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Sasikumar Elumalai,1 John Grabber,2 Dino Ress3, Yuki Tobimatsu3, Yimin Zhu3, Christy L. Davidson3, John Ralph,1,3 and Xuejun Pan.1,†

1Department of Biological Systems Engineering, 460 Henry Mall; 2U.S Dairy Forage Research Centre, USDA - ARS, 1925 Linden Drive, Madison, WI 53706; 3Department of Biochemistry, 433 Babcock Drive, University of Wisconsin-Madison, Madison, WI 53706

†E-mail: xpan@wisc.edu

Lignin, one of the major three components of lignocellulosic biomass, is considered to be a major source of recalcitrance to enzymatic release of the fermentable sugars from the biomass. One of the greatest challenge in producing fuels, chemicals, and fibers from biomass is the efficient removal of lignin. Lignin malleability provides opportunities for engineering its chemical structure and linkages. It has been demonstrated that, perturbing single genes in the monolignol pathway can lead to dramatic shifts in the proportions of monolignols polymerized into lignin, and that other (novel monolignols) monomers can incorporate with lignins. Incorporation of ester conjugates or other related diphenolics would improve lignin extraction during chemical pretreatments via cleavage of interunit ester linkages to depolymerize lignin. We believe that polyphenolic epicatechins, epicatechin gallate and quercetin derivatives, and ferulates might be attractive targets for lignin bioengineering because their copolymerization in lignin could form weak linkages (easily cleavable with alkali/acid), reduce lignin hydrophobicity and cross-linking to polysaccharides, or facilitating delignification by chemical pretreatments.

The following are the objectives of this research : • Development of protocol for the microscale analysis of chemical constituents of the lignified cell walls. • Evaluating the response of lignified cell walls to chemical pretreatments (with acid and base), and

subsequent enzymatic hydrolysis.

Mansfield, S.D. et al. Substrate and enzyme characteristics that limit cellulose hydrolysis. Biotechnol Prog, 1999,15, 804-816. Ralph, J. et al. Lignins: natural polymers from oxidative coupling of 4-hydroxyphenylpropanoids. Phytochem Rev, 2004, 3, 29-60. Boerjan, W. et al. Lignin Biosynthesis. Annu Rev Plant Biol, 2003, 54, 519-549. Grabber, J.H. et al. Coniferyl ferulate incorporation into lignin enhances the alkaline delignification and enzymatic degradation of cell walls. Biomacromol,

2008, 9, 2510-2516. Sasikumar E, et al. Evaluating the response of artificially lignified cell wall to pretreatments and enzymatic hydrolysis. Proceedings of the 16th ISWFPC,

China. 2011, 6, 963-967.

Fig. 2 Chemical composition of nonlignified cell wall before pretreatment

Experimental

Isolated maize cell walls were artificially lignified with normal monolignols (coniferyl alcohol and sinapyl alcohol at 1.0-1.3 mM level) and novel lignin precursors including epicatechin gallate/quercetin derivatives, ferulates, and guaiacyl butenol (at 0.60 -0.75 mM level) (Fig. 1).

Dilute sulfuric acid (DA) and sodium hydroxide (NaOH) pretreatments were conducted at microscale (250 mg cell wall) and at mild conditions (10% chemical loading on cell wall, w/w; 100°C for 1 h).

Cell wall constituents such as, carbohydrates, uronic acid and lignin were determined at a microscale level (10 mg size). Subsequent enzymatic hydrolysis was performed according to standard procedure (NREL LAP 009) using cellulase

(Novozymes, NC) supplemented with enzyme complex (containing hemicellulases and xylanase activities) at 15 FPU/g glucan and 15 PGU/ g cell wall.

Carbohydrate contents were measured by High Performance Ion Chromatography (Dionex DX-3000, CA). Total uronic acids were colorimetrically determined using m-hydroxydiphenyl reagent by UV/Vis-Spectrophotometery (Cary 50, PA).

Lignin content was determined as acetyl bromide soluble lignin (ABSL), and/or as acid-insoluble lignin by the Klason lignin method.

Fig. 1 Chemical structure of lignin monomer replacements for improving biomass processing. Red bonds can be readily cleaved during chemical pretreatment of biomass.

Lignin precursors

Does novel precursors influence delignification? Some monolignols like HP and OGH enhanced the delignification with NaOH under the investigated conditions, while

the others did not. Are the pretreatment conditions employed strong enough to cleave the ester bonds in lignin? Yes, epicatechin gallate/quercetin derivatives showed considerable delignification with NaOH, but not to others. Does incorporated novel precursors enhance substrate conversion? Yes, considerable improvement was observed with epicatechin gallates/quercetins, but further investigation is

needed under varied conditions. Research on the way: Would further investigation under severe conditions cleave the ester & amide bonds, and enable to

distinguish the cell walls? How did incorporated novel precursors influence bioconversion?

Fig. 3 Acetyl bromide soluble lignin (ABSL) content of cell walls before pretreatment. Results are presented as means (n=4) with LSD (P=0.05).

Acknowledgements The authors would like to thank the Global Climate and Energy Project (GCEP) for funding this research.

Experimental Rationale and Objectives

SUMMARY

Acknowledgements

References

Results

Compositional analysis of lignified cell walls

Epicatechin gallate / quercetin derivatives

Ferulates

NL-nonlignifed ; LLC-low lignin control; HLC-high lignin control; EGCG-epigallocatechin gallate; HP-hyperoside; OGH-2”-O-galloylhyperin; ETG-ethyl gallate; COG-corilagin; PGG-pentagalloylglucose; FER-ferulate; GUB-guaiacyl butenol; METF-methoxytyramine ferulate.

Lignification with novel precursors did not show significant impact on cell wall components like cellulose and hemicelluloses (Fig.2), except lignin content. The amount of lignin formed in cell walls was varied (16-26% of dry weight) depending on the type of monolignol substitute added (Fig. 3). It was observed that lignin content determined by acetyl bromide method was 10-30% higher than that by Klason method (acid insoluble lignin).

In general, NaOH pretreatment showed higher substrate (>75%) and carbohydrate (>80%) recovery than DA pretreatment (~10-15 points high). About 8-55% delignification was achieved with alkaline pretreatment, and was observed to be varied depending on the type of monolignol substitutes added (Fig. 4).

Fig. 4 Delignification result of cell walls with NaOH and DA pretreatments. Results are presented as means (n=4) with LSD (P=0.05).

Incorporation of novel monolignols significantly improved the enzymatic digestibility of cell walls (~10-15 points higher than the controls). Almost equal rate of sugar production was observed with alkali and acid pretreated cell walls (±1point), though the acid pretreated ones showed high substrate conversion efficiency (~3-7 points higher than

NaOH). However, enzymatic digestibility of cell walls were depending on the residual lignin content (Fig. 5). Cell walls ETG and COG showed highest substrate conversion among the lignified cell walls (>90% glucose conversion yield and >60% TSY with DA). A two pool exponential model well represented the enzymatic hydrolysis kinetics.

Response of lignified cell walls to chemical pretreatments

Response of lignified cell walls to enzymatic hydrolysis

Fig. 5 Comparative chart of total saccharification (polysaccharides-to-saccharides) yield (TSY) of pretreated cell walls after 48 h hydrolysis. (Inset) Enzymatic hydrolysis profile of ETG cell wall with and without pretreatment. Results are presented as means (n=4) with LSD (P=0.05).

Results

Arabinose 19%

Galactose 9%

Glucose 32%

Xylose 14%

Mannose 1%

Uronic acid 9% Lignin

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𝑌 = 𝐴 × 1 − 𝑒 𝑘1𝑧1 + 𝐵 × 1 − 𝑒 𝑘2𝑧2

Lignin content