3.4 The impacts of ChCl-DHBA DES pretreatments on the physicochemical properties of lignin
The characteristics of the fractionated lignin from biomass pretreatment are important to achieve a successful biorefinery strategy. Therefore, the properties of lignin are as important as its recovery. Moreover, the changes in lignin reveal how the pretreatment chemicals and solvents transformed biomass components. Lignin was recovered from the DESs after the pretreatment of WT and TG sorghum stover. Molecular weight distribution, the composition of aromatic units, and abundance of interunit linkages were characterized by GPC and 2D HSQC NMR analyses. Detailed NMR spectra and corresponding structures are shown inFigure S2 . By assigning peaks based on previous studies,45,46 cross-signals corresponding to syringyl (S), guaiacyl (G), p -hydroxyphenyl (H), p -coumaric acid (PCA), ferulic acid (FA) and tricin (T) could be observed in the aromatic regions of the spectra (Figure S2b ). Signals correlated to condensed S units were also detected but only at a low level. In the aliphatic regions, peaks of β–O–4 ether (A) and methoxy groups (OMe) were identified, as well as a low level of β-5 linkages (Figure S2a ). Semiquantitative analysis results based on S, G, and H units of the NMR spectra are shown in Figure 4 . Overall, the lignins fractionated from WT and TG sorghum stover by the same ternary DES, either ChCl-DHBA-EG and ChCl-DHBA-W, were structurally similar, while binary DES resulted in a different abundance of β–O–4 linkage with two biomass. As shown in Figure 4a , lignin isolated from ChCl-DHBA DES pretreated WT biomass had 9/100Ar β–O–4 linkage, which was similar to lignin fractionated by ChCl-DHBA-EG DES pretreatment. However, for the TG biomass, β–O–4 linkage only accounted for 4/100Ar in the fractionated lignin from TG biomass after ChCl-DHBA DES pretreatment. It is worth noting that ChCl-DHBA DES pretreatment removed much less amount of lignin from TG biomass (9%) than from WT biomass (22%). Examining the abundances of subunits (Figure 4b ) shows that lignin isolated from binary ChCl-DHBA DES pretreatment had higher hydroxycinnamates (PCA and FA) than those fractionated by the ternary ChCl-DHBA DESs pretreatments. Combining with the lower delignification of the binary DES than the ternary DESs, it can be inferred that binary ChCl-DHBA DES mostly extracted the aromatic units incorporated in lignin via ester linkages which are susceptible to cleavage,47,48 rather than extracting the bulk lignin. The higher amounts of hydroxycinnamates observed in binary DES pretreated WT lignin compared to TG lignin indicate that binary DES was less effective on the engineered sorghum stover.
Different DES compositions resulted in varied alterations of lignin interunit linkages and aromatic subunits. In both WT and TG sorghum, lignin fractionated by ChCl-DHBA-W pretreatment had the highest β–O–4 ether content, being approximately 15/100Ar. The impact of ternary DESs on lignin did not show significance between the WT and TG biomass, unlike the binary DES, for both interunit linkages and aromatic subunits (Figure 4 ). Another important observation from the 2D HSQC NMR spectra is cross-signals corresponding to DHBA, identified with the peaks at δCH=116.3/7.3 ppm, 121.6/7.3 ppm, 121.6/6.8 ppm, and 114.8/6.8 ppm (overlapping with G5), as indicated in Figure S2 . The appearance of DHBA in the recovered lignin could be caused by the accumulation of DHBA in planta due to genetic modification and/or arose from residue DES. Nonetheless, our previous study has demonstrated the feasibility of selectively recovering the aromatic component from isolated lignin via depolymerization.33 The recovery of DHBA in lignin to facilitate a sustainable biorefinery process will need further studies.
The weight-average molecular weight (MW), number-average molecular weight (Mn), and dispersity (Đ) are shown inTable 3 , while the molecular weight distributions are displayed in Figure 5 . The average molecular weights and Đ of the fractionated lignins showed a narrow range, indicating that lignins had relatively uniform size distribution after pretreatment. The molecular weights and Đ of lignin fractionated from the two sorghum stover by the aforementioned ChCl-DHBA DESs were similar, with standard deviations of 240 g/mol and 0.1, respectively. Lignin fractionated from WT sorghum stover by ChCl-DHBA-W showed the lowest molecular weight of 1078 g/mol, while ChCl-DHBA-EG pretreatment of the TG biomass resulted in the highest lignin molecular weight of 1776 g/mol, which could be attributed to the depolymerization and repolymerization of lignin during the DES pretreatments. Lignin fractionated by binary DES pretreatment showed lower molecular weights than those fractionated by ternary DESs, which is consistent to the 2D HSQC NMR results.