STRUCTURAL CHARACTERIZATION OF ALKALINE HYDROGEN PEROXIDE (AHP)

PRETREATED BIOMASS

By

Muyang Li

A THESIS

Submitted to Michigan State University

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Chemical Engineering

2011

ABSTRACT

STRUCTURAL CHARACTERIZATION OF ALKALINE HYDROGEN PEROXIDE (AHP)

PRETEATED BIOMASS

By

Muyang Li

Alkaline hydrogen peroxide (AHP) pretreatment is exceptionally well-suited

to grasses, yielding high digestibilities at low enzyme loadings, while generating

relatively few fermentation inhibitors. For AHP pretreatment, the question of how

structural and chemical compositional changes within the plant cell wall correlate to

pretreatment effectiveness has not been effectively resolved, while knowledge of how

selective modification of lignin during pretreatment may improve digestibility may

yield insights into both improving pretreatments and tailoring plant cell walls for

deconstruction. This study presents a comprehensive chemical and structural

characterization of the changes in plant cell walls associated with switchgrass

(Panicum virgatum cv. Cave-In-Rock), corn stovers (a commercial hybrid and inbred

brown midrib lines bm1 and bm3) and sugar maple (Acer saccharum) and that were

AHP pretreated at varied severities. Both the remaining solids and solubilized

biomass hydrolysates were subjected to a number of characterizations including total

polysaccharide composition, lignin content as both Klason and acetyl bromide lignin,

and the ratio of H/G/S monolignols as determined by thioacidolysis

GC/MS. Solid-state 13

C CP/MAS NMR and HSQC 2D NMR were applied to

determine changes associated with intra-lignin, carbohydrate-carbohydrate,

carbohydrate-lignin linkages and functional groups. Apparent molecular weight

distributions of soluble polymeric lignins and hemicellulose aggregates were

determined by HP-SEC. Pyrolysis-GC/MS was utilized to characterize changes in the

volatilized compounds before and after pretreatment implying pretreatment-induced

alterations in pyrolysis-labile monolignol linkages.

iv

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................ vi LIST OF FIGURES .......................................................................................... vi ABBREVIATIONS ....................................................................................... viii 1. INTRODUCTION .................................................................................... 1

1.1 LIGNOCELLULOSIC BIOMASS ...................................................... 1

1.2 BIOMASS CONVERSION PROCESSES .......................................... 3 1.3 ALKALINE HYDROGEN PEROXIDE (AHP) PRETREATMENT . 4

1.4 OBJECTIVES ...................................................................................... 6 2. STRUCTRAL CHANGES OF RESIDUE CELL WALL ........................ 9

2.1 INTRODUCTION ............................................................................... 9

2.2 MATERIALS AND METHODS ....................................................... 11

2.2.1 Sampling ................................................................................. 11

2.2.2 AHP Pretreatment ................................................................... 12

2.2.3 Milling..................................................................................... 12

2.2.3 NREL Compositional Analysis............................................... 13

2.2.5 ABSL Analysis ....................................................................... 13

2.2.6 Enzymatic Hydrolysis ............................................................. 13

2.2.7 Bacterial Cellulose Solid-state NMR Study ........................... 14 2.2.8 Residual Cell Wall Solid-state NMR Study............................ 14 2.2.9 Residue Cell Wall 2D HSQC NMR Profiling ........................ 15

2.3 RESULTS AND DISCUSSIONS ...................................................... 15

2.3.1 NREL Compositional Analysis and Digestibility Evaluation 15 2.3.2 ABSL Analysis ....................................................................... 18

2.3.3 Correlation between Lignin Content and Glucan Digestibility20

2.3.4 Bacterial Cellulose Peak Assignment ..................................... 21

2.3.5 Residual Cell Wall Solid-state NMR Study............................ 22 2.3.6 Residue Cell Wall 2D HSQC NMR Profiling ........................ 25

3. SOLUBILIZED COMPOUNDS IN HYDROLYSATE ......................... 27

3.1 INTRODUCTION ............................................................................. 27

3.2 MATERIAL AND METHODS ......................................................... 28

3.2.1 Sample Concentration ............................................................. 28

3.2.2 Compositional Analysis of Hydrolysate ................................. 28

v

3.2.3 Hydrolysate NMR Profiling .................................................... 29

3.2.4 SEC Study on Hydrolysate ..................................................... 29

3.3 RESULTS AND DISCUSSION ........................................................ 30

3.3.1 Compositional Analysis of Hydrolysate ................................. 30 3.3.2 Hydrolysate NMR Profiling .................................................... 32

3.2.3 1H NMR characterization of Hydrolysate ............................... 33 3.3.4 SEC Study on Hydrolysate ..................................................... 36

4. PHYSIOCHEMICAL CHARACTERIZATION OF LIGNIN ............... 39

4.1 INTRODUCTION ............................................................................. 39

4.2 MATERIALS AND METHODS ....................................................... 41

4.2.1 Thioacidolysis ......................................................................... 41

4.2.2 Analytical Pyrolysis ................................................................ 42

4.3 RESULTS AND DISCUSSIONS ...................................................... 43

4.3.1 Pretreatment, Lignin Composition and Digestibility .............. 43 4.3.2 Pyrogram Peak Assignment .................................................... 44

4.3.3 Pyrolyzable Compound Comparison ...................................... 48 4.3.4 Lignin Composition Based on Abundant Pyrolyzable Compounds

...................................................................................................................... 50 4.3.5 Lignin Compositional Changes by Pretreatment .................... 51 4.3.6 S/G Variation .......................................................................... 53

5. CONCLUSIONS..................................................................................... 54 APPENDIX ...................................................................................................... 57 BIBLIOGRAPHY ............................................................................................ 61

vi

LIST OF TABLES

Table 2.1: Definitions and conditions of AHP pretreatment ........................................... 12

Table 2.2 Weight remaining of four grasses after AHP pretreatments ............................ 15

Table 3.1 The ratio of H located in aromatic ring and carbon backbone based on the composition analysis results. ................................................................................... 35

Table 3.2 Proposed biopolymers molecular weight distribution in low severity AHP

pretreatment hydrolysate (Switchgrass) ................................................................... 37

Table 4.1 Results of ABSL, Thioacidolysis and glucan digestibility .............................. 43

Table 4.2 Py-GC/MS compound library of hybrid corn stover under AHP pretreatments.................................................................................................................................. 44

Table 4.3 Py-GC/MS compound library of inbred bm1 stover under AHP pretreatments

.................................................................................................................................. 45

Table 4.4 Py-GC/MS compound library of inbred bm3 stover under AHP pretreatments.................................................................................................................................. 46

Table A.1 Peak assignments and results from the spectral fitting of cellulose 13C NMR

spectra for bacterial cellulose ................................................................................... 60

vii

LIST OF FIGURES

Figure 2.1 Compositional analysis of four grasses under different AHP pretreatments . 16

Figure 2.2 Glucan digestibilities of four grasses under different AHP pretreatments ..... 16

Figure 2.3 Correlation between ABSL and Klason lignin ............................................... 19

Figure 2.4 Correlation between Klason lignin and glucan digestibility by species ......... 20

Figure 2.5 Correlation between ABSL and glucan digestibility by treatments ............... 20

Figure 2.6 900 MHz Solid-state NMR spectra for bacterial cellulose (Numbers on the peaks refer to 6 carbon atoms on the hexose ring) ................................................... 21

Figure 2.7 Solid-state CP/MAS 13C NMR spectra of switchgrass under AHP

pretreatments with different NaOH concentration ................................................... 22

Figure 2.8 Solid-state CP/MAS 13C NMR spectra of switchgrass under AHP pretreatments with different NaOH concentration (15-40 ppm) .............................. 23

Figure 2.9 Solid-state CP/MAS 13C NMR spectra of switchgrass under AHP

pretreatments with different NaOH concentration (110-150 ppm) .......................... 23

Figure 2.10 Gel-state HSQC 2D NMR spectrum of ball-milled low severity AHP pretreated switchgrass with a mixture solvent DMSO-d6 and Pyridine-d5 (4:1 v/v).................................................................................................................................. 25

Figure 3.1 Flowchart of Low NaOH AHP pretreatment (Switchgrass)........................... 30

Figure 3.2 Mass balance of solid (s) and liquid (l) phase before (1) and after (2) low NaOH

AHP pretreatment (Switchgrass) ............................................................................. 30

Figure 3.3 HSQC 2D NMR spectrum of AHP Pretreatment liquor with solvent DMSO-d6 and a series of depressed water peak in 1H at 3.5 ppm ........................................... 32

Figure 3.4 Labeled 1H NMR spectrum of AHP pretreatment liquor with solvent

DMSO-d6 ................................................................................................................. 33

Figure 3.5 Labeled 1H NMR spectrum of AHP pretreatment liquor with solvent D2O after 0.22 µm pore size ultra-filtering. ............................................................................. 34

viii

Figure 3.6 SEC result of low severity AHP pretreatment liquor (Switchgrass) .............. 36

Figure A.1 C4 region peak deconvolution of solid-state 13C NMR spectra for bacterial cellulose ................................................................................................................... 59

ix

ABBREVIATIONS

2D two-dimensional ABSL acetyl bromide soluble lignin AFEX ammonia fiber expansion AHP alkaline hydrogen peroxide CAD cinnamyl alcohol dehydrogenase COMT caffeic acid O-methyl transferase CS corn stover DMSO dimethylsulfoxide DP degree of polymerization FA ferulic acid G guaiacyl GC gas chromatography H p-hydroxyphenyl HMQC heteronuclear multiple quantum coherence HPLC high-performance liquid chromatography HSQC heteronuclear single quantum coherence LCC lignin-carbohydrate complex MS mass spectroscopy MW molecular weight NMR nuclear magnetic resonance NREL national renewable energy laboratory pCA para-coumaric acid PCW plant cell wall

S syringyl SEC size exclusion chromatography SEM scanning electron microscope SG switchgrass SM sugar maple

1

1. INTRODUCTION

1.1 LIGNOCELLULOSIC BIOMASS

The worldwide emerging demand of fossil fuel replacements leads to the development

of alternative renewable energy resource such as bioenergy, solar and wind energy. Among

them, bioenergy based on conversion of the natural biological materials plays an important role

in clean liquid transportation fuel production. Bioenergy can be produced by either

biochemical methods including hydrolysis and fermentation, or thermochemical processes

including gasfication, pyrolysis and depolymerization. Since ethanol is a neat transportation

fuel can be blend with gasoline to reduce emissions and increase octane [1], production of

ethanol from renewable sources of lignocellulosic biomass is a potential energy supply to meet

the current U.S. Renewable Fuels Standard (RFS) which is 36 billion gallons of biofuel and

biodiesel production per year by 2022 [2].

First generation biofuels are generally made by agricultural products including

biodiesel produced from soybean, palm oil or canola oil, and bioethanol produced from corn

starch and sugarcane. Second generation biofuels are based on lignocellulosic biomass

including energy crops, forestry and agricultural waste, which is more abundant and not in

competition with human food. Compared to the first generation bioethanol production, which

is mainly enzymatic hydrolysis followed by fermentation of cornstarch, or fermentation of

sucrose from sugarcane, the heterogeneous structure of lignocellulosic biomass needs an acid,

2

thermal or alkaline pretreatment process before the more complex enzymatic hydrolysis, in

order to break down the ultrastructure of biomass. Lignocellulosic biomass represents one of

the highest potential replacements of fossil fuel because of its high sugar content and high yield

of liquid clean fuel conversion.

Plant cell walls (PCWs), which mainly present in plants transport tissues, are the

majority of lignocellulosic biomass. The low bulk density, high moisture content and

heterogeneous structure of PCWs result in limitations including relative low productivity,

collection, transportation, and fractionation of the agriculture-based bioenergy. The three

major components of PCWs are cellulose, hemicellulose and lignin [3]. The organization of

typical PCW is primary cell wall around a lumen in the center, and then the adjacent secondary

cell wall. In the primary cell wall, cellulose exists as the crystalline structure form, and

hemicelluloses coalesce with the surface of cellulose microfibrils [4]. In the secondary cell

wall, lignin grows surrounding the primary cell wall forming a middle lamella layer to resist

water and enhance rigidity [5, 6]. Lignification is regarded as the main barrier to biomass

conversion.

The hydrogen bonded matrix structure consists of cellulose microfibrils and

hemicelluloses provides the major strength of PCWs [7]. Native cellulose has crystalline and

amorphous structures, which are both polysaccharide chains composed of β-1-4 linked

D-glucosyl units. In nature, the crystalline structure has distinct but coexist crystallite forms, Iα

and Iβ [8]. Hemicelluloses are branched polysaccharides which consist of different unmodified

sugars or modified sugars including glucose, xylose, arabinose, mannose and galactose, with

random and amorphous structures. Lignins are complex polymers composed of 3 monolignols

3

(coniferyl, sinapyl, and p-coumaryl alcohols) and up to 11 types of linkages [9]. The

composition properties of both hemicelluloses and lignin vary by species.

1.2 BIOMASS CONVERSION PROCESSES

The conversion of biomass is mainly about breaking down the ultrastructure of biomass,

overcoming the lignin preventing access to polysaccharides for enzymes, and depolymerizing

crystalline cellulose to fermentable monosugars. In order to be effective, biomass conversion

processes requires both physical effects and chemical effects. At a molecular level, the physical

effect is disrupting the higher order structure such as increasing the surface area to let chemical

or enzyme better penetrate into plant cell walls, as well as the chemical effect of changing the

solubility of macromolecules, or depolymerizing and breaking the crosslinking between the

macromolecules.

Targeting at the crystalline structure of cellulose, the enzymatic hydrolysis primarily

needs the synergy of three types of enzymes [10]. Endoglucanases first split the crystalline

structure of cellulose and increase the porosity and surface area of the substrate.

Cellobiohydrolases next cut the end of cellulose chain to produce cellobiose. This is followed

by β-glucosidase conversion of cellobiose to two glucose units. The physical properties of

biomass including porosity and accessible surface area have impact on enzyme access to

cellulose microfibrils [11]. Quantitative models were set up describing enzyme absorbance and

inhibition by substrates features. Studies showed increasing degree of polymerization

decreases cellulose solubility and the availability of chain ends, and the crystallinity index and

enzymatic hydrolysis have negative correlations with cellulase accessibility [12].

4

Major factors influencing the digestibility of lignocellulosic biomass include porosity,

cellulose crystallinity, lignin content and hemicellulose content [13]. Various pretreatments

have different effects on these factors such as changing the lignin structure, partially removing

hemicelluloses and interrupting cellulose crystallinity, which increase the accessible area of

cellulose microfibrils. One of the best studied pretreatments is dilute acid pretreatment

performed between 160°C and 220°C [14]. Since the hydrothermal pretreatment requires high

temperature and has drawbacks in lignin globule impediment and fermentation inhibitor

formation, a series of alternative pretreatments are being studied and developed, such as

organic solvent [15], ionic liquid [16], oxidation and alkaline pretreatments [17, 18].

1.3 ALKALINE HYDROGEN PEROXIDE (AHP) PRETREATMENT

An effective pretreatments applied in biomass conversion are through chemical

modification and physical redistribution of the supramolecular structure of the raw material in

order to achieve higher digestibility. One class of pretreatments including acidic hydrothermal

pretreatment utilizing the acid to hydrolyze and solubilize xylan, melt and redistribute lignin,

increases the enzymatic accessible surface area of the raw material to improve digestibility,

while another class of pretreatment approaches such as kraft pulping and bleaching is based on

changing lignin solubility besides the solubilization of xylan as well.

Alkaline hydrogen peroxide (AHP) pretreatment is originally a bleaching process

widely applied in the paper industry for pulping. Kraft pulping is cooking wood chips at high

temperature (170°C) in sodium hydroxide and sodium sulfide solution to remove the majority

of the lignin [19], the beaching step is to utilize hydrogen peroxide to decolor the remaining

5

kraft lignin which is structurally distinct from native lignin. AHP pretreatment is based on

hydrogen peroxide oxidation of lignin under alkaline condition in room temperature, while

alkali saponification reactions with hemicellulose esters and lipids. The solubilization and

cleavage of lignin and hemicelluloses from cellulose can effectively increase the hydrolysis

conversion of residual PCWs.

During AHP pretreatment, hydrogen peroxide is decomposed to several important ions

acting with lignin, including hydrogen peroxide anion (HOO-), hydroxyl (•OH) and superoxide

anion (•O2-) radicals. Reactions are as follows:

And the sum reaction is:

In the alkaline environment, the hydrogen peroxide decomposition rate can reach the

maximum at pH=11.5 at 25°C. HOO- as a strong nucleophile species only attacks lignin end

groups either free phenolic or terminal aliphatic regions of lignin without depolymerizing,

forming an active lignin anion, which may further react with superoxide anion radical from

decomposed hydrogen peroxide to initiate the cleavage of lignin ring structure,

lignin/carbohydrate linkages and some methoxyl groups, resulting in depolymerization and

nucleophilic substitution [20]. The reaction was studied as a first order reaction and the rate

constant corresponds with the concentration of hydroxyl ion [21].

2 2

2 2 2 2

H O HOO H

H O HOO OH O H O

− +

− −

⇔ +

+ → • + • +

2 2 2 22H O OH O H O H− +→ • + • + +

6

1.4 OBJECTIVES

The heterogeneous macromolecular structure of plant cell wall limits the enzymatic

hydrolysis of biomass. To better understand the interaction between pretreatment and

enzymatic hydrolysis, many studies based on characterizing the impact of various structural

features of biomass on cellulose enzymatic hydrolysis have been carried out in recent years. A

study using confocal microscopy showed xylan content decreasing in center of cell wall while

remaining in the lumen and middle lamella layer during dilute acid pretreatment, which

indicates the hydrophobic nature of lignin retards the solubilization of xylan associated to

lignin [22]. A study using a fluorescence-labeled cellulase to probe and examine the enzyme

binding with the substrates showed that the amorphous forms of the celluloses are more

digestible, which support the idea that the reaction extent of hydrolysis depends on the higher

order structure of the substrate [23]. Also, attempts have been made to link delignification with

cellulose enzymatic digestibility, and it has been found that the delignification is not

necessarily linear with the digestibility, since lignin location is more important than PCW’s

bulk composition [14]. Those three studies above all support the idea that digestibility is set by

the higher order structures of PCW. A model has been developed to predict enzymatic

hydrolysis of biomass based on structural features including crystallinity index, acetyl content

and lignin content [24]. Experimental data showed the rate of hydrolysis depends on

parameters related to biomass structural features independent from the cellulose and enzyme

concentrations and accessible surface fraction.

Current leading pretreatment technologies including dilute acid and steam explosion

7

have the limitation of enzymatic accessibility due to lignin relocalization and globule

formation, while alkaline pretreatments including lime and aqueous ammonia show better

lignin removal and some cell wall swelling [25]. Alkaline hydrogen peroxide pretreatment is a

pretreatment method well-suited to grasses [26] which yields relatively high digestibility and

generates low fermentation inhibitors [27]. One advantage of AHP pretreatment is the

capability to be performed under room temperature and pressure [28, 29], which could

significantly reduce the capital costs of a biorefinery for ethanol production. AHP as an

oxidative pretreatment has the disadvantage of the high cost of commercial hydrogen peroxide

since studies have shown the ratio of hydrogen peroxide to substrate should be at least 0.25 to

get good delignification and digestibility [28]. Also, since the density of lignocellulosic

feedstock is relatively low, the limited solid loading is another concern of pretreatment and

enzymatic hydrolysis [30] leading to significant effects on processing costs [31, 32]. Those

problems can be solved in the future by optimizing reaction procedures to reduce reagents

consumption or applying catalysts to speed up lignin oxidation [19].

The complexity of the biological macromolecules results in difficulties in biomass

assessment using traditional analytical methods. In order to further understand the mechanism

of the pretreatment impact on digestibility, this study is designed to comprehensively

understand the structural change during AHP pretreatment by physiochemical characterization

using a number of analytical methods and tools. The length scale of physical structure of

cellulose microfibrils and hemicellulose matrix is of the 10-4

to 10-6 meter, and the chemical

structure of the polysaccharides is in the 10-7

m to 10-9 m order of magnitude. Accurate

modeling and direct assessment of changes in PCW structure is the primary challenge due to

8

the complexity of the biomass and the complicated reaction mechanism of the cellulase system.

The monomer composition of biomass can be determined by NREL two-stage sulfuric acid

hydrolysis [33], trifluoroacetic acid (TFA) hydrolysis [34]. Detergent fiber analysis is a

method to determine the composition of three main components [35]. However, due to the

complexity of PCWs and the experimental errors, the uncertainty of compositional analysis is

significant. For instance, NREL method has 4% to 10% total relative standard deviation [36].

Many modern analytical instruments have been applied to characterize the chemical

composition including NMR [37, 38], NIR/PLS (near infrared reflectance spectroscopy) [39],

TGA (thermogravimetric analysis) [40] and pyrolysis GC/MS [41]. Also, the physical

structural characteristics of biomass such as the porosity and crystallinity have been profiled

respectively by SEM [42] and solid-state NMR. The lignin content, composition and functional

group distribution have been studied by 13

C, HSQC, and 31

P NMR spectroscopy [43].

This study is focused on the pretreatment effects on structural changes of grasses, and

how the structural changes impact glucan digestibility in the subsequent enzymatic hydrolysis.

This study uses ball-milled switchgrass (Panicum virgatum cv. Cave-In-Rock), corn stovers (a

commercial hybrid and inbred brown midrib lines bm1 and bm3), sugar maple (Acer

saccharum) that were AHP pretreated at varied conditions as research subjects. The remaining

solids and solubilized biomass pretreatment liquor were subjected to total polysaccharide

compositional analysis and solid-state 13

C CP/MAS NMR and HSQC 2D NMR profiling,

which have the potential to determine changes associated with carbohydrate-carbohydrate,

carbohydrate-lignin linkages and functional groups. Apparent molecular weight distributions

of soluble polymeric lignins and proposed hemicellulose aggregates were determined by

9

HP-SEC. Lignin content was quantified by both Klason and acetyl bromide lignin methods,

and the ratio of H/G/S monolignols has determined by thioacidolysis GC/MS and Pyrolysis

GC/MC. Pyrolysis GC/MS was utilized to characterize changes in the volatilized compounds

before and after pretreatment implying alterations in pyrolysis-labile monolignol linkages

which are induced by pretreatment. The relationship between digestibility and compositional

features of biomass were discussed.

2. STRUCTRAL CHANGES OF RESIDUE CELL WALL

2.1 INTRODUCTION

Alkaline pretreatment disrupts cell walls by somewhat swelling cellulose microfibrils

[44], dissolving hemicellulose and cleaving ester bonds between hemicellulose and lignin [45].

Hemicelluloses contain many esters bonded substitution groups including acetate and ferulic

acid which are alkali-labile and thus increase the alkali solubility of hemicelluloses after their

cleavage by alkali. The alkali vulnerable ferulate ester crosslinks between xylan-xylan,

xylan-lignin and lignin-lignin has been found unique in grasses [46]. Those features enable

AHP to be either an effective pretreatment [27] or a pre-treatment delignification step [45] by

playing a role in overcoming the recalcitrance of biomass especially grasses.

Composition analysis is usually required before enzymatic hydrolysis since the amount

of enzymes is determined by the sugar content. One type of composition analysis is to convert

the cellulose and hemicelluloses to monosugars under high severity conditions such as

two-stage acid hydrolysis, and then analyze the sugar concentrations in solution via

10

chromatography [33]. The acid insoluble portion is regarded as Klason lignin. Acetyl Bromide

Soluble Lignin (ABSL) provides quantitative information of total lignin content by UV

absorbance of aromatic rings in lignin [47].

In order to understand important features of lignocellulosic biomass other than sugar

composition, such as crystallinity index, degree of polymerization and lignin carbohydrate

linkages, previous research has applied many instruments and analytical methods including

SEM, XRD and so on. Among them, Nuclear Magnetic Resonance (NMR) is a method

commonly used in recent years for studying the structural characteristics of lignocellulosic

biomass. It is based on the principle that certain types of magnetic nuclei with spin properties in

an applied magnetic field absorb electromagnetic radiation at a frequency depending on the

local chemical environment, which makes it possible to provide information of the molecules

in which they are contained through exploiting the magnetic properties of the certain nuclei.

The properties of 13

C and 1H can provide information of the macromolecules in which they are

contained.

Native cellulose exists as cellulose microfibrils each contains 36 cellulose chains, and

coalesce as two distinct crystalline forms Iα and Iβ [8]. The ratio of crystalline and amorphous

cellulose varies by species. In order to further investigate the chemical changes during biomass

conversion, the morphology of cellulose including crystallinity is one of the target properties of

structural characterization. Solid-state NMR method is a way to analyze chemical structure in a

native state, which is effective for biobased macromolecular samples which usually have

limited solubility. 13

C-cross-polarisation magic angle spinning (CP/MAS) NMR spectrum

contains information of amount and structures of different cellulose, which make it possible to

11

study the cellulose morphology including the degree of crystallinity and accessible surface area

through spectrum fitting[48-51].

Solid-state 13

C CP/MAS NMR can provide information of polysaccharides through

characterization of chemical environment of carbon nuclei. 2D NMR methods including

HSQC (Heteronuclear Single Quantum Correlation) and HMQC (heteronuclear multiple

quantum coherence) through correlating C and H nuclei on the complex biopolymers of PCWs

provide structural information including intra-lignin, carbohydrate-carbohydrate,

carbohydrate-lignin linkages and functional groups [52, 53], which are also important

properties associated with pretreatment effectiveness. For investigation on structural change

associated with pretreatment, previous studies have showed no distinction between intact and

degraded cellulose microfibrils [54], the different pulping conditions influence the crystallinity

of cellulose differently [51], and hydrolysis does not alter apparent crystallinity [42]. This

chapter is focusing on structural information provided by NMR indicating pretreatment

effectiveness. The cellulose crystallinity and functional groups was studied by 13

C CP/MAS

NMR. The carbohydrate-carbohydrate and carbohydrate-lignin linkages were determined by

2D HSQC NMR.

2.2 MATERIALS AND METHODS

2.2.1 Sampling

In this study, several types of biomass have been used including switchgrass (Panicum

virgatum cv. Cave-In-Rock), corn stovers (a commercial hybrid and inbred brown midrib lines

bm1 and bm3) and sugar maple (Acer saccharum). Corn stover and switchgrass are common

12

bioenergy feedstock and sugar maple is a common hardwood in the state of Michigan. The

brown midrib (bmr) mutants are well known as having lower lignin content and higher

digestibility than normal phenotypes in corns and have been studied for more than 50 years[55].

Specific mutants with potential in structural research on lignin include bm1 line with lower

level of cinnamyl alcohol dehydrogenase (CAD) resulting in reduced lignin, ferulic acid (FA)

and para-coumaric acid (pCA) esters as well as enriched C-C monolignol linkages, and bm3

line deficient in caffeic acid O-methyl transferase (COMT) resulting in decreased total lignin

and syringyl monolignol incorporation [56, 57].

2.2.2 AHP Pretreatment

The 2 mm screen biomass samples were treated by a series of aqueous solutions with

distinct sodium hydroxide concentrations and fixed hydrogen peroxide concentration as 2%

solid loading in 100 mL total volume for 24 hours at room temperature. As shown in Table 2.1,

the samples are categorized by high NaOH, low NaOH and pH=11.5 adjusted pretreated

according to different sodium hydroxide adding amounts. The untreated biomass is regarded as

control. Pretreatments were performed in duplicate.

Solid loading (g biomass/g total)

NaOH (g/g biomass)

H2O2

(g/g biomass) H2O (mL)

Low NaOH 2% 0.1 0.125 100 High NaOH 2% 1.1 0.125 100 pH=11.5 2% pH=11.5 0.125 100

Table 2.1: Definitions and conditions of AHP pretreatment

2.2.3 Milling

The pretreated biomass was grinded by QIAGEN TissueLyser II equipped with 25 mL

stainless steel jars and 20 mm Ø balls in 25 Hz for 2 minutes with liquid nitrogen cooling

13

interval.

2.2.3 NREL Compositional Analysis

NREL compositional analysis method is a two-stage acid hydrolysis followed by

HPLC (Aminex HPX-87H column and Refractive Index detector) to get the monosaccharide

concentration. 0.1 g biomass sample was measured and put into pressure tube equipped with a

glass rob, 1 mL 72% H2SO4 was added and the pressure tube was immersed into water bath set

at 30°C for 1 hour with stirring every five minutes. 28 mL deionized water was added to adjust

the concentration of H2SO4 to 4% which was then autoclaved at 121°C for 1 hour. 1mL of the

solution was neutralized by CaCO3, filtered by 0.22 mm syringe filter and transferred to HPLC

vial. Five standard solutions of D(+)glucose, D(+)xylose, L(+) arabinose with concentration

gradient were prepared. Standards were injected with samples at the same time and the sugar

concentrations were calculated by peak integration of demand ingredients. The remaining

liquid was filtered to get acid insoluble lignin (AIL) content as Klason lignin [58].

2.2.5 ABSL Analysis

2mg ball-milled biomass sample was reacted with 250 µL fresh 25% acetyl bromide in

acetic acid solution at 50°C for 3 hours [59]. After centrifugation, 100 µL of the reaction

solution was transferred into an empty 2 ml volumetric flask, then 400 µL 2 M NaOH and 75

µL fresh 0.5 M hydroxylamine hydrochloride solutions were added, and the volume was added

up to 2 mL with acetic acid. Triplicate samples were tested with 96-well UV spectrophotometer

at 280 nm absorbance.

2.2.6 Enzymatic Hydrolysis

Pretreated samples were incubated at 50°C for 24 hours with Accellerase 1500 enzyme

14

provided by Genencor at a loading of 50 mg protein/g glucan, which is higher than the

theoretical enzyme requirement in order to achieve highest possible conversion and evaluate

only substrate effects. The conditions were 10% solid loading at 5 mL total volume and with

0.05 M Na-citrate buffer pH 5. The digestibility was determined by the HPLC analyzable

glucose concentration after incubation divided by the original glucan content in the pretreated

samples.

2.2.7 Bacterial Cellulose Solid-state NMR Study

Bacterial cellulose G. xylium was cultivated in Hestrin and Schramm (HS) medium

containing 5 g/L peptone, 5 g/L yeast extract, 2.7 g/L Na2HPO4, 1.15 g/L citric acid and 20 g/L

glucose [60] for 7 days in room temperature. The NMR instrument is a Bruker Avance 900

MHz superconducting NMR Spectrometer equipped with a TCI triple resonance inverse

detection cryoprobe.

2.2.8 Residual Cell Wall Solid-state NMR Study

The 2 mm screen biomass samples were treated by three conditions of AHP

pretreatment based on different NaOH loadings, 0.05 g/g, 0.1 g/g and 0.15 g/g biomass, and the

same H2O2 concentration, 0.125 g/g biomass at 30°C for 24 hours. The samples were air-dried

after pretreatment and 300 mg of each sample was packed into solid-state NMR tube. The 13

C

CP/MAS solid-state NMR instrument is a Varian 400 MHz superconducting

NMR-Spectrometer operating at 399.745 MHz interfaced with a Sun Microsystems Ultra5

UNIX console. Spectrum were acquired over 4 hours and normalized to the height of the C6

cellulose peak. Peak identifications were taken from figure 2.6.

15

2.2.9 Residue Cell Wall 2D HSQC NMR Profiling

For HSQC 2D NMR study, the pre-milled switchgrass under low severity AHP

pretreatment was extracted with distilled water (ultrasonication 1 hour for three times) and 80%

ethanol (ultrasonication 1 hour for three times) to remove the metal ions induced from the

stainless ball and jar during pre-milling. Then after drying, the metal ions free sample was

ball-milled by QIAGEN TissueLyser II in 2 mL polycarbon tubes with 2 mm Ø zirconium

dioxide (ZrO2) balls (25 Hz, 2 minutes, 5 times) with liquid nitrogen cooling interval. 50 mg

ball-milled sample were transferred into a 5 mm diameter x 8” length NMR tube, where the

sample powder was distributed as well as possible along the sides of the tube. 1 mL mixture

solvent of DMSO-d6 and pyridine-d5 with a ratio 4:1 v/v was previously prepared, and was

carefully added along the side of the tube. The NMR tubes were placed in a sonicator and

sonicated for 5 hours until the gel became homogenous[52]. The HSQC 2D NMR instrument is

a Varian 600 MHz superconducting NMR-Spectrometer operating at 599.892 MHz interfaced

with a Sun Microsystems Ultra2 UNIX console, capable for probes pretuned for 1H,

13C, and

15N. HSQC spectrum was acquired over 4 hours.

2.3 RESULTS AND DISCUSSIONS

2.3.1 NREL Compositional Analysis and Digestibility Evaluation

Biomass Untreated Low NaOH AHP High NaOH AHP Hybrid Corn Stover 100% 77% 47% Inbred bm1 Stover 100% 66% 36% Inbred bm3 Stover 100% 68% 36%

Switchgrass 100% 76% 53%

Table 2.2 Weight remaining of four grasses after AHP pretreatments

16

Figure 2.1 Compositional analysis of four grasses under different AHP pretreatments

Figure 2.2 Glucan digestibilities of four grasses under different AHP pretreatments

0.0

0.2

0.4

0.6

0.8

1.0

Co

ntr

ol

low

hig

h

Co

ntr

ol

low

pH

11

.5

hig

h

Co

ntr

ol

low

pH

11

.5

hig

h

Co

ntr

ol

low

hig

h

Hybrid Corn

Stover

Inbred bm1 Stover Inbred bm3 Stover Switchgrass

Co

mp

osi

tio

n W

t F

ract

ion

Unquantified Klason Lignin Hemicellulose Cellulose

0%

20%

40%

60%

80%

100%

Pioneer Hybrid

Stover

Inbred bm1 StoverInbred bm3 Stover Switchgrass (cv.

Cave-In-Rock)

Glu

can

Dig

est

ibil

ity

Untreated

Low NaOH AHP

pH 11.5 AHP

High NaOH AHP

17

Grasses are much more difficult to be chemical analyzed than woody plants due to the

higher content of material other than cell wall structural polymers such as minerals, proteins

and soluble sugars [61]. Compositional analysis result of the untreated control samples is

shown in Figure 2.1, and the weight loss during pretreatment is shown in Table 2.2. For

example, pioneer hybrid corn stover is composed of 41% cellulose, 33% hemicellulose and 15%

lignin in a dry basis, and cv. cave –in-rock switchgrass contains lower portions of cellulose

(37%) and hemicellulose (31%) and high level of lignin (21%). After low severity AHP

pretreatment, 76% switchgrass and 77% hybrid corn stover remained in the solid phase, and

after high severity pretreatment, only 53% switchgrass and 47% hybrid corn stover remained.

For compositional analysis of four types of grasses shown in Figure 2.1, lignin and

hemicellulose content of samples decreases when pretreatment severity increases, which

shows significant lignin and hemicellulose removal by AHP pretreatment. According to Table

2.2, Figure 2.1 and Figure 2.2, stovers including hybrid corn stover, inbred lines bm1 and bm3

have higher weight loss, higher hemicellulose and lignin removal, and higher glucan

digestibility than switchgrass. Among three stovers, inbred lines bm1 and bm3 have higher

weight loss, and higher digestibility than hybrid corn stover, indicating their higher suitability

for AHP pretreatment and as potential feedstocks for ethanol production. The varied

digestibilities of the samples under the same pretreatment between stover species suggests

that the structural differences of grass lignins as shown in 2.2.1 have impacts on AHP

pretreatment effectiveness towards glucan digestibility. The samples pretreated with pH11.5

adjusted AHP pretreatment have significantly higher digestibility while lower hemicellulose

and lignin removal than those under low NaOH pretreatment, which suggested effective

18

oxidation of lignin may contribute to pretreatment effectiveness.

2.3.2 ABSL Analysis

Klason method is based on removing carbohydrates through two stage acid hydrolysis

to get a brown colored acid insoluble residue as lignin product [9]. Acetyl bromide soluble

lignin (ABSL) method is to solubilize lignin as brominated derivatives and determine the lignin

content using an extinction coefficient by UV spectroscopy. The ABSL content was

determined by the following equation:

ABSL= �

�×��×500

(Equation 1)

Where

a = UV absorbance at 280 nm, A

PL = path length, cm

m = weight, g

k = extinction coefficient, A cm-1 g

-1, estimate equals to 17.75 for grasses [62].

19

Figure 2.3 Correlation between ABSL and Klason lignin

ABSL usually overestimates lignin content, which is due to the reasons that the

phenolic compounds existed in proteins or other PCW components, the carbohydrates

degradation products also have UV absorbance, and the extinction coefficient is estimated and

varies by species. Figure 2.3 shows the content of ABSL and Klason lignin has a linear

correlation with an R value equals to 0.9573, and ABSL is about 5%~10% higher than Klason

lignin for four types of grasses. Among three stovers, inbred line bm1 shows the highest lignin

content in both ABSL and Klason.

y = 0.8224x - 0.0303

R² = 0.9573

0%

10%

20%

0% 10% 20% 30%

Kla

son

Lig

nin

(g

Lig

nin

/g b

iom

ass

)

Acetyl Bromide SolubleLignin (g ABSL/g Biomass)

20

2.3.3 Correlation between Lignin Content and Glucan Digestibility

Figure 2.4 Correlation between Klason lignin and glucan digestibility by species

Figure 2.5 Correlation between ABSL and glucan digestibility by treatments

0%

20%

40%

60%

80%

100%

0.000 0.050 0.100 0.150 0.200 0.250

Glu

can

Dig

est

ibil

ity

Klason Lignin (Mass Fraction of Cell Wall)

Pioneer Hybrid Stover

bm1 Inbred Stover

bm3 Inbred Stover

Switchgrass (cv. Cave-In-Rock)

y = -4.51x + 1.334

R² = 0.9225

0%

20%

40%

60%

80%

100%

0% 10% 20% 30%

Dig

est

ibil

ity

(g

luca

n c

on

ve

rsio

n)

ABSL(g ABSL/g biomass)

Untreated and

Low NaOH

pH=11.5

High NaOH

21

The sigmoid negative correlation shown in Figure 2.4 between Klason lignin and

digestibility by species shows that in both very high and very low lignin content grasses, the

increase in digestibility due to decreased lignin content is less significant than in the middle

range of lignin content, indicating structural changes occurs due to different pretreatments.

When plotted by types of pretreatment, the glucan digestibility directly depends on lignin

content of ABSL as well as Klason at the same slope for the untreated samples and the mild

pretreated samples. However, pH-adjusted and high NaOH pretreated samples are of different

slope of correlation between lignin content and digestibility which implies structural changes

and needs detailed investigation.

2.3.4 Bacterial Cellulose Peak Assignment

Figure 2.6 900 MHz Solid-state NMR spectra for bacterial cellulose (Numbers on the peaks

refer to 6 carbon atoms on the hexose ring)

5565758595105115

ppm

C1 C4 C2,3,5 C6

22

Bacterial cellulose synthesized by Acetobacter species with ultra-fine fiber network

structure with high mechanical strength, water absorption and crystallinity, has been looked

as a novel commercial biochemical with applications in food science, tissue engineering and

paper industry [63]. Peaks referred to carbons on the hexose ring of bacterial cellulose

microfibrils have been assigned in Figure 2.6 according to Udhardt et al. [64]. Since the 900

MHz facility equipped with cyroprobe gives high resolution spectrum, the clearly defined

peak assignment can be applied to other 13

C solid-state NMR results. The detailed

crystallinity study of bacterial cellulose is shown in the Appendix.

2.3.5 Residual Cell Wall Solid-state NMR Study

Figure 2.7 Solid-state CP/MAS 13

C NMR spectra of switchgrass under AHP pretreatments

with different NaOH concentration

1535557595115135

ppm

Control

0.05g NaOH/g SG

0.1g NaOH/g SG

0.15g NaOH/g SG

23

Figure 2.8 Solid-state CP/MAS 13

C NMR spectra of switchgrass under AHP pretreatments

with different NaOH concentration (15-40 ppm)

Figure 2.9 Solid-state CP/MAS 13

C NMR spectra of switchgrass under AHP pretreatments

with different NaOH concentration (110-150 ppm)

1520253035

ppm

Control

0.05g NaOH/g SG

0.1g NaOH/g SG

0.15g NaOH/g SG

110115120125130135140145150

ppm

Control 0.05g NaOH/g SG

0.1g NaOH/g SG 0.15g NaOH/g SG

24

Solid-state 13

C CP/MAS NMR can provide information of polysaccharides structural

properties. However, the restriction is the sensitivity of the probe equipped in the 600 MHz

NMR facility is relatively low to investigate the crystallinity properties of macromolecules in

biomass. Also, the coexisting hemicelluloses make the amorphous region much intense and

thus attenuate the sharpness of cellulose crystalline peaks. The spectra were normalized based

on the height of C6 peak since the an assumption had been made that crystalline cellulose is not

changed during relative low NaOH concentration AHP pretreatment, so called “mild” alkali

pretreatment, and only C6 is contained in cellulose as well as C1 through C5 are either from

cellulose or hemicelluloses. Figure 2.8 shows signal of aliphatic region in lignin at 27-40 ppm

is slightly reduced after mild pretreatment, and acetyl group at 21 ppm is significantly

decreased. Figure 2.9 shows aromatic lignin region at 148 ppm is gradually reduced as

pretreatment severity increased [65]. Signal reduction at 60 ppm implies the methoxyl groups

from either lignin or 4-O-methyl-glucuronoxylan are partially removed. Those signal

reductions suggest an idea the abundant ester or ether crosslinked substitutions in

hemicelluloses backbone such as acetate and methoxyl are primarily cleaved during mild AHP

pretreatment (0.1 g NaOH and 0.125 g H2O2 / g switchgrass), and the cell wall building blocks

lignin and hemicelluloses are partially removed by mild AHP pretreatment.

25

2.3.6 Residue Cell Wall 2D HSQC NMR Profiling

Figure 2.10 Gel-state HSQC 2D NMR spectrum of ball-milled low severity AHP pretreated

switchgrass with a mixture solvent DMSO-d6 and Pyridine-d5 (4:1 v/v)

2D NMR provides finger prints for plant cell walls. However, it is not as fast or

sensitive as other analytical methods due to the limitation of NMR in analyzing bio-based

macromolecules. In Figure 2.10, most of the correlations are in the δC/δH=60-105/3.2-4.5

ppm region, which belong to polysaccharides components [52] and no apparent correlation in

lignin region. The carbons corresponding to xylan and glucan were identified [52]. Three

hydrogen atoms each located at either β-D-Glcp (2), (3), and (4) or β-D-Xylp (2), (3), and (4)

have the same carbon atom shift with different H shifts. Two hydrogen atoms at β-D-Xylp (5)

26

and one hydrogen atoms at β-D-Glcp (6) have the same carbon atom shift with different

hydrogen shifts. β-D-Glcp (1) and β-D-Xylp (1) have one unique shift. The lack of cryoprobe

leads to low S/N (signal to noise) in the experiment and low resolution spectra. Kim et al. [52]

stated the importance of milling of the biobased macromolecules in order to homogenously

suspend them in solvent as a gel state, and the time length and cycle number of milling depend

on the biomass species and treatment. The Retsch Mixer Mill MM400 mentioned in his paper

provides rotary grinding and results in smaller particles. After appropriate milling, the

spectrum was able to be obtained in only two hours. Thus milling is another possibility that low

resolution and only carbohydrate region shows up in the result. However, since the

hemicelluloses have more amorphous structure than cellulose, the better swelling and

suspending in the solvent lead to stronger carbon-hydrogen correlation of xylan shown in the

spectrum, although the concentration of xylan is considered to be lower than glucan.

27

3. SOLUBILIZED COMPOUNDS IN HYDROLYSATE

3.1 INTRODUCTION

Ester/ether linkages between lignin and hemicellulose forming lignin carbohydrate

complex (LCC) are one of the major contributions to recalcitrance to enzymatic accessibility

[46], which makes mild alkaline treatment effective way to extract hemicelluloses and lignin

from biomass [26]. Depending on the conditions, alkaline pretreatments partially solubilize

polysaccharides into the liquid phase and some of them are degraded to toxic or nontoxic

byproducts including organic acids, aldehydes and phenols [66]. Pretreatment also could be a

pre-extraction step results in the removal of extractives including lipids, proteins and

inorganics which are contained in plant tissues. The toxicity of the hydrolysate of AHP

pretreatment was investigated in order to demonstrate if a detoxify step is indispensible for

following enzymatic hydrolysis and fermentation [67].

Lignin carbohydrate complexes (LCCs) were introduced in 1950s [68] and were found

in many organic extracted or alkaline extracted woods [69]. Study has shown the aggregate

formation of glucuronoxylan from alkali extracted aspen by detecting the glucuronic acid

distribution along the xylan chains [70]. For grasses, alkali extracted LCCs were also observed

and found to contain the similar ratio of monosaccharide residues as those in the untreated cell

wall [71, 72] with a reported DP at around 50 [73].

Last chapter showed AHP pretreatment effects on fractionation of lignin and

hemicelluloses. In order to investigate the pretreatment hydrolysate, the mass balance of

soluble and insoluble phases was performed in this chapter, and molecular weight distribution

28

of hemicelluloses and lignin in the hydrolysate was estimated by Size Exclusion

Chromatography (SEC). Similar to gel-state NMR for residue cell walls, solution-state HSQC

NMR were utilized for profiling the hydrolysate. Meanwhile, solution-state 1H NMR is

another fast method to obtain the information about compounds contained in the hydrolysate

[38].

3.2 MATERIAL AND METHODS

3.2.1 Sample Concentration

140 mL low severity switchgrass pretreatment liquor was filled into a 15” x 3” dialysis

tube with closures in both ends and the air was removed out of the tubing. The dialysis tubing

was immersed into a basin filled with deionized water overnight in 4°C. Transfer the liquor to a

round bottom flask connected with rotary evaporator and set the water bath to 50°C to let the

liquor concentrate until the volume become around 30 mL(5 times concentrated).

3.2.2 Compositional Analysis of Hydrolysate

10 mL concentrated liquor was taken. pH was measured to calculate the amount of 72%

H2SO4 needed to bring the pH equals to the pH of 4% H2SO4. The liquor and that amount of

72% H2SO4 were transferred to pressure tube to autoclave at 121°C for 1 hour. 1 mL of the

autoclaved solution was filtered by 0.22 mm microfilter and transferred to HPLC vial. Five

standard solutions of D(+)glucose, D(+)xylose, L(+) arabinose with concentration gradient

were prepared and injected with the samples at the same time and the peak area of the demand

ingredients in chromatogram were integrated to calculate sugar concentrations.

29

3.2.3 Hydrolysate NMR Profiling

10mL concentrated sample was taken and filled into five 2 mL sarstedt tubes,

lyophilized overnight. One of those tubes was taken and 100 µL deionized water was added to

dissolve the dried hydrolysate, then 900 µL DMSO-d6 was added to vortex. The mixture was

injected to a 5 mm diameter x 8” length NMR tube. The HSQC 2D NMR instrument is a Varian

600 MHz superconducting NMR-Spectrometer operating at 599.892 MHz interfaced with a

Sun Microsystems Ultra2 UNIX console, capable for probes pretuned for 1H,

13C, and

15N.

HSQC spectrum was acquired in 14 hours [53]. The 1H NMR experiments were using the same

instrument as HSQC 2D NMR and the spectrum were obtained in 10 minutes.

3.2.4 SEC Study on Hydrolysate

0.5 mL of concentrated liquor was combined with 0.5 mL mobile phase and injected to

HPLC sample vial. The size exclusion chromatography utilizes 0.1 M sodium nitrate and 0.01

M sodium hydroxide solution as the mobile phase. The column is Waters ultrahydrogel 250

column, 6 µm, 7.8 x 300 mm, which is applicable for molecular weight range from 1,000 Da to

80,000 Da. The experimental condition is 20 µL injection and 0.6 mL/min flow rate with both

detectors of Refractive Index Detector and UV Diode Array Detector. Six standard 2 g/L

dextran solutions are utilized and the apparent molecular weights are 1,000, 5,000, 12,000,

25,000, 50,000 and 80,000 Da. A calibration curve was set up and the apparent molecular

weight distribution of the sample was calculated via the calibration curve.

30

3.3 RESULTS AND DISCUSSION

3.3.1 Compositional Analysis of Hydrolysate

Figure 3.1 Flowchart of Low NaOH AHP pretreatment (Switchgrass)

Figure 3.2 Mass balance of solid (s) and liquid (l) phase before (1) and after (2) low NaOH

AHP pretreatment (Switchgrass)

Figure 3.1 shows untreated switchgrass consists of approximately 37% glucan, 26%

xylan and 21% lignin. After alkali fractionation, about 10% extractives are extractable, 10%

lignin along with 5% xylan and 1% glucan is removed to the liquid phase, and 10% of solid

phase is not quantified. Approximately 5% acetate from hemicelluloses was neither quantified

in compositional analysis of the solid before pretreatment, nor quantified in the pretreatment

Glucan(s)

Glucan(s)

Xylan(s)

Xylan(s)

Lignin(s)

Lignin(s)

Others(s)

Others(s)

0% 20% 40% 60% 80% 100%

2

1

Composition Wt. Fraction

Glucan(l) Xylan(l) Lignin(l) Others(l) Glucan(s) Xylan(s) Lignin(s) Others(s)

100 kg switchgrass containing 37 kg glucan, 29 kg xylan, 23 kg lignin

Low NaOH AHP Pretreatment at 2% Solid Loading

22 kg liquid containing 1 kg glucan, 4 kg xylan, 4 kg lignin

78 kg solid containing 34 kg glucan, 22 kg xylan, 11 kg lignin

31

liquor due to the removal of low molecular weight compounds during dialysis. The glucan in

the liquid phase could be derived from either the minor cell wall components such as starch,

sucrose or xyloglucan hemicellulose.

Different pretreatments have diverse decomposition products in hydrolysates.

Literatures showed during ammonia fiber expansion (AFEX) and dilute acid pretreatment of

corn stover, water soluble decomposition products were identified by LC/MS and GC/MS

including carboxylic acids, furans, carbohydrates, lignin derived aromatics [74]. Those

compounds in hydrolysate have different impacts on fermentation. AFEX pretreated biomass

is fermentable with no detoxification or external nutrient supplementation necessary. Dilute

acid pretreatment forms furans in high severity conditions as considered fermentation

inhibitors. Unlike these, alkali pretreatment significantly fractionates lignin and hemicelluloses

into solution as recoverable polymers, which are further quantified by SEC in 3.3.4.

32

3.3.2 Hydrolysate NMR Profiling

Figure 3.3 HSQC 2D NMR spectrum of AHP Pretreatment liquor with solvent DMSO-d6 and a

series of depressed H2O peak in 1H at 3.5 ppm (For interpretation of the references to color in

this and all other figures, the reader is referred to the electronic version of this thesis.)

The lyophilized hydrolysate was very slightly soluble in DMSO-d6. Due to the

dilemma that adding more solvent may cause better solublization as well as more diluted and

weaker signal, a small portion of deionized water was added to dissolve the dried material. The

water peak was depressed in 1H at 3.5 ppm in Figure 3.2. The absence of cryoprobe resulted in

longer acquisition time.

In Figure 3.2, the carbons corresponding to xylan and methoxyl group were identified

33

according to Kim et al. [52]. Three hydrogen atoms each located at β-D-Xylp (2), (3), and (4)

have the same carbon atom shift with different hydrogen shifts. Two hydrogen atoms at

β-D-Xylp (5) have the same carbon atom shift with different hydrogen shifts. β-D-Xylp (1)

have one unique carbon and hydrogen shift. The methoxyl group peak shown in hydrolysate is

either in lignin or 4-O-methyl-glucuronoxylan, which is consistent with the significantly

reduction signal of methoxyl group shown in Figure 2.7, the solid-state 13

C NMR result of

residual cell wall.

3.2.3 1H NMR characterization of Hydrolysate

Figure 3.4 Labeled 1H NMR spectrum of AHP pretreatment liquor with solvent DMSO-d6

34

Figure 3.5 Labeled 1H NMR spectrum of AHP pretreatment liquor with solvent D2O after 0.22

µm pore size ultra-filtering.

The peak identification was referred to the Spectral Database for Organic Compounds

(SDBS). The 1H NMR spectra showed the ratio of

1H located in aromatic region and

1H

located in carbon backbone region is 27.77% and 72.23% before ultra-filtration, 18.43% and

81.57% after ultra-filtration. According to the S, G and H lignin monomer ratio in

thioacidolysis results in 4.3.1 for both untreated and treated switchgrass, the monolignol ratio

in hydrolysate is calculable. Along with the mass ratio of xylan and lignin in hydrolysate in

3.3.1, the ratio of two category of hydrogen atom is calculated and shown in Table 3.1.

35

Aromatic

H # (Ha)

Backbone

H # (Hb)

Mass Ratio

Average M.W.

Molar Ratio (f) Ha x f Hb x f

H Lignin 5 5 0.120 150 0.013 0.067 0.067 G Lignin 3 7 0.429 180 0.395 1.185 2.764 S Lignin 2 10 0.080 210 0.063 0.126 0.629 Xylan 0 5 0.480 150 0.529 0.000 2.645

Sum: 1.377 6.104 Ratio: 18.41% 81.59%

Table 3.1 The ratio of H located in aromatic ring and carbon backbone based on the

composition analysis results.

Ha: The number of hydrogen atom located in aromatic rings of lignin

Hb: The number of hydrogen atom located in carbon backbones of lignin or xylan.

f: Molar ratio of components on a basis of total mass equals to one.

Since 0.22 µm pore size ultra-filtering is also a sample preparation step before sugar

analysis using HPLC described in last chapter, the consistent result of the ratio of two types of

1H gives a strong evidence that solution-state

1H NMR can be used as a quantitative tool for

PCW complexes assessment. Figure 3.2 and Figure 3.3 show that the ultra-filtration removed

majority of protein and lipids as well as a portion of lignin with higher molecular weight.

36

3.3.4 SEC Study on Hydrolysate

Figure 3.6 SEC result of low severity AHP pretreatment liquor (Switchgrass)

Molecular weight distribution is able to be represented by the number average

molecular weight (�) and the weight average molecular weight (�). � describes the

average of the molecular weights of the individual macromolecules, while � is the average

weight of the polymer which a random monomer belongs to. Another characteristic called

polydispersity index (PD) which equals to �/� reveals the non-uniformity of polymers.

The SEC principle is

t × u = log(M.W. ) (Equation 2)

where

t = time of elution, min

u = volumetric flow rate, mL/min

0

400

800

1200

10 100 1000 10000 100000 1000000

Re

lati

ve

Ab

un

da

nce

Molecular Weight (Da)

RID

UV

37

M.W. = molecular weight, Da

Number average molecular weight M� =∑����∑��

(Equation 3)

Weight average molecular weight M� = ∑�����

∑���� (Equation 4)

Polydispersity index PD = ∑�!

∑�"

(Equation 5)

where

#= molecular weight of an individual polymer, Da

$#=fraction of an individual polymer

Type of polymer MN (kDa) MW (kDa)

Polydispersity Index

Hemicellulose oligomers 4.7 7.0 1.48 Hemicellulose aggregations 64.5 88.4 1.37

Lignin 6.2 10.2 1.64

Table 3.2 Proposed biopolymers molecular weight distribution in low severity AHP

pretreatment hydrolysate (Switchgrass)

According to the double peaks in the Figure 3.5, a hypothesis about AHP effects has

been proposed, which is shown and calculated in Table 2.2. Hemicelluloses in the hydrolysate

exist as low molecular weight oligomers and high molecular weight aggregations with number

average molecular weight 4.7x103 Da and 64.5x10

3 Da (31 and 430 DP), polydispersity

indexes of 1.48 and 1.37. Lignin exists as low molecular weight polymers with number average

molecular weight 6.2 x103 Da (40 DP) and polydispersity index 1.64. A few second delay

between the UV detector (DAD) and Refractive Index detector (RID) results in the almost

38

overlapping peaks on chromatograms, indicating low molecular weight hemicelluloses and

lignin are linked together and hence flow through the column simultaneously.

As the hypothesis described above, the alkaline pretreatment partially solubilizes

hemicelluloses and lignin from biomass into the solution, where a portion of lignin and

hemicelluloses are still linked together as lignin-carbohydrate complexes. The DP range of

lignin and hemicelluloses is consistent with literature data and shows the aggregation

formation of hemicelluloses with a small fraction of associated lignin as well as the low

molecular weight hemicelluloses with a large fraction of associated lignin, indicating the

potential of low severity AHP pretreatment changing the solubility of hemicelluloses.

However, there are also other possibilities to interpret the perfectly overlapping peaks

from both chromatograms of UV and RI. One explanation is that, since lignin has both UV

and RI signal, both of the peaks are referred to lignin, and then the high molecular weight peak

in RI may be just original hemicelluloses either with or without the aggregate formation.

Another possibility is that the overlapping peaks refers to lignin in UV and hemicellulose in

RI, since they have similar molecular weight range but don’t crosslink between each other,

which means LCCs structures do not exist in the AHP hydrolysate.

39

4. PHYSIOCHEMICAL CHARACTERIZATION OF LIGNIN

4.1 INTRODUCTION

Lignin as the most complicated compound plays a resistance role in plant cell wall

materials. It is a web structure composed of three types of p-hydroxycinnamyl alcohols

(monolignols) and up to 11 types of linkages [9]. Based on the number of methoxyl group on

the aromatic rings, the three monolignols are defined as p-coumaryl alcohol, coniferyl alcohol

and sinapyl alcohol. The linkages between lignin units are primarily labile β-O-4 and α-O-4

ether bonds and a smaller fraction of “C-C” including β–5, β–β, and 5–5 and biphenyl ether

including 4-O-5 and 5-O-4 bonds which are called condensed structure and resistant to

chemical degradation [75]. Lignin composition and structural organization in monocot grasses

are significantly different from herbaceous and woody dicots or gymnosperm lignins. Instead

of branched softwood lignins and β-O-4 linkage rich hardwood lignins, ferulates and

p-coumarate comprise a considerable fraction in grass lignins via ester crosslinks and makes

the lignin highly condensed with higher phenolic hydroxyl content [76], which increases the

solubilization of grass lignins in alkali [75]. Also, many alkali-labile esters bonds existing in

hemicelluloses such as diferulate ester is unique in grasses [77]. Those characteristics of grass

lignins yield high alkali solubility and make alkaline hydrogen peroxide pretreatment

well-suited to grasses.

Studies have shown strong negative correlation between lignin content and digestibility

of different types of biomass [78, 79], however, impacts of lignin composition on digestibility

are varied by type of biomass and types of pretreatments. During dilute acid hydrolysis of

40

woody biomass from the populus family, both lignin content and S/G ratio effects its sugar

yield, and slightly lower S/G ratio yield significantly higher rate of hydrolysis [80]. However,

after hot water pretreatment, sugar release was higher for natural populus with higher S/G

ratios [78]. For alfalfa biomass under dilute acid pretreatment, large differences in enzymatic

saccharification efficiencies were observed between various lines, and with those lines have

high H content, S/G ratio alone doesn’t correlate with sugar yield [79]. For arabidopsis tissue

after hot water pretreatment, cell walls with higher S/G ratio gave a much higher glucose yield

[81].

In order to obtain the detailed information of the lignin structure in biomass, different

methods can be used. Klason and ABSL described in the residual cell wall analysis chapter are

methods estimating the total lignin content. However, since lignin composition and structures

differ among softwood, hardwood and grasses, methods are being developed to determine the

ratio of monomers, the linkage composition and distribution. This chapter mainly discussed

two methods estimating the lignin composition. The first method called thioaciolysis is based

on the principle of cleaving the β-O-4 inter-unit linkage after lignin isolation from

polysaccharides by dioxane, then adding volatile groups on monolignols to test by GC/MS.

Since the β-O-4 content is varied by species and only composes 40% of grass lignin

crosslinkings, thioacidolysis usually yields only 10%-20% monolignols. Analytical pyrolysis

based on GC/MS is another direct method of lignin content measurement with advantages

including easy sample preparation, short analysis times and small sample sizes [82-84]. When

heating biomass at high temperature between 300 and 600°C in the absence of oxygen, the

carbohydrate molecules are rapidly depolymerized to anhydroglucose units that further react to

41

provide a tarry pyrolyzate [85]. Vapor phase cracking products were identified based on

GC/MS chromatogram and primary pyrolysis pathway was discussed by Evans et al [86].

Pyrolysis phenolic products are able to classify precursor lignins as either guaiacyl type or

syringyl type [87]. Correlations between pyrolysis lignin and Klason lignin content have been

analyzed in softwood and hardwood [82]. Among the major pyrolysis lignin product of

monocot grasses, 4-vinylphenol and 4-vinylguaiacol have been identified to be derived from

p-coumaric acid and ferulic acid residues [88].

4.2 MATERIALS AND METHODS

4.2.1 Thioacidolysis

Thioacidolysis is the method being used to estimate the H/G/S ratio of lignin based on

the cleavage of β-O-4 bond, which is to extract lignin using dioxane and add trymethylsilyl

groups to volatilize monolignols for GC/MS quantitative determination. The procedure is to

weigh 2 mg ball-milled biomass sample, react with 200 µL 2.5% BF3, 10% EtSH dioxane

solution at 100°C for 4 hours. After cooling down the sample, 150 µL 0.4 M sodium

bicarbonate, 10 µL tetracosane standard solution (5 mg/mL EtOH, internal standard), 1 mL

water and 0.5 mL EtOAc were added in sequence, then the samples were vortexred to separate

phases. 150 µL EtOH phase of each sample was transferred to GC/MS vial, then dried to

evaporate ethanol and washed with acetone for twice, followed by adding 400 µL EtOAc, 20

µL pyridine and 100 µL N-O-bis (trymethylsilyl) acetamide to react at room temperature for 2

hours before sample injection to GC/MS. To get a quantitative result, 50 µL of 1 mg/mL

Bisphenol E was used as an internal standard before the 4-hour reaction, and 1000 µL dioxane

42

mixture was added to instead of 200 µL to complete the extraction.

4.2.2 Analytical Pyrolysis

Ball-milled control and pretreated samples 50-100 µg was pyrolyzed in a quartz tube in

a Pyroprobe 120 (Chemical Data Systems) at 600°C for 10 seconds using helium as the carrier

gas with a flow rate of 1 mL/min. The sample was carried onto a 60 m x 0.25 µm x 0.25 µm

Restek 1701 column fitted in a Shimadzu GCMS-QP5050 with a 100 split ratio. The

temperature was programmed to rise from 40°C to a final temperature of 260°C at 8°C/min, and

held at that temperature for a total run time of 35 minutes.

43

4.3 RESULTS AND DISCUSSIONS

4.3.1 Pretreatment, Lignin Composition and Digestibility

Type Treatment ABSL S/G Klason Digestibility Pioneer Stover

Untreated (Extractive-free) 23.0% 0.861 15.0% 23.9% Low NaOH Pretreated 18.1% 1.266 11.6% 51.7% High NaOH Pretreated 6.0% 0.781 1.3% 73.2%

bm1 Untreated (Extractive-free) 24.0% 1.358 16.4% 30.4% Low NaOH Pretreated 21.3% 2.100 14.8% 37.1%

pH=11.5 Pretreated 15.0% 2.094 11.3% 67.4% High NaOH Pretreated 5.2% 1.136 0.9% 84.7%

bm3 Untreated (Extractive-free) 22.7% 0.209 14.6% 27.9% Low NaOH Pretreated 21.2% 0.236 11.6% 45.2%

pH=11.5 Pretreated 14.4% 0.227 9.3% 77.8% High NaOH Pretreated 5.6% 0.125 2.9% 98.1%

Switchgrass Untreated (Extractive-free) 26.4% 0.378 21.1% 14.5% Low NaOH Pretreated 21.3% 0.516 14.6% 40.8% High NaOH Pretreated 14.2% 0.466 10.5% 84.0%

Table 4.1 Results of ABSL, Thioacidolysis and glucan digestibility

Table 4.1 shows in all grasses tested in this study, S/G ratio increases in mild condition

pretreated samples and then goes down in high severity pretreated samples. One reason

associated with degree of lignification is the β-O-4 linked G lignin with less free phenolic

hydroxyl groups is less condensed [75] and more alkali-labile than S lignin and decreases first

in mild alkali pretreatment. Also, the incorporation of coniferyl ferulate in grass lignins

increased lignin extractability in alkaline environment indicates that the ester conjugates

improves alkaline delignification [89].

44

4.3.2 Pyrogram Peak Assignment

No. Precursor Untreated (%) Low (%) High (%) 1 C,HC 7.21 12.0 17.0 2 HC 19.1 8.32 8.37 3 C,HC 0.55 6.56 4.40 4 C 2.57 5.46 7.61 5 H 6.38 4.47 0.27 6 P 4.19 3.42 2.50 7 C,HC 3.15 3.25 3.19 8 G 3.91 2.91 0.66 9 C,HC 2.38 2.67 3.43 10 C,HC 2.29 2.61 4.73 11 C,HC 1.64 2.26 2.24 12 C,HC 1.64 2.16 1.56 13 S 1.95 1.94 0.87 14 HC 1.82 1.90 1.92 15 C,HC 0.56 1.74 0.66 16 G 1.87 1.70 0.84 17 HC 0.59 1.39 2.26 18 C,HC 1.69 1.12 1.06 19 HC 0.83 1.10 1.32 20 G 1.01 1.03 0.46 21 HC 0.46 0.94 0.78 22 C,HC 0.92 0.80 0.89 23 C,HC 0.51 0.77 0.71 24 0.35 0.75 0.66 25 0.72 0.73 0.29 26 S 0.81 0.68 0.32 27 C 1.19 0.68 0.54 28 0.47 0.64 1.30 29 C,HC 1.24 0.63 0.72 30 H 0.78 0.60 0.29 31 C,HC 0.42 0.58 1.01 32 G 0.75 0.57 0.11 33 C,HC 0.36 0.51 0.57 34 0.68 0.51 0.36 35 C 0.46 0.46 0.81 36 C 0.48 0.46 0.87 37 G 0.79 0.40 0.23 38 C 0.37 0.40 0.87 39 G 0.56 0.37 0.13

Table 4.2 Py-GC/MS compound library of hybrid corn stover under AHP pretreatments

45

No. Precursor Untreated (%) Low (%) pH11.5 (%) High (%) 1 C,HC 22.4 13.2 17.2 26.2 2 HC 11.1 10.2 7.24 7.66 3 C,HC 5.14 7.09 3.27 4.02 4 C 4.22 4.46 6.93 9.93 5 H 8.38 8.04 6.45 0.00 6 P 5.63 5.23 3.79 4.50 7 C,HC 1.74 2.92 2.61 4.30 8 G 4.82 3.79 2.90 0.00 9 C,HC 2.78 3.69 2.71 4.94 10 C,HC 3.07 3.30 4.59 5.37 11 C,HC 3.17 2.88 2.13 3.58 12 C,HC 2.66 3.94 2.03 3.07 13 S 1.61 1.26 2.04 0.00 14 HC 1.89 2.32 2.60 2.76 16 G 2.67 1.98 1.90 0.00 18 C,HC 2.17 1.65 1.69 1.65 19 HC 1.13 1.01 0.86 1.52 20 G 1.40 0.91 0.97 0.00 21 HC 0.00 1.40 0.00 0.00 22 C,HC 0.81 2.25 0.00 1.89 25 0.00 1.76 0.00 0.00 26 S 0.78 0.72 0.59 0.00 27 C 2.12 5.62 1.98 5.22 28 0.53 0.58 0.65 0.87 32 G 0.45 0.81 0.97 0.00 35 C 0.99 0.81 1.01 1.41 37 G 0.69 0.45 0.56 0.00 30 H 1.91 0.90 1.39 0.00

Table 4.3 Py-GC/MS compound library of inbred bm1 stover under AHP pretreatments

46

No. Precursor Untreated (%) Low (%) pH11.5 (%) High (%) 1 C,HC 12.2 15.7 20.3 20.1 2 HC 23.1 8.82 7.67 3.78 3 C,HC 5.01 6.07 4.73 5.19 4 C 5.15 6.37 8.72 7.71 5 H 9.55 7.87 5.43 0.00 6 P 5.57 4.82 4.61 4.71 7 C,HC 1.68 3.28 3.72 3.17 8 G 5.83 4.87 4.21 0.00 9 C,HC 3.36 3.84 4.46 4.89 10 C,HC 3.04 3.94 4.71 4.35 11 C,HC 2.37 2.96 2.73 4.10 12 id Lo2.12 2.12 3.16 2.44 4.63 13 S 0.78 0.65 0.68 0.00 14 HC 2.00 2.31 3.02 2.04 16 G 3.13 2.44 2.47 0.00 18 C,HC 2.08 1.58 1.50 1.35 19 HC 1.17 1.09 1.23 1.58 20 G 0.71 0.52 0.00 0.00 21 HC 0.00 1.26 1.19 2.23 22 C,HC 0.00 1.72 1.35 3.76 23 C,HC 0.00 0.59 0.97 0.00 25 1.20 2.08 0.00 0.00 27 C 0.00 3.97 2.61 17.4 28 0.66 0.71 0.71 0.41 30 H 2.00 1.06 1.15 0.00 32 G 0.95 1.15 0.80 0.00 35 C 1.08 1.02 1.23 0.00 37 G 0.52 0.00 0.70 0.00

Table 4.4 Py-GC/MS compound library of inbred bm3 stover under AHP pretreatments

(1. Acetol; 2. Acetic Acid; 3. Methylglyoxal; 4. cyclopropyl carbinol(CPMO); 5.

4-viny-phenol; 6. 1-Nitro-2-propanone; 7. Biacetyl; 8. 4-viny-guaiacol; 9. Butanedial; 10.

Maple Lactone; 11. 6-Oxa-bicyclo[3.1.0]hexan-3-one; 12. Ethyl pyruvate; 13. Syringol; 14.

Furfural; 15. Hydroxyacetaldehyde; 16. Guaiacol; 17. 2-Hydroxy-gamma-butyrolactone; 18.

Acetol acetate; 19. Furfuryl alcohol; 20. Acetoveratrone; 21. 2(5H)-Furanone; 22. Hexanoic

acid, 5-hydroxy-3-methyl-, delta-lactone; 23. Propanoic acid; 24. 1-Octene; 25. Guanosine;

47

26. Methoxyeugenol; 27. Levoglucosan; 28. 3-ethyl-2-hydroxy-2-Cyclopenten-1-one; 29.

Butanone 30. Phenol; 31. Butyrolactone; 32. Creosol; 33. Acetylpropionyl; 34. Propylene

Carbonate; 35. 3-methyl-2-Cyclopenten-1-one; 36. 2-Propanol,1-isopropoxy; 37.

p-Ethylguaiacol; 38. (S)-(+)-2',3'-Dideoxyribonolactone; 39. (E)-Isoeugenol)

Pyrolysis-GC/MS results were analyzed using the compound library of GCMS

Solutions installed within the Py-GC/MS system. Majority of the pyrolytic compounds in those

chromatograms were identified with similarities larger than 80%. Those compounds were

subjected to different possible precursors including cellulose (C), hemicelluloses (H), S lignin

(S), G lignin (G) and H lignin (H) by the number and location of carbon atom. Mass percentage

of each compound was calculated by peak area integration and the total peak area was regarded

as total mass.

Table 4.2 to 4.4 show the mass percentage change of abundant pyrolyzable compounds

in three stovers associated with AHP pretreatment. The precursors of the compounds were

assigned. Cellulose derived compounds are mainly increased excludes one pyrolysis product

levoglucosan. The trends of pyrolytical compounds possibly either from cellulose or

hemicellulose are varied. Compounds derived from monolignols decreases gradually as the

NaOH loading of the pretreatment increases. The trends are similar to those described in the

residual cell wall analysis chapter.

48

4.3.3 Pyrolyzable Compound Comparison

Figure 4.1 Pyrolysis products correlation between types of biomass under the same

pretreatment conditions (well-correlated, especially for the mild condition)

0.1%

1.0%

10.0%

100.0%

0.1% 1.0% 10.0% 100.0%

Co

rn S

tov

er

(Pio

ne

er

Hy

bri

d 3

6H

56

)

Fra

ctio

n v

ola

tili

zed

py

roly

sis

pro

du

cts

Switchgrass (cv. Cave-In-Rock)

Fraction volatilized pyrolysis products

Untreated

Low NaOH

High NaOH AHP

49

Figure 4.2 Pyrolysis products correlation between pretreated biomass and untreated biomass

(not well-correlated)

The correlation of relative abundant pyrolysis products between species under different

types of AHP pretreatments are shown in Figure 4.2 and Figure 4.3. Figure 4.2 shows pyrolysis

products are nearly identical between different species under the same pretreatment, especially

for low NaOH pretreated samples. And pyrolysis products are not well correlated between

pretreatments for the same species is shown in Figure 4.3. It can be concluded that structure

changes in lignocellulosic biomass vary among pretreatment conditions. Due to a portion of

non-ester/ether bonded G and S lignin, namely “condensed”, is high recalcitrance, mild

condition AHP pretreatment may affect different grasses similarly to alkaline extraction, which

0.1%

1.0%

10.0%

100.0%

0.1% 1.0% 10.0% 100.0%

AH

P-p

retr

ea

ted

Bio

ma

ss

Fra

ctio

n p

yro

lysi

s v

ola

tili

zed

pro

du

cts

Untreated Biomass

Fraction volatiliized pyrolysis products

Switchgrass, Low NaOH

Switchgrass, High NaOH

Corn Stover, Low NaOH

Corn Stover, High NaOH

50

mainly only pulls off lignin-carbohydrate complexes from cellulose microfibrils without lignin

oxidation. The significant decreases of lignin content in high NaOH samples are possibly due

to the lignin oxidation by oxidative radicals generated by hydrogen peroxide in alkali adequate

environment.

4.3.4 Lignin Composition Based on Abundant Pyrolyzable Compounds

Figure 4.3 Lignin composition based on pyrolysis GC/MS of five types of biomass

The pyrograms of pyrolysis-GC/MS can be used to estimate S/G ratio and characterize

the changes to lignin composition associated with pretreatment. Compared with fragments

0%

20%

40%

60%

80%

100% Syringaldehyde

3,4,5-

TrimethoxytolueneMethylsyringol

Methoxyeugenol

Syringol

Methoxyeugenol

(E)-Isoeugenol

p-Ethylguaiacol

Creosol

Acetoveratrone

Guaiacol

Phenol

4-vinyl-guaiacol

4-vinyl-phenol

51

from syringyl lignin and guaiacyl lignin that are identifiable in sugar maple, which is the only

hardwood sampled in the study, four pools of lignin monomers are identifiable in three stovers

and switchgrass including 4-vinyl-guaiacol and 4-vinyl-phenol, which are respectively derived

from ferulic acid (FA) and p-coumaric acid (pCA) other than S and G lignin. Though FA and

pCA only account for 3-5% of grass lignins, Figure 4.4 showing significant levels of

4-vinyl-guaiacol and 4-vinyl-phenol implies the easier cracking by pyrolysis of ester bonds

crosslinked hydroxycinnamic acids.

4.3.5 Lignin Compositional Changes by Pretreatment

Figure 4.4 Four categories of pyrolyzable lignin components (S, G, FA, pCA) changes with

pretreatments (1)

0%

20%

40%

60%

80%

100%

Control Low

NaOH

High

NaOH

Control Low

NaOH

pH 11.5

(12.5%

H2O2)

pH 11.5

(25%

H2O2)

pH 11.5

(50%

H2O2)

High

NaOH

switchgrass Pioneer Hybrid Stover

Vo

lati

lize

d A

rom

ati

cs

Syringyl Lignins Guaiacyl Lignins Ferulic Acid p-coumaric acid

52

Figure 4.5 Four categories of pyrolyzable lignin components (S, G, FA, pCA) changes with

pretreatments (2)

Figure 4.5 and Figure 4.6 show the how the pyrolyzable lignin composition of four grass

species changes during different conditions of AHP pretreatment. For switchgrass and corn

stover, the percentage of FA and pCA in four pools of grass lignins decrease significantly as

the NaOH and H2O2 loading increases, respectively. In AHP pretreated inbred bm1 and bm3

stovers, the pyrolysis lignin composition changes much more slightly than switchgrasss and

hybrid corn stover. The results indicate the hydroxycinnamic acids including FA and pCA are

more alkali-labile than other lignin building blocks thus could be better removed in AHP

pretreatments. However, the removal of hydroxycinnamic acids is depended on the structural

characteristic of grass lignins and differs by species.

0%

20%

40%

60%

80%

100%

control Low

NaOH

pH 11.5 High

NaOH

Control Low

NaOH

pH 11.5 High

NaOH

Inbred bm1 Stover Inbred bm3 Stover

Vo

lati

lize

d A

rom

ati

csSyringyl Lignins Guaiacyl Lignins Ferulic Acid p-coumaric acid

53

4.3.6 S/G Variation

Figure 4.6 Comparison of S/G ratio between Thioacidolysis and Pyrolysis GC/MS

Figure 4.6 shows most of the species have apparently higher S/G by thioacidolysis than

pyrolysis, especially for bm1 stover. Since thioacidolysis only reflects the frequency of β-O-4

linked H, G or S units, one reasoning could be related to the grass lignin structure and the

incorporation of coniferyl aldehyde and S lignin, which is a frequent β-O-4 linked structure in

grasses, may cause higher S lignin release amount during thioacidolysis [90]. Besides,

pyrolysis primarily breaks ether and ester bonds, thus the C-C linked condensed lignin could

not be quantified in pyrograms, which can also explain why hydroxycinnamic acid derivatives

shown in Figure 4.4 are much more abundant than their actual content in grass lignins.

0

0.5

1

1.5

2

2.5

0 0.5 1 1.5 2

Sy

rin

gy

l:G

ua

iacy

l Li

gn

in

(Th

ioa

cid

oly

sis

GC

/MS

)

Syringyl:Guaiacyl Lignin

(Pyrolysis GC/MS)

pioneer stover

bm1

bm3

switchgrass

sugar maple

54

5. CONCLUSIONS

Cellulose, hemicellulose and lignin as three major components in plant cell walls were

investigated in the study respectively for the physical and chemical properties including

crystallinity, molecular weight distribution, composition and content. The structural changes of

plant cell wall and the composition of hydrolysate associated with AHP pretreatment were

assessed by a series of analytical techniques. Compositional analysis of the residual cell wall

and the pretreatment liquor was performed using HPLC method developed by NREL. Plant

cell wall residues were profiled in a native state by 13

C solid-state NMR and HSQC gel-state

NMR. Pretreatment hydrolysate was profiled by SEC, HSQC solution-state NMR and 1H

solution-state NMR. Lignin composition and content was studied through chemical and

physical approaches including ABSL, thioacidolysis and pyrolysis-GC/MS.

Low NaOH concentration mild AHP pretreatment performs similarly to alkaline

extraction, which partially solubilizes hemicelluloses and lignin while LCCs structures

possibly maintain. SEC results indicated the hydrolysate either contains hemicelluloses

aggregation and low molecular weight LCCs, or high molecular weight hemicellulose

oligomers as well as low molecular weight lignin. Solid-state 13

C NMR result of the residual

cell walls showed reduction of acetate and methoxyl substitutional groups on hemicelluloses

and the decreasing of methoxyl groups and the aromatics on lignin. For the analysis on lignin,

the content of ABSL and Klason lignin in biomass has a linear correlation, while ABSL is

around 5%~10% higher than Klason lignin. The digestibility significantly increased by the

increasing amount of lignin removal in different pretreatment conditions, which also has been

55

shown in flowthrough dilute acid pretreatment [14]. Thioacidolysis results showed G lignin

drops first in mild alkali pretreatment, because β-O-4 linked G lignin with less free phenolic

hydroxyl groups is less condensed [75] and more alkali-labile than S lignin and dissociates first

in mild alkali pretreatment.

In order to further investigate the pretreatment impacts on structural changes related to

pretreatment effectiveness, two inbred brown midrib stovers bm1 and bm3 were utilized in

studying of pretreatment effects on lignin composition and glucan digestibility. CAD down

regulated bm1 shows the highest lignin content in both ABSL and Klason results among three

stovers. Since sigmoid alike correlations were found between lignin content and glucan

digestibility for different grasses, structural changes differ with respect to pretreatment

conditions. Plotting of pyrolysis products correlation between conditions and species shown in

Figure 4.2 and Figure 4.3 also implies that mild pretreatment removes cell wall components

without notably changing lignin structure, while high NaOH pretreatment modifies lignin

structures or selectively removes lignin with certain structural features. In monocot grasses,

non-ester/ether bonded lignin portion is called condensed structure and high recalcitrance [75],

mild AHP pretreatment pulls off a certain amount of lignin as LCCs. In high NaOH or pH=11.5

adjusted AHP pretreated samples, the dramatically decreasing lignin content are possibly due

to the lignin oxidation by oxidative radicals generated by hydrogen peroxide in alkali adequate

environment.

The effectiveness of AHP pretreatment on grasses is demonstrated by the observation

that besides three major lignin building blocks, the majority of pyrolyzable grass lignins are

alkali-liable p-coumeric acid and ferulic acid. The incorporation of coniferylaldehyde and S

56

lignin in grasses was shown by the higher level of S lignin releasing in thioacidolysis than

pyrolysis. Higher xylan and lignin removal of stovers than switchgrass showed stovers might

be more promising feedstock for bioethanol production, and different digestibilities of mutant

bm1 and bm3 stovers revealed the lignin composition plays important role in AHP

pretreatment speeding enzymatic hydrolysis. Less condensed G lignin abundant bm3 has the

highest digestibility under different pretreatments among stovers indicates lignin structure has

impact on the glucan digestibility.

57

APPENDIX

58

Bacterial cellulose sample was examined by subjecting appropriate regions of CP/MAS

13C NMR spectra to non-linear least-squares fitting of Lorentzian and Gaussian peaks. The

mathematical model is [49]:

S(ω) = ∑ w()�

(*+ G((ω) + ∑ w(.�

(*+ L((ω)

(Equation 6)

G((ω) =+

0�√23exp(7

(878�)�

20�� ) (Equation 7)

L((ω) =+3

29�+:;(878�)�9�

� (Equation 8)

σ = +9=>�(2?@)

(Equation 9)

where

S (ω): The sum of a series of Gaussian and Lorentzian peaks

G (ω): Gaussian function of an individual peak

Li (ω): Lorentzian function of an individual peak

WiG

, WiL: The weights of the Gaussian and the Lorentzian function of individual peaks

σi, τi: The spread of an individual peak

ωi: The chemical shift of an individual peak

59

The peaks at the chemical shift of 84-94 could be interpreted as C-4 signal and assigned

to different domains corresponding to different types of crystalline structures [15]. After Excel

programming and least squares fitting, the spectra is separated to 10 Gaussian and Lorentzian

peaks, of each indicating one structural form of cellulose.

Figure A.1 C4 region peak deconvolution of solid-state 13

C NMR spectra for bacterial

cellulose

848688909294

ppm

Cellulose Iα

Paracrystalline

Cellulose

Cellulose Iα + Iβ

Cellulose Iβ

Amorphous

Cellulose

Cellulose

Microfibril Surface

Cellulose

Microfibril Surface

Cellulose

Oligomers

60

Assignment Chemical Shift (ppm)

FWHH (ppm)

Intensity (%) Line Type

Cellulose Iα 92.35 1.07 14.8 Lorentz Cellulose Iα+Iβ 91.47 0.94 21.2 Lorentz Paracrystalline Cellulose

91.47 3.92 21.2 Gauss

Cellulose Iβ 90.90 1.07 14.8 Lorentz Amorphous Cellulose

88.62 7.85 7.4 Gauss

Cellulose Microfibril Surface

86.78 1.57 4.2 Gauss

Cellulose Microfibril Surface

85.99 1.57 6.9 Gauss

Cellulose Oligomers 85.25 1.18 1.1 Gauss

Table A.1 Peak assignments and results from the spectral fitting of cellulose 13

C NMR spectra

for bacterial cellulose

The spectra of bacterial cellulose suggested a crystallinity index (δ 89-94 ppm/ δ84-94

ppm) equals to 77%. After curves deconvolution, eight morphological assignments were

subjected as well as two additional peaks are unknown. The intensity indicating the ratio of

different crystalline structures, which suggested 13

C CP/MAS NMR has the potential for

non-destructive structural characterization of pretreated PCWs.

61

BIBLIOGRAPHY

62

1. Wyman, C.E., Ethanol from lignocellulosic biomass: Technology, economics, and

opportunities. Bioresource Technology, 1994. 50(1): p. 3-15.

2. A USDA Regional Roadmap to Meeting the Biofuels Goals of the Renewable Fuels

Standard by 2022. 2010, U.S. Department of Agriculture (USDA).

3. Muhlethaler, K., Ultrastructure and Formation of Plant Cell Walls. Annual Review of

Plant Physiology, 1967. 18(1): p. 1-24.

4. Hayashi, T., M.P.F. Marsden, and D.P. Delmer, Pea Xyloglucan and Cellulose: V.

Xyloglucan-Cellulose Interactions in vitro and In vivo. Plant Physiology, 1987. 83(2): p.

384-389.

5. Micic, M., et al., Study of the lignin model compound supramolecular structure by

combination of near-field scanning optical microscopy and atomic force microscopy.

Colloids and Surfaces B: Biointerfaces, 2004. 34(1): p. 33-40.

6. Terashima, N., Formation of macromolecular lignin in ginkgo xylem cell walls as

observed by field emission scanning electron microscopy? Comptes Rendus Biologies,

2004. 327(9-10): p. 903-910.

7. Whitney, S.E.C., et al., Roles of cellulose and xyloglucan in determining the

mechanical properties of primary plant cell walls. Plant Physiology, 1999. 121(2): p.

657-663.

8. Atalla, R.H. and D.L. Vanderhart, NATIVE CELLULOSE - A COMPOSITE OF 2 DISTINCT

CRYSTALLINE FORMS. Science, 1984. 223(4633): p. 283-285.

9. Adler, E., Lignin chemistry—past, present and future. Wood Science and Technology,

1977. 11(3): p. 169-218.

10. Mosier, N., et al., Reaction Kinetics, Molecular Action, and Mechanisms of Cellulolytic

Proteins, in Recent Progress in Bioconversion of Lignocellulosics. 1999, Springer Berlin

/ Heidelberg. p. 23-40.

11. Himmel, M.E., et al., Biomass Recalcitrance: Engineering Plants and Enzymes for

Biofuels Production. Science, 2007. 315(5813): p. 804-807.

12. Zhang, Y.-H.P. and L.R. Lynd, Toward an aggregated understanding of enzymatic

hydrolysis of cellulose: Noncomplexed cellulase systems. Biotechnology and

Bioengineering, 2004. 88(7): p. 797-824.

63

13. McMillan, J.D., Pretreatment of Lignocellulosic Biomass, in Enzymatic Conversion of

Biomass for Fuels Production, M.E. Himmel, J.O. Baker, and R.P. Overend, Editors.

1994. p. 292-324.

14. Yang, B. and C.E. Wyman, Effect of xylan and lignin removal by batch and

flowthrough pretreatment on the enzymatic digestibility of corn stover cellulose.

Biotechnology and Bioengineering, 2004. 86(1): p. 88-98.

15. Sannigrahi, P., S.J. Miller, and A.J. Ragauskas, Effects of organosolv pretreatment and

enzymatic hydrolysis on cellulose structure and crystallinity in Loblolly pine.

Carbohydrate Research, 2010. 345(7): p. 965-970.

16. Zhao, H., G.A. Baker, and J.V. Cowins, Fast Enzymatic Saccharification of Switchgrass

After Pretreatment with Ionic Liquids. Biotechnology Progress, 2010. 26(1): p.

127-133.

17. Gupta, R. and Y.Y. Lee, Pretreatment of hybrid poplar by aqueous ammonia.

Biotechnology Progress, 2009. 25(2): p. 357-364.

18. Kim, J., Y. Lee, and S. Park, Pretreatment of wastepaper and pulp mill sludge by

aqueous ammonia and hydrogen peroxide. Applied Biochemistry and Biotechnology,

2000. 84-86(1): p. 129-139.

19. Weinstock, I.A., et al., A new environmentally benign technology for transforming

wood pulp into paper. Engineering polyoxometalates as catalysts for multiple

processes. Journal of Molecular Catalysis A: Chemical, 1997. 116(1-2): p. 59-84.

20. Xiang, Q. and Y. Lee, Oxidative cracking of precipitated hardwood lignin by hydrogen

peroxide. Applied Biochemistry and Biotechnology, 2000. 84-86(1): p. 153-162.

21. Ji, Y., E. Vanska, and A. van Heiningen, New kinetics and mechanisms of oxygen

delignification observed in a continuous stirred tank reactor. Holzforschung, 2009.

63(3): p. 264-271.

22. Brunecky, R., et al., Redistribution of xylan in maize cell walls during dilute acid

pretreatment. Biotechnology and Bioengineering, 2009. 102(6): p. 1537-1543.

23. Jeoh, T., et al., Cellulase digestibility of pretreated biomass is limited by cellulose

accessibility. Biotechnology and Bioengineering, 2007. 98(1): p. 112-122.

64

24. Zhu, L., et al., Structural features affecting biomass enzymatic digestibility.

Bioresource Technology, 2008. 99(9): p. 3817-3828.

25. Donohoe, B.S., et al., Surface and ultrastructural characterization of raw and

pretreated switchgrass. Bioresource Technology. In Press, Corrected Proof.

26. Scalbert, A., et al., Ether linkage between phenolic acids and lignin fractions from

wheat straw. Phytochemistry, 1985. 24(6): p. 1359-1362.

27. Saha, B.C. and M.A. Cotta, Ethanol production from alkaline peroxide pretreated

enzymatically saccharified wheat straw. Biotechnology Progress, 2006. 22(2): p.

449-453.

28. Gould, J.M., ALKALINE PEROXIDE DELIGNIFICATION OF AGRICULTURAL RESIDUES TO

ENHANCE ENZYMATIC SACCHARIFICATION. Biotechnology and Bioengineering, 1984.

26(1): p. 46-52.

29. Gould, J.M., Studies on the mechanism of alkaline peroxide delignification of

agricultural residues. Biotechnology and Bioengineering, 1985. 27(3): p. 225-231.

30. Kristensen, J., C. Felby, and H. Jørgensen, Yield-determining factors in high-solids

enzymatic hydrolysis of lignocellulose. Biotechnology for Biofuels, 2009. 2(1): p. 1-10.

31. Zacchi, G. and A. Axelsson, Economic evaluation of preconcentration in production of

ethanol from dilute sugar solutions. Biotechnology and Bioengineering, 1989. 34(2):

p. 223-233.

32. Wingren, A., M. Galbe, and G. Zacchi, Techno-Economic Evaluation of Producing

Ethanol from Softwood: Comparison of SSF and SHF and Identification of Bottlenecks.

Biotechnology Progress, 2003. 19(4): p. 1109-1117.

33. Sluiter, J.B., et al., Compositional Analysis of Lignocellulosic Feedstocks. 1. Review

and Description of Methods. Journal of Agricultural and Food Chemistry, 2010.

58(16): p. 9043-9053.

34. Yu Ip, C.C., et al., Carbohydrate composition analysis of bacterial polysaccharides:

Optimized acid hydrolysis conditions for HPAEC-PAD analysis. Analytical Biochemistry,

1992. 201(2): p. 343-349.

35. Sarath, G., et al., Internode structure and cell wall composition in maturing tillers of

switchgrass (Panicum virgatum. L). Bioresource Technology, 2007. 98(16): p.

2985-2992.

65

36. Templeton, D.W., et al., Compositional Analysis of Lignocellulosic Feedstocks. 2.

Method Uncertainties. Journal of Agricultural and Food Chemistry, 2010. 58(16): p.

9054-9062.

37. Kiemle, D.J., A.J. Stipanovic, and K.E. Mayo, Proton NMR methods in the

compositional characterization of polysaccharides, in Hemicelluloses: Science and

Technology, P. Gatenholm and M. Tenhanen, Editors. 2004, Amer Chemical Soc:

Washington. p. 122-139.

38. Mittal, A., et al., Quantitative analysis of sugars in wood hydrolyzates with 1H NMR

during the autohydrolysis of hardwoods. Bioresource Technology, 2009. 100(24): p.

6398-6406.

39. Hames, B.R., et al., Rapid biomass analysis - New tools for compositional analysis of

corn stover feedstocks and process intermediates from ethanol production. Applied

Biochemistry and Biotechnology, 2003. 105: p. 5-16.

40. Renneckar, S., et al., Compositional analysis of thermoplastic wood composites by

TGA. Journal of Applied Polymer Science, 2004. 93(3): p. 1484-1492.

41. Liu, Q., et al., Pyrolysis of wood species based on the compositional analysis. Korean

Journal of Chemical Engineering, 2009. 26(2): p. 548-553.

42. Zhao, H., et al., Studying cellulose fiber structure by SEM, XRD, NMR and acid

hydrolysis. Carbohydrate Polymers, 2007. 68(2): p. 235-241.

43. Samuel, R., et al., Structural Characterization and Comparison of Switchgrass

Ball-milled Lignin Before and After Dilute Acid Pretreatment. Applied Biochemistry

and Biotechnology, 2009. 162(1): p. 62-74.

44. Westman, L. and T. Lindstroem, Swelling and mechanical properties of cellulose

hydrogels. V. Swelling in concentrated alkaline solutions. 1983. Medium: X; Size:

Pages: 363-376.

45. Sun, R., J.M. Lawther, and W.B. Banks, Influence of alkaline pre-treatments on the

cell wall components of wheat straw. Industrial Crops and Products, 1995. 4(2): p.

127-145.

46. Hatfield, R.D., J. Ralph, and J.H. Grabber, Cell wall cross-linking by ferulates and

diferulates in grasses. Journal of the Science of Food and Agriculture, 1999. 79(3): p.

403-407.

66

47. Iiyama, K. and A.F.A. Wallis, Determination of lignin in herbaceous plants by an

improved acetyl bromide procedure. Journal of the Science of Food and Agriculture,

1990. 51(2): p. 145-161.

48. Larsson, P.T. and P.-O. Westlund, Line shapes in CP/MAS 13C NMR spectra of

cellulose I. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy,

2005. 62(1-3): p. 539-546.

49. Larsson, P.T., K. Wickholm, and T. Iversen, A CP/MAS13C NMR investigation of

molecular ordering in celluloses. Carbohydrate Research, 1997. 302(1-2): p. 19-25.

50. Hult, E.-L., P.T. Larsson, and T. Iversen, A comparative CP/MAS 13C-NMR study of

cellulose structure in spruce wood and kraft pulp. Cellulose, 2000. 7(1): p. 35-55.

51. Maunu, S., et al., 13C CPMAS NMR investigations of cellulose polymorphs in different

pulps. Cellulose, 2000. 7(2): p. 147-159.

52. Kim, H. and J. Ralph, Solution-state 2D NMR of ball-milled plant cell wall gels in

DMSO-d6/pyridine-d5. Organic & Biomolecular Chemistry, 2010. 8(3): p. 576.

53. Kim, H., J. Ralph, and T. Akiyama, Solution-state 2D NMR of Ball-milled Plant Cell Wall

Gels in DMSO-d 6. BioEnergy Research, 2008. 1(1): p. 56-66.

54. Josefsson, T., H. Lennholm, and G. Gellerstedt, Steam explosion of aspen wood.

Characterisation of reaction products. Holzforschung, 2002. 56(3): p. 289-297.

55. Barrière, Y. and O. Argillier, Brown-midrib genes of maize: a review. Agronomie, 1993.

13(10): p. 865-876.

56. Marita, J.M., et al., Variations in the Cell Wall Composition of Maize brown midrib

Mutants. Journal of Agricultural and Food Chemistry, 2003. 51(5): p. 1313-1321.

57. Barriere, Y., et al., Genetic variation for organic matter and cell wall digestibility in

silage maize. Lessons from a 34-year long experiment with sheep in digestibility

crates. MYDCAH 2004. 49: p. 115-126.

58. Sluiter, A., et al., Determination of Structural Carbohydrates and Lignin in Biomass.

NREL Analytical Procedure, 2004. National Renewable Energy Laboratory, Golden,

CO.

59. Hatfield, R.D., et al., Using the Acetyl Bromide Assay To Determine Lignin

Concentrations in Herbaceous Plants:  Some Cautionary Notes. Journal of Agricultural

and Food Chemistry, 1999. 47(2): p. 628-632.

67

60. Hestrin, S. and M. Schramm, SYNTHESIS OF CELLULOSE BY

ACETOBACTER-XYLINUM .2. PREPARATION OF FREEZE-DRIED CELLS CAPABLE OF

POLYMERIZING GLUCOSE TO CELLULOSE. Biochemical Journal, 1954. 58(2): p.

345-352.

61. Torget, R., et al., Dilute acid pretreatment of short rotation woody and herbaceous

crops. Applied Biochemistry and Biotechnology, 1990. 24-25(1): p. 115-126.

62. Fukushima, R.S. and R.D. Hatfield, Comparison of the Acetyl Bromide

Spectrophotometric Method with Other Analytical Lignin Methods for Determining

Lignin Concentration in Forage Samples. Journal of Agricultural and Food Chemistry,

2004. 52(12): p. 3713-3720.

63. Iguchi, M., S. Yamanaka, and A. Budhiono, Bacterial cellulose—a masterpiece of

nature's arts. Journal of Materials Science, 2000. 35(2): p. 261-270.

64. Udhardt, U., S. Hesse, and D. Klemm, Analytical Investigations of Bacterial Cellulose.

Macromolecular Symposia, 2005. 223(1): p. 201-212.

65. Willis, J.M. and F.G. Herring, 13C CP/MAS nuclear magnetic resonance study of the

peroxide bleaching of ultra high yield chemimechanical pulp produced from sound

and spruce budworm killed balsam fir. Wood Science and Technology, 1987. 21(4): p.

373-380.

66. Klinke, H.B., et al., Characterization of degradation products from alkaline wet

oxidation of wheat straw. Bioresource Technology, 2002. 82(1): p. 15-26.

67. Qureshi, N., et al., Removal of fermentation inhibitors from alkaline peroxide

pretreated and enzymatically hydrolyzed wheat straw: Production of butanol from

hydrolysate using Clostridium beijerinckii in batch reactors. Biomass and Bioenergy,

2008. 32(12): p. 1353-1358.

68. Bjorkman, A., Lignin and Lignin-Carbohydrate Complexes. Industrial & Engineering

Chemistry, 1957. 49(9): p. 1395-1398.

69. Lundquist, K., R. Simonson, and K. Tingsvik, LIGNIN CARBOHYDRATE LINKAGES IN

MILLED WOOD LIGNIN PREPARATIONS FROM SPRUCE WOOD. Svensk

Papperstidning-Nordisk Cellulosa, 1983. 86(6): p. R44-R47.

70. Roubroeks Johannes, P., et al., Contribution of the Molecular Architecture of

4-Contribution of the Molecular Architecture of 4-O-Methyl Glucuronoxylan to Its

Aggregation Behavior in Solution, in Hemicelluloses: Science and Technology. 2003,

American Chemical Society. p. 167-183.

68

71. Morrison, I.M., Structural invesiigations on the lignin-carbohydrate complexes of

Lolium perenne. The Biochemical journal, 1974. 139(1): p. 197-204.

72. Morrison, I.M., Isolation and analysis of lignin-carbohydrate complexes from Lolium

multiflorum. Phytochemistry, 1973. 12(12): p. 2979-2984.

73. Alam, M. and R.J. McIlroy, A xylan from perennial rye-grass (Lolium perenne). Journal

of the Chemical Society C: Organic, 1967: p. 1577-1580.

74. Chundawat, S.P.S., et al., Multifaceted characterization of cell wall decomposition

products formed during ammonia fiber expansion (AFEX) and dilute acid based

pretreatments. Bioresource Technology, 2010. 101(21): p. 8429-8438.

75. Grabber, J.H., et al., Genetic and molecular basis of grass cell-wall degradability.

I. Lignin-cell wall matrix interactions. Comptes Rendus Biologies, 2004. 327(5): p.

455-465.

76. Higuchi, T., Y. Ito, and I. Kawamura, p-hydroxyphenylpropane component of grass

lignin and role of tyrosine-ammonia lyase in its formation. Phytochemistry, 1967.

6(6): p. 875-881.

77. Iiyama, K., T.B.T. Lam, and B.A. Stone, Phenolic acid bridges between polysaccharides

and lignin in wheat internodes. Phytochemistry, 1990. 29(3): p. 733-737.

78. Studer, M.H., et al., Lignin content in natural Populus variants affects sugar release.

Proceedings of the National Academy of Sciences, 2011. 108(15): p. 6300-6305.

79. Chen, F. and R.A. Dixon, Lignin modification improves fermentable sugar yields for

biofuel production. Nature Biotechnology, 2007. 25(7): p. 759-761.

80. Davison, B.H., et al., Variation of S/G ratio and lignin content in a Populus family

influences the release of xylose by dilute acid hydrolysis. Applied Biochemistry and

Biotechnology, 2006. 130(1-3): p. 427-435.

81. Li, X., et al., Lignin monomer composition affects Arabidopsis cell-wall degradability

after liquid hot water pretreatment. Biotechnology for Biofuels, 2010. 3(1): p. 1-7.

82. Alves, A., et al., Analytical pyrolysis as a direct method to determine the lignin

content in wood. Journal of Analytical and Applied Pyrolysis, 2006. 76(1-2): p.

209-213.

69

83. Alves, A., et al., Analytical pyrolysis as a direct method to determine the lignin

content in woodPart 2: Evaluation of the common model and the influence of

compression wood. Journal of Analytical and Applied Pyrolysis, 2007.

84. Alves, A., et al., Analytical pyrolysis as a direct method to determine the lignin

content in woodPart 3. Evaluation of species-specific and tissue-specific differences in

softwood lignin composition using principal component analysis. Journal of Analytical

and Applied Pyrolysis, 2009. 85(1-2): p. 30-37.

85. Shafizadeh, F., Introduction to pyrolysis of biomass. Journal of Analytical and Applied

Pyrolysis, 1982. 3(4): p. 283-305.

86. Evans, R.J. and T.A. Milne, Molecular characterization of the pyrolysis of biomass.

Energy & Fuels, 1987. 1(2): p. 123-137.

87. Obst, J.R., Analytical pyrolysis of hardwood and softwood lignins and its use in

lignin-type determination of hardwood vessel elements. Journal Name: J. Wood

Chem. Technol.; (United States); Journal Volume: 3:4, 1983: p. Medium: X; Size:

Pages: 377-397.

88. Ralph, J. and R.D. Hatfield, Pyrolysis-GC-MS characterization of forage materials.

Journal of Agricultural and Food Chemistry, 1991. 39(8): p. 1426-1437.

89. Grabber, J.H., et al., Coniferyl Ferulate Incorporation into Lignin Enhances the

Alkaline Delignification and Enzymatic Degradation of Cell Walls. Biomacromolecules,

2008. 9(9): p. 2510-2516.

90. Grabber, J.H., S. Quideau, and J. Ralph, p-coumaroylated syringyl units in maize lignin:

Implications for [beta]-ether cleavage by thioacidolysis. Phytochemistry, 1996. 43(6):

p. 1189-1194.