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).