignin droplets. For TH51, CS was also cracked, showing the internal cellulose structure (Fig. 2e). Moreover, the fiber bundles can be seen in TH52 pretreated CS (Fig. 2f), which significantly increased the accessibility of cellulose to the enzyme, contributing to the highest reducing sugar yield. With the aggravation of reaction conditions, TH54 emerged a slurry structure (Fig. 2g), it was worth noting that although it retained the cellulose, its structure was not as loose as the TH52, which explained the cause of reducing sugar yield decrease.
Component change and mass loss of the CS
The change of CS structure after pretreatment will inevitably lead to the change of component and the loss of mass. Therefore, the analysis of component changes and mass loss before and after pretreatment was conducted.
The CS used in this study consists of 34.9% of cellulose, 38.5% of hemicellulose, 13.9% of lignin, and 12.7% of other non-organic and organic components [24]. As shown in Additional file 1: Figure S3, compared with the untreated CS, lignin and hemicellulose in the solid residual decreased from 13.87 to 10.02% and from 38.36 to 13.8%, respectively, with the increase of reaction time at 120 °C. It indicated that lignin and hemicellulose is continuously dissolved in THF–H2O system. The removal of lignin and hemicellulose also gradually increased at 150 °C. The content of lignin and hemicellulose in CS residue after pretreatment decreased from 12.8 to 6.59% and from 30.68 to 10.21%, respectively. Due to the removal of lignin, the content of cellulose was correspondingly increased. It changed from 47.71 (TH21) to 55.68% (TH24) and from 52.99 (TH51) to 71.71% (TH54), respectively. However, the highest cellulose content of 76.11% occurred in the TH52, which was best to the production of reducing sugar from TH52, the result was in line with the reducing sugar yield.
The mass loss of treated CS was all increase from TH21 to TH24 and TH51 to TH54. The data were 36.14%, 50.54%, 52.00% and 48.11%, 55.11%, 67.46% under 120 °C and 150 °C, respectively (Fig. 3). The mass loss of CS under TH21 was the minimum of 36.14%. In addition, based on the analysis of the reducing sugar yield and the morphology of the CS, it can be inferred that the mass loss of CS is mainly attributed by the removal of a small amount of lignin and other components like hemicellulose by THF–H2O co-solvent system [42]. Most of the lignin formed droplets and deposited on the surface of the CS residue. Compared with TH21, the mass losses of TH22 and TH24 were markedly to 50.5% and 52% respectively, which were resulted from most of the hemicellulose and lignin removal (Additional file 1: Figure S3). Similarly, with the increase of reaction time, the more lignin was removed, the increasing mass loss was observed at 150 °C. Moreover, under TH54, some cellulose was degraded after lignin removal, which resulted in the mass loss reach to 67.46%, and it was unacceptable in the practical application.
Therefore, in order to maximize the yield of the reducing sugar from biomass, it is very important to optimize the pretreatment conditions. Compared with the other THF pretreatment [43, 44], in our work, the pretreatment method adopted the lower temperature (120 °C), and we also obtained the satisfying result. The most important reason we selected THF–H2O co-solvent as the pretreatment solvent is that the pretreatment liquid can be directly to prepare lignin nanoparticles [20, 26] rather than discarded.
Formation of LCPs
The current utilization of biomass resources is involved in the utilization of both cellulose and lignin due to its unique structure. Therefore, in addition to the high reducing sugar yield obtained from CS by THF–H2O pretreatment, we directly used the pretreatment supernatant to prepare LCPs via introducing water into it without other chemical modification, which realized the efficient utilization of lignin in biomass. Different concentrations of colloid solution were formed based on the supernatants obtain from pretreatment under six different reaction conditions (Additional file 1: Figure S2). To understand the characteristics of the colloid solution, we adopted SEM to observe the morphology of LCPs, nano size analyzer to get information of particle size distribution statistics, Uv–vis to measure the concentration of colloid solution, and FTIR to analyze the functional groups of lignin particles.
Morphology and Size analysis of LCPs
All the prepared LCPs were spherical and rich, concatenate (Fig. 4). The phenomenon is similar to other researches [27, 45], resulting from the electrostatic interaction. The particle size increased with the increase of the reaction time under the temperatures of 120 °C and 150 °C. LCP21 were very small and had a small number. The particle size mainly distributes between 164 and 295 nm, and the average particle size was 202 nm. The size distribution of LCP22 was 164–342 nm, which was a little bigger than that of LCP21. Its average particle size was 243 nm and the dispersion of particles was better than that of LCP21. LCP24 had a particle size distribution of 255–615 nm with the average particle size of 385 nm. Besides, it could be observed that the minimum particle size of LCP24 is larger than the average particle size of LCP21 and LCP22. Moreover, the particle sizes of LCP51, LCP52, and LCP54 were gradually increased. The corresponding particle sizes were 190–342, 459–712, 615–955 nm with the average particle sizes of 259, 547 and 732 nm, respectively. These results indicated that the lignin particle size gradually increased with the increase of reaction intensity. In addition, some particles of LCP54 even reached about ~ 1000 nm. The explanation for this result might be related to the lignin structure dissolved in different pretreatment solution. In addition, the LCPs prepared in this work was comparative to the other lignin particles with various shapes and sizes prepared by different methods (Additional file 1: Table S1).
To confirm the different particle size of LCPs was caused by the structure of lignin, we next adopted the UV wavelength scanning to observe the if the formation interaction changes.
Uv–vis analysis of LCPs
Uv–vis was used to compare the lignin in the pretreatment solution with the corresponding LCPs to analyze the formation of LCPs. Previous studies have discussed that the LCPs are formed by the π–π interaction of orbitals of non-chemical bonds between aromatic rings in lignin molecule [20, 46]. Before and after the formation of colloid particles, the adsorption peak would shift under the π–π interaction [46]. As displayed in Fig. 5, the absorption peak of typical benzene compounds (~ 280 nm) was observed in the lignin and LCPs solution, suggesting the aromatic ring structure of lignin has not destroyed in the process of LCPs formation. It was consistent with the conclusion that π-π interaction in lignin caused the hydrophobic effect which brought about the formation of lignin colloid in solution [20]. The different shifts of maximum λ at ~ 280 nm indicated the weakening or strengthening of aromatic groups in π-stacking from lignin [20]. Lignin consist of three units including guaiacyl, syringyl, and phydroxyphenyl, their aromatic structure is different [19]. Therefore, the variation in π-stacking may be attributed to the different aromatic structures of lignin dissolved in the pretreatment liquid. Thus, we will conduct the effect research of lignin structure on LCPs.
Moreover, we found the LCP24, LCP52, and LCP54 had a higher absorption peak at maximum λ, which were related to their high concentration that can be seen in Additional file 1: Figure S2. The higher concentration of LCPs depended on the increase in lignin dissolution with the increase of pretreatment reaction degree.
FTIR analysis of LCPs
The structural properties of materials determine their application values. Therefore, FTIR was used to characterize the functional groups of LCPs. As shown in Fig. 6, the abundant functional groups were visually observed in the LCPs prepared from pretreatment solutions at 120 °C and 150 °C. The broad absorption peak at ~ 3400 cm−1 and the band at ~ 1379 cm−1 represent –OH stretching of phenolic and aliphatic moieties. The bands at 2918 cm−1 and 2849 cm−1 represent C-H stretching [47]. Aromatic skeleton stretching occurs at ~ 1600 cm−1, ~ 1515 cm−1, ~ 1426 cm−1. The C=O stretching in guaiacyl (G) unit appeared at ∼ 1267 cm−1 [48]. All these location peaks suggested that there is no significant change in the aromatic structure of lignin in the process of LCPs formation; and the particle surface modified with high phenolic hydroxyl and carboxyl functional groups is good for the solubility and stability of LCPs. In addition, LCPs have extra function groups of C=O (at ~ 1330 cm−1 and ~ 1265 cm−1) and C–O–C (at ~ 1165 cm−1) compared with the lignin [49]. In recent years, the LCPs with plenty of functional groups have been reported to play an important role in the adsorption materials applied in the environment field and other composite materials preparation for biomedical or cosmetic fields [18, 50]. Therefore, the LCPs in our study have the potential to be applied in these fields.
Influence of lignin on the LCPs
Spherical LCPs with different sizes were prepared in different pretreatment solution. The formation of LCPs is closely related to the structure and concentration of dissolved lignin [28], therefore, we not only performed component analysis, but also extracted the lignin from the liquid and analyzed their molecular weight and structural changes by GPC, FTIR, and 2D-HSQC-NMR to elucidate the impact mechanism of lignin on the formation of LCPs.
GPC analysis
The analysis of the changes in lignin molecular weight through GPC can be used to characterize the degree of lignin polymerization after THF–H2O pretreatment. The deconstruction of the macromolecular lignin can be characterized via three indexes including weight-average molecular weight (Mw), number-average molecular weight (Mn), and dispersive poly-dispersity (PDI).
Figure 7 displayed the Mw and PDI of lignin extracted from different pretreatment solutions. The lignin extracted from the raw CS had the highest Mw (9742 Da) and PDI (2.57), indicating the high polymerizability and complexity of lignin in CS [51]. Compared with the raw lignin, the Mw of lignins in the pretreatment solution drastically decreased. The Mw and PDI increased from 2670 and 1.95 of lignin-TH21 to 3527 and 2.38 of lignin-TH24, respectively. In addition, the highest Mw (3644) and PDI (2.42) were obtained in lignin-TH51, then, decrease to 2988 and 2.16 of lignin-TH54. Combined with the above “The release of reducing sugar” section analysis, it can be inferred that lignin with small molecule weight was initially dissolved by THF–H2O under TH21, and the molecular weight distribution (MWD) is narrow; Then, more and more lignin with large molecule weight was dissolved, the corresponding MWD gradually widened. The molecule weight of dissolved lignin from TH51 reached to the maximum. Next, the dissolved lignin started to depolymerize as the reaction condition continued to intensify. However, some of depolymerized lignin might repolymerize under severe condition [52, 53]. Therefore, the Mw of lignin-TH54 was a little higher than that from lignin-TH52. It seems to explain the increased size of LCPs was because of the increase of Mw from lignin-TH21 to lignin-TH24. However, it was not suitable to the trend from LCP-TH51 to LCP-TH54. Thus, it is necessary to further analyze the specific changes of lignin structure and ensure the form of lignin units in THF–H2O.
FTIR analysis
Based on the reducing sugar yield and the characteristics of the prepared LCPs, the lignin-TH22 and lignin-TH52 were selected for FTIR and 2D-HSQC-NMR analysis to further investigate the effect of lignin structure on the size of LCPs.
Obviously, FTIR spectra (Fig. 8) showed that most of the characteristic peaks of lignin-TH22 and lignin-TH52 still remained in these two samples [54, 55]. The peaks at 1617 cm−1, 1513 cm−1, and 1426 cm−1 were related to the vibration of aromatic ring skeleton, and the peak at 1462 cm−1 represented the C-H deformation of aromatic ring skeleton. These peaks indicated the existence of benzene structure in lignin-TH22 and lignin-TH52 [24]. However, compared with lignin-TH22, the peak at 1320 cm−1 which represented C=O stretching in syringyl (S) derivatives disappeared in lignin-TH52. In addition, the G ring breathing with C=O stretching (at 1270 cm−1) significantly decrease from lignin-TH52 [56]. Moreover, the peak at 1164 cm−1 which was attributed to the C–O–C vibration only remained in lignin-TH22. The peak at 1227 cm−1 associated with C–C, C–O, and C=O stretching only appeared in lignin-TH52. These changes might be caused by the break of linkage bonds due to the severe pretreatment. The destruction of linkages resulted in the changes of lignin structure units (i.e. S and G), which might be the key factors to explain the formation of LCPs with different size. In order to further confirm the speculation, we adopted the 2D-HSQC-NMR analysis to illustrate the structural changes of lignin.
2D-HSQC-NMR analysis
Generally, the lignin consists of three basic units (S, G and p-hydroxyphenyl (H)) and linked by β-O-4, β-β, β-5 etc. [57]. All these signals can be reflected through the 2D-NMR spectra. The signal information of side chain region (δC/δH 50–90/2.5–6.0) and aromatic region (δC/δH 100–135/5.5–8.5) of lignin-TH22 and lignin-TH52 was displayed in Fig. 9. And the main cross-signals of lignin related to HSQC spectra were listed in Additional file 1: Table S2.
In the side chain region, the signals of β-O-4 and β-5 bonds in lignin-TH22 is stronger than that of lignin-TH52, indicating the lignin in the dissolved process was severely destroyed under TH52. In the aromatic region, the S-type and G-type of lignin-TH52 significantly decreased, and the signals of H-type lignin completely disappeared. In contrast, the signals of all three basic lignin units (S, G and H-type) remained in lignin-TH22. Specifically, the G-type lignin-TH22 were much higher than that of lignin-TH52. It is suggested that THF–H2O can dissolve the three types of lignin initially, and H lignin was not destroyed in lignin-TH22. However, the portion of G-type lignin was depolymerized in lignin-TH52. It is worth noting that it is easier for high content of G-type or low S/G lignin to form small size LCPs [21]. Therefore, in order to achieve the preparation of LCPs with smaller particle size, the reaction solvent must have the ability to dissolve more G-type lignin or control the deconstruction of G-type lignin via the suitable reaction environment. In our work, the size of LCP22 was lower than that of LCP52, suggesting the THF–H2O solvent under TH22 efficiently dissolved more types of lignin, the G-type lignin under TH52 was not as much as TH22 due to its depolymerization.
The concentration of lignin in the supernatant
The concentration of lignin depends on the amount of lignin removed after pretreatment. Lignin removal was increased with the increase of the reaction intensity, what was demonstrated by the component change (Additional file 1: Figure S3). Component analysis showed that the order of lignin content removed from the CS was TH54 > TH52 > TH24 > TH52 > TH22 > TH21. The highest concentration was obtained under TH54. Besides, lignin concentration could be directly observed from the color and turbidity of the LCPs solution (Additional file 1: Figure S2). Size analysis of LCPs indicated that the change of particle size is consistent with the change of lignin concentration. And the largest size of LCPs was obtained under TH54, which may be because the gather between particles. At the same volume, the particle size in the solution with low concentration of lignin is smaller due to the less content of lignin.
Lignin from the pretreatment solution under TH52 cannot form good LCPs even if the maximum yield of reducing sugar was obtained. However, pretreatment under TH22 realize the preparation of LCPs with good properties and high reducing sugar yield. Therefore, the pretreatment under TH22 was more practical in the actual biorefinery. The schematic of the sugar conversion and the mechanism of the formation of LCPs was fabricated as shown in Fig. 10. This strategy provides a practical guiding significance for the design of biomass pretreatment and the preparation of LCPs.