Mechanistic insight of pseudo-lignin formation and its impact on enzymatic hydrolysis
Fan Hu, Arthur J. Ragauskas. School of Chemistry and Biochemistry, Institute of Paper Science and Technology
Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
Abstract
Pseudo-lignin, which can be broadly defined as aromatic material that yields a positive
Klason lignin value and is not derived from native lignin, has been recently reported to form
during dilute acid pretreatment of poplar holocellulose. To investigate the chemistry of
pseudo-lignin formation, GPC, FT-IR and 13C NMR have been utilized to characterize
pseudo-lignin isolated from dilute acid pretreated α-cellulose and holocellulose. The results
show that pseudo-lignin consisting of carbonyl, carboxylic, aromatic and aliphatic
structures can be produced from both dilute acid pretreated cellulose and hemicellulose. In
addition, pseudo-lignin extracted from pretreated holocellulose had similar molecular
weights and structural features. The presence and structure of pseudo-lignin is important
to bioethanol production since it is shown in this study that pseudo-lignin can significantly
decrease the enzymatic conversion yield of cellulose (from 7 to 42% compared to dilute
acid pretreated poplar holocellulose).
Experimental Hybrid poplar milled to a pass a 2 mm screen was obtained from Oakridge National
Laboratory.
The sample was air-dried and extractives were removed by Soxhlet extractions with
ethanol/benzene. Holocellulose and α-cellulose were isolated from the extractive-free
poplar according to the literature methods.
Two-step dilute acid pretreatments were performed on both poplar holocellulose and α-
cellulose. The pretreatment conditions are listed in Table 1.
Pseudo-lignin was isolated from pretreated holocellulose and α-cellulose by p-
dioxane/water mixture according to the literature methods.
Several pseudo-lignin on holocellulose samples were prepared, and the glucose yields of
enzymatic hydrolysis of these samples were compared with dilute acid pretreated poplar
and holocellulose.
Conclusions and
Future work
Pseudo-lignin consists of
carbonyl, carboxylic, aromatic
and aliphatic structures.
Formation of pseudo-lignin
should be avoided since it can
significantly inhibit enzymatic
hydrolysis of cellulose.
Investigation of the impact of
pseudo-lignin versus dilute
acid pretreated lignin on
enzymatic deconstruction of
cellulose.
Preliminary Results and Discussions
Table 1. The pretreatment conditions .
The proportion of pseudo-lignin in the pretreated solids increases as the pretreatment
severity increases, while the proportion of carbohydrates retained in the solids shows the
inverse trend.
Pseudo-lignin can be produced from both acid pretreated cellulose and hemicellulose.
The methoxy group of pseudo-lignin may come from 4-O-methyl-D-glucuronic acid in the
hemicellulose of poplar.
Hydrolysis of polysaccharides to the corresponding monosaccharides, and the
subsequent dehydration of sugars, leading to the formation of furfural and 5-
hydroxymethylfurfural (HMF), took place during pretreatment.
Further rearrangement of furfural and/or HMF to yield other aromatic compounds (such as
1,2,4-benzenetriol and 3,8-dihydroxy-2-methylchromone) occurred during pretreatment.
Polycondensation and/or polymerization reactions led to the formation of pseudo-lignin
during pretreatment.
Table 2. Molecular weight analysis.
Fig. 1. FT-IR spectra of pseudo-lignin and starting
materials.
Sample 1st step condition
(same for all
samples)
2nd step condition
Holocellulose A Soaking (5% solids)
while stirring in 0.1 M
H2SO4 at room
temperature for 4 h
180 °C, 0.1 M H2SO4, 40
min
Holocellulose B 180 °C, 0.2 M H2SO4, 60
min
α-Cellulose A 170 °C, 0.1 M H2SO4, 20
min
α-Cellulose B 180 °C, 0.1 M H2SO4, 40
min
Pseudo-lignin
isolated from
Mn (g/mol)
Mw (g/mol)
PDI
α-Cellulose B
1.08 x 103
3.44 x 103
3.17
Holocellulose A 1.24 x 103
5.08 x 103
4.09
Holocellulose B
1.19 x 103
5.97 x 103
5.00
Fig. 2. 13C NMR spectra of pseudo-lignin.
Fig. 3. Proposed reaction pathways.
Fig. 4. SEM images of holocellulose (top) and
pseudo-lignin on holocellulose (bottom).
Fig. 5. Time course of glucose yield of various
samples after 48 h of enzymatic hydrolysis.
Acknowledgement This work was supported by the DOE office of Biological and
Environmental Research through the BioEnergy Science
Center (BESC).