STRUCTURAL CHARACTERIZATION OF ALKALINE HYDROGEN PEROXIDE (AHP) PRETREATED BIOMASS. Muyang Li

Transcription

1 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

2 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

3 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.

4 TABLE OF CONTENTS LIST OF TABLES... vi LIST OF FIGURES... vi ABBREVIATIONS... viii 1. INTRODUCTION LIGNOCELLULOSIC BIOMASS BIOMASS CONVERSION PROCESSES ALKALINE HYDROGEN PEROXIDE (AHP) PRETREATMENT OBJECTIVES STRUCTRAL CHANGES OF RESIDUE CELL WALL INTRODUCTION MATERIALS AND METHODS Sampling AHP Pretreatment Milling NREL Compositional Analysis ABSL Analysis Enzymatic Hydrolysis Bacterial Cellulose Solid-state NMR Study Residual Cell Wall Solid-state NMR Study Residue Cell Wall 2D HSQC NMR Profiling RESULTS AND DISCUSSIONS NREL Compositional Analysis and Digestibility Evaluation ABSL Analysis Correlation between Lignin Content and Glucan Digestibility Bacterial Cellulose Peak Assignment Residual Cell Wall Solid-state NMR Study Residue Cell Wall 2D HSQC NMR Profiling SOLUBILIZED COMPOUNDS IN HYDROLYSATE INTRODUCTION MATERIAL AND METHODS Sample Concentration Compositional Analysis of Hydrolysate iv

5 3.2.3 Hydrolysate NMR Profiling SEC Study on Hydrolysate RESULTS AND DISCUSSION Compositional Analysis of Hydrolysate Hydrolysate NMR Profiling H NMR characterization of Hydrolysate SEC Study on Hydrolysate PHYSIOCHEMICAL CHARACTERIZATION OF LIGNIN INTRODUCTION MATERIALS AND METHODS Thioacidolysis Analytical Pyrolysis RESULTS AND DISCUSSIONS Pretreatment, Lignin Composition and Digestibility Pyrogram Peak Assignment Pyrolyzable Compound Comparison Lignin Composition Based on Abundant Pyrolyzable Compounds Lignin Compositional Changes by Pretreatment S/G Variation CONCLUSIONS APPENDIX BIBLIOGRAPHY v

6 LIST OF TABLES Table 2.1: Definitions and conditions of AHP pretreatment Table 2.2 Weight remaining of four grasses after AHP pretreatments Table 3.1 The ratio of H located in aromatic ring and carbon backbone based on the composition analysis results Table 3.2 Proposed biopolymers molecular weight distribution in low severity AHP pretreatment hydrolysate (Switchgrass) Table 4.1 Results of ABSL, Thioacidolysis and glucan digestibility Table 4.2 Py-GC/MS compound library of hybrid corn stover under AHP pretreatments Table 4.3 Py-GC/MS compound library of inbred bm1 stover under AHP pretreatments Table 4.4 Py-GC/MS compound library of inbred bm3 stover under AHP pretreatments Table A.1 Peak assignments and results from the spectral fitting of cellulose 13C NMR spectra for bacterial cellulose vi

7 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 Figure 2.3 Correlation between ABSL and Klason lignin Figure 2.4 Correlation between Klason lignin and glucan digestibility by species Figure 2.5 Correlation between ABSL and glucan digestibility by treatments Figure MHz Solid-state NMR spectra for bacterial cellulose (Numbers on the peaks refer to 6 carbon atoms on the hexose ring) Figure 2.7 Solid-state CP/MAS 13C NMR spectra of switchgrass under AHP pretreatments with different NaOH concentration Figure 2.8 Solid-state CP/MAS 13C NMR spectra of switchgrass under AHP pretreatments with different NaOH concentration (15-40 ppm) Figure 2.9 Solid-state CP/MAS 13C NMR spectra of switchgrass under AHP pretreatments with different NaOH concentration ( ppm) 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) 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 3.3 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 Figure 3.4 Labeled 1H NMR spectrum of AHP pretreatment liquor with solvent DMSO-d Figure 3.5 Labeled 1H NMR spectrum of AHP pretreatment liquor with solvent D2O after 0.22 µm pore size ultra-filtering vii

8 Figure 3.6 SEC result of low severity AHP pretreatment liquor (Switchgrass) Figure A.1 C4 region peak deconvolution of solid-state 13C NMR spectra for bacterial cellulose viii

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

10 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, 1

11 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, I α and I β [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 2

12 (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]. 3

13 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 4

14 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 ( O 2 ) radicals. Reactions are as follows: H O HOO + H 2 2 H O + HOO OH + O + H O And the sum reaction is: + 2H O OH + O + H O + H 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]. 5

15 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 6

16 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 7

17 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 8

18 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 9

19 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 1 H 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 I α and I β [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 10

20 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 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 11

21 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] 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) H 2 O 2 (g/g biomass) H 2 O (ml) Low NaOH 2% High NaOH 2% ph=11.5 2% ph= Table 2.1: Definitions and conditions of AHP pretreatment 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 12

22 interval 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% H 2 SO 4 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 H 2 SO 4 to 4% which was then autoclaved at 121 C for 1 hour. 1mL of the solution was neutralized by CaCO 3, 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] 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 Enzymatic Hydrolysis Pretreated samples were incubated at 50 C for 24 hours with Accellerase 1500 enzyme 13

23 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 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 Na 2 HPO 4, 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 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 H 2 O 2 concentration, 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 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

24 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 (ZrO 2 ) 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-d 6 and pyridine-d 5 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 MHz interfaced with a Sun Microsystems Ultra2 UNIX console, capable for probes pretuned for 1 H, 13 C, and 15 N. HSQC spectrum was acquired over 4 hours. 2.3 RESULTS AND DISCUSSIONS 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 15

25 1.0 Unquantified Klason Lignin Hemicellulose Cellulose Control low high Control low ph11.5 high Control low ph11.5 high Control low high Composition Wt Fraction Hybrid Corn Inbred bm1 Stover Inbred bm3 Stover Switchgrass Figure 2.1 Compositional analysis of four grasses under different AHP pretreatments Glucan Digestibility 100% 80% 60% 40% 20% Untreated Low NaOH AHP ph 11.5 AHP High NaOH AHP 0% Pioneer Hybrid Stover Inbred bm1 StoverInbred bm3 Stover Switchgrass (cv. Cave-In-Rock) Figure 2.2 Glucan digestibilities of four grasses under different AHP pretreatments 16

26 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 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 17

27 oxidation of lignin may contribute to pretreatment effectiveness 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 for grasses [62]. 18

28 Klason Lignin (g Lignin/g biomass) 20% 10% y = x R²= % 0% 10% 20% 30% Acetyl Bromide SolubleLignin (g ABSL/g Biomass) 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 , 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. 19

29 2.3.3 Correlation between Lignin Content and Glucan Digestibility Glucan Digestibility 100% 80% 60% 40% Pioneer Hybrid Stover bm1 Inbred Stover bm3 Inbred Stover Switchgrass (cv. Cave-In-Rock) 20% 0% Klason Lignin (Mass Fraction of Cell Wall) Figure 2.4 Correlation between Klason lignin and glucan digestibility by species Digestibility (glucan conversion) 100% 80% 60% 40% 20% y = -4.51x R²= Untreated and Low NaOH ph=11.5 High NaOH 0% 0% 10% 20% 30% ABSL(g ABSL/g biomass) Figure 2.5 Correlation between ABSL and glucan digestibility by treatments 20

30 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 Bacterial Cellulose Peak Assignment C1 C4 C2,3,5 C ppm Figure MHz Solid-state NMR spectra for bacterial cellulose (Numbers on the peaks refer to 6 carbon atoms on the hexose ring) 21

31 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 Residual Cell Wall Solid-state NMR Study Control 0.05g NaOH/g SG 0.1g NaOH/g SG 0.15g NaOH/g SG ppm Figure 2.7 Solid-state CP/MAS 13 C NMR spectra of switchgrass under AHP pretreatments with different NaOH concentration 22

32 Control 0.05g NaOH/g SG 0.1g NaOH/g SG 0.15g NaOH/g SG ppm Figure 2.8 Solid-state CP/MAS 13 C NMR spectra of switchgrass under AHP pretreatments with different NaOH concentration (15-40 ppm) ppm Control 0.05g NaOH/g SG 0.1g NaOH/g SG 0.15g NaOH/g SG Figure 2.9 Solid-state CP/MAS 13 C NMR spectra of switchgrass under AHP pretreatments with different NaOH concentration ( ppm) 23

33 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 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 g H 2 O 2 / g switchgrass), and the cell wall building blocks lignin and hemicelluloses are partially removed by mild AHP pretreatment. 24

34 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-d 6 and Pyridine-d 5 (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 δ C /δ H =60-105/ 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) 25

35 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. 26

36 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 27

37 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 1 H NMR is another fast method to obtain the information about compounds contained in the hydrolysate [38]. 3.2 MATERIAL AND METHODS 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) Compositional Analysis of Hydrolysate 10 ml concentrated liquor was taken. ph was measured to calculate the amount of 72% H 2 SO 4 needed to bring the ph equals to the ph of 4% H 2 SO 4. The liquor and that amount of 72% H 2 SO 4 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. 28

38 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-d 6 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 MHz interfaced with a Sun Microsystems Ultra2 UNIX console, capable for probes pretuned for 1 H, 13 C, and 15 N. HSQC spectrum was acquired in 14 hours [53]. The 1 H NMR experiments were using the same instrument as HSQC 2D NMR and the spectrum were obtained in 10 minutes 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. 29

39 3.3 RESULTS AND DISCUSSION Compositional Analysis of Hydrolysate 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 Figure 3.1 Flowchart of Low NaOH AHP pretreatment (Switchgrass) Glucan(l) Xylan(l) Lignin(l) Others(l) Glucan(s) Xylan(s) Lignin(s) Others(s) 1 Glucan(s) Xylan(s) Lignin(s) Others(s) 2 Glucan(s) Xylan(s) Lignin(s) Others(s) 0% 20% 40% 60% 80% 100% Composition Wt. Fraction 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 30

40 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