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 δC/δH=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.