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Extraction of hemicelluloses from sugar maple chips after biotreatment with Ceriporiopsis subvermispora

Dissertation
Author: Vincent A. Barber
Abstract:
The effect that biotreatment with Ceriporiopsis subvermispora has on sugar maple (Acer saccharum ) chips, and its ability to enhance the extraction of hemicellulose was investigated. The effect of the biotreatment on the chip was analyzed to assess selectivity towards the removal of the non-carbohydrate mass as well as the physical and chemical changes in the chips, which can affect the removal of hemicelluloses. The loss of lignin from the chips increased with the loss of mass during biotreatment and represented the majority of the mass loss. Klason lignin losses were 12%, 29.8%, and 41.1% in biotreated chips with total mass losses of 3.8%, 10.6%, and 18.6%. Images taken with a scanning electron microscope show degradation and erosion of the cell walls, which result in a more open structure. The opening of the structure was confirmed by analysis of the mass of water in a given volume of saturated biotreated chips. The pH of water extract of the chips was also seen to decrease after the biotreatment process dropping from 5.29 for untreated wood to 3.26, and 3.23 for biotreated chips with mass losses of 10.6% and 18.6% respectively. The benefits of biotreatment on the alkali extraction or hydrolysis of hemicellulose was then evaluated. The hemicelluloses that can be extracted with 25% KOH from the maple wood chips increased with even moderate biotreatment. Xylan is the primary hemicellulose found in hardwood and was measured as 19.4% of the mass of the untreated wood. The mass of xylan that was extracted increased from 2.9% for untreated chips to 6.7%, 7.1%, and 7.6% of the raw wood mass, for biotreated wood with mass losses of 3.8%, 10.6% and 18.6%, respectively. Autohydrolysis of the maple chips with temperatures in the range of 140ºC to 180ºC was used to extract hemicellulose from the maple chips. The total sugars and the xylose in the extract correlated well to the total non-volatile solids content of the extract for both biotreated and untreated chips at all temperatures and times used for the experiments. From these correlations a predicted maximum xylose content equivalent to 10.6% of the mass of the raw wood can be removed and retained in the extract. The rate of removal from the biotreated chips was found to be twice as fast as from the untreated chips at each of the temperatures used. Key words . Ceriporiopsis subvermispora , white-rot fungi, bio-delignification, sugar maple, hemicellulose, xylan, carbohydrate, alkali extraction, autohydrolysis

Table of Contents

ABSTRACT........................................................................................................................1 ACKNOWLEDGEMENTS................................................................................................3 Chapter 1: Introduction.......................................................................................................4 1.1 Outline of study...................................................................................................6 Chapter 2: Literature Review..............................................................................................9 2.1 Cellulose...........................................................................................................11 2.2 Lignin................................................................................................................11 2.3 Hemicelluloses..................................................................................................15 2.3.1 Properties..................................................................................................15 2.3.2 Uses...........................................................................................................18 2.4 Hemicellulose Extraction..................................................................................19 2.4.1 Hydrolysis.................................................................................................20 2.4.2 Alkaline Extraction...................................................................................25 2.5 Bio-treatment....................................................................................................29 2.5.1 Biomechanical pulping.............................................................................32 2.5.2 Biochemical pulping.................................................................................32 2.5.3 Biobleaching.............................................................................................33 Chapter 3: Biotreatment of maple wood...........................................................................35 3.1 Methods and Materials......................................................................................37 3.1.1 Maple chips...............................................................................................37 3.1.2 Bio-treatment............................................................................................37 3.1.3 NMR analysis of sugar content of wood samples.....................................39 3.1.4 Concentration of xylan determined by NMR analysis..............................45 3.1.5 Void volume of biotreated wood..............................................................46 3.1.6 Acid Insoluble Lignin...............................................................................47 3.1.7 Acid Soluble Lignin..................................................................................47 3.1.8 pH..............................................................................................................48 3.1.9 Scanning Electron Microscope.................................................................48 3.2 Results and Discussion.....................................................................................49 Chapter 4: Enhancement of KOH extraction of xylan through the bio-treatment of wood chips..................................................................................................................................59 4.1 Methods and Materials......................................................................................61 4.1.1 KOH extraction.........................................................................................61 4.1.2 Bulk Hemicellulose Dialysis.....................................................................62 4.1.3 NMR Analysis of Bulk Hemicellulose.....................................................62 4.1.4 Acid Insoluble Lignin...............................................................................67 4.1.5 Acid Soluble Lignin..................................................................................67 4.1.6 Molecular Weight Determination.............................................................67 4.2 Results and Discussion.....................................................................................71 Chapter 5: Effects of biotreatment on the hot water extraction of Hemicellulose............83 5.1 Methods and Materials......................................................................................85 5.1.1 Maple Chips..............................................................................................85 5.1.2 Biotreatment..............................................................................................85 5.1.3 Hot water extraction of hemicellulose......................................................85

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5.1.4 Non-volatile solids in the extract..............................................................87 5.1.5 NMR analysis of extract...........................................................................87 5.1.6 Titration of untreated maple chip extract to pH of biotreated extract.......90 5.2 Results and Discussion.....................................................................................91 Chapter 6: Conclusions...................................................................................................110 Chapter 7: Future Work..................................................................................................113 Chapter 8: Appendix.......................................................................................................117 Appenxix A: Chapter 3...............................................................................................117 Appendix B: Chapter 4...............................................................................................119 Appendix C: Chapter 5...............................................................................................121 References.......................................................................................................................129 Vita..................................................................................................................................136

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List of Tables

Table 2-1: Common lignin linkages and their occurrences in milled wood lignin (compiled from [1])...................................................................................................13 Table 2-2: Reported molecular weights of xylan from various sources...........................18 Table 3-1: Conditions of NMR system and analysis of samples composed from Kiemle et al. [106].....................................................................................................................40 Table 3-2: Standard deviation in NMR analysis of two different excessively hydrolyzed samples......................................................................................................................45 Table 3-3: Mass loss of maple wood during biotreatment................................................49 Table 3-4: The NMR sugar analysis of wood chips after biotreatment............................53 Table 3-5: Polysaccharide content of biotreated maple wood samples............................54 Table 4-1: Sugar content of precipitate from alkali extract of biotreated and milled maple wood..........................................................................................................................77 Table 4-2: Sugar content of precipitate from alkali extract of biotreated maple chips.....77 Table 4-3: Xylan extracted from biotreated maple chips and milled wood......................78 Table 4-4: Xylose concentration in sugars precipitated from alkali extract of biotreated maple wood...............................................................................................................79 Table 4-5: Molecular weights of hemicellulose extracted with alkali from biotreated maple wood determined using SEC with RI and MALLS.......................................82 Table 4-6: Molecular weights of hemicellulose extracted with alkali from biotreated maple wood determined using GPC with pullulan standards...................................82 Table 5-1: Hot water extraction sample times..................................................................86 Table 8-1: Location and ratio of sugar peaks in the 1 H NMR spectra of sugars in acidic D 2 O (composed from [107])...................................................................................117 Table 8-2: Location of non-sugar peaks in the 1 H NMR spectra of sugars in acidic D 2 O .................................................................................................................................118 Table 8-3: Klason lignin of biotreated maple chips........................................................118 Table 8-4: Acid soluble lignin content of biotreated maple chips..................................118 Table 8-5: Klason lignin in alkali extracted biotreated maple chips..............................119 Table 8-6: Acid soluble lignin content of alkali extracted biotreated maple chips........119 Table 8-7: Mass of alkali extract from biotreated maple precipitated with ethanol.......119 Table 8-8:Uncorrected values of sugar precipitated from biotreated and milled maple wood........................................................................................................................120 Table 8-9: Uncorrected values of sugar precipitated from biotreated and milled maple wood........................................................................................................................120 Table 8-10: Sugar analysis of 140ºC hot water extraction of sugar maple chips...........121 Table 8-11: Sugar analysis of 140ºC hot water extract from biotreated maple chips.....121 Table 8-12: Sugar analysis of 160ºC hot water extract from sugar maple chips............122 Table 8-13: Sugar analysis of 160ºC hot water extract from biotreated sugar maple chips. .................................................................................................................................122 Table 8-14: Sugar analysis of 180ºC hot water extract from sugar maple chips............123 Table 8-15: Sugar analysis of 180ºC hot water extract from biotreated maple chips.....123 Table 8-16: Error analysis of total sugars values calculated with increasing non-volatile solids.......................................................................................................................124

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Table 8-17: Errors analysis for total sugars values calculated with decreasing non-volatile solids.......................................................................................................................125 Table 8-18: Errors analysis of xylose values calculated with increasing non-volatile solids.......................................................................................................................126 Table 8-19; Errors analysis for xylose content values calculated with decreasing non- volatile solids..........................................................................................................127 Table 8-20: Titration of untreated maple wood cold water extract to pH of biotreated maple wood extract.................................................................................................128

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List of Figures Figure 2-1: Wood fiber schematic. This schematic depicts the makeup of both the living and dead cell. [1].......................................................................................................10 Figure 2-2: Transmission electron micrograph of softwood tracheids showing: middle lamella (M), primary wall (P), and outer (S 1 ), middle (S 2 ), and inner (S 3 ) secondary wall layers [2]...........................................................................................................10 Figure 2-3: Lignin precursors: (1) coumaryl alcohol, (2) coniferyl alcohol, (3) sinapyl alcohol [1].................................................................................................................12 Figure 2-4: Most common linkages between phenylpropane units in lignin [1]..............12 Figure 2-5: Proposed benzyl ether bond between lignin and hemicellulose ([5])............13 Figure 2-6: Proposed benzyl ester bond between lignin and hemicellulose ([5]).............14 Figure 2-7: Proposed glycoside bond between lignin and hemicellulose ([5])................14 Figure 2-8: Proposed acetal bond between lignin and hemicellulose ([5])......................15 Figure 2-9: Structure of O-acetyl-4-O-methylglucuronoxylan (xylan) from hardwood [14] ...................................................................................................................................17 Figure 2-10: Mechanisms involved in the hydrolysis of glycosidic bonds [35]...............21 Figure 2-11: Formation of furfural, hydroxymethylfurfural, levulinic acid and formic acid from xylose and glucose in acidic medium [4].........................................................23 Figure 3-1: Flow diagram of maple log chipping and biotreatment.................................38 Figure 3-2: Configuration of typical biotreatment apparatus...........................................38 Figure 3-3: 1 H NMR spectra of maple wood in acidic D 2 O.............................................42 Figure 3-4: 1 H NMR spectra of biotreated maple chips in acidic D 2 O.............................43 Figure 3-5: Klason lignin remaining in maple wood after biotreatment..........................50 Figure 3-6: Acid soluble lignin remaining in wood chips after biotreatment...................51 Figure 3-7: %Mass loss of xylose and glucose after biotreatment with C. subvermispora. ...................................................................................................................................52 Figure 3-8: Increased water retention of biotreated chips................................................55 Figure 3-9: Titration of room temperature water extract from 100g of biotreated milled wood with 0.1N NaOH.............................................................................................56 Figure 3-10: SEM image of non-biotreated sugar maple..................................................57 Figure 3-11: SEM image of sugar maple biotreated 2weeks/3.8% mass loss..................57 Figure 3-12: SEM image of sugar maple biotreated 4weeks/10.6% mass loss................58 Figure 3-13: SEM image of sugar maple biotreated 18weeks/18.6% mass loss..............58 Figure 4-1: Apparatus used in the alkali extraction of hemicellulose from maple wood, nitrogen was injected at the bottom of the flask to remove oxygen. ......................62 Figure 4-2: 1 H NMR spectra of ethanol precipitate of KOH extracted maple chips........64 Figure 4-3: 1 H NMR spectra of ethanol precipitate from KOH extract of biotreated maple chips..........................................................................................................................65 Figure 4-4: 1 H NMR spectra of dialyzed, ethanol precipitate of KOH extract from untreated maple wood...............................................................................................66 Figure 4-5: Klason lignin remaining after biotreatment and KOH extraction..................71 Figure 4-6: Effects of biotreatment on the acid soluble lignin content before and after extraction...................................................................................................................72 Figure 4-7: Bulk hemicellulose precipitated with ethanol from KOH extract of bio-treated milled wood and wood chips....................................................................................74 Figure 4-8: Total sugars in hydrolyzed bulk hemicellulose..............................................76

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Figure 4-9: xylose in bulk hemicellulose..........................................................................76 Figure 4-10: Xylan in ethanol precipitates of KOH extract, from biotreated maple chips and milled wood........................................................................................................78 Figure 4-11:Molecular weight ( M w) of hemicellulose extracted from biotreated maple wood calculated using SEC-MALLS........................................................................81 Figure 4-12: Molecular weight ( M w) of hemicellulose extracted from biotreated maple wood calculated using SEC-RI method....................................................................81 Figure 5-1: Schematic of M&K digester used is autohydrolysis......................................86 Figure 5-2: 1 H NMR spectra of 1hr 160ºC extract from untreated maple chips...............88 Figure 5-3: 1 H NMR spectra of sugars from 1hr 160ºC extract from biotreated maple chips..........................................................................................................................89 Figure 5-4: Non-volatile solids buildup in hot water extract of maple chips...................92 Figure 5-5: Non-volatile solids buildup in hot water extract of biotreated maple chips..93 Figure 5-6: Non-volatile solids buildup in 140ºC hot water extracts of biotreated and untreated maple chips...............................................................................................94 Figure 5-7: Non-volatile solids buildup in 160ºC hot water extracts of biotreated and untreated maple chips...............................................................................................95 Figure 5-8: Non-volatile solids buildup in 180ºC hot water extract of treated and untreated maple chips...............................................................................................95 Figure 5-9: Total sugar content of extract from treated and untreated sugar maple wood. ...................................................................................................................................97 Figure 5-10: Total sugars content vs. non-volatile solids content of extract from biotreated and untreated maple wood.......................................................................99 Figure 5-11: Comparison between total sugar content and non-volatile solids content of extract. Linear trend lines have been fit to the data points to model sugars for both increasing and decreasing total solids content..........................................................99 Figure 5-12: Xylose content of hot water extracts from untreated and biotreated maple chips........................................................................................................................100 Figure 5-13: Xylose content vs. Non-volatile solids in extracts of biotreated and untreated maple chips extracted at 140ºC, 160ºC, and 180ºC................................................103 Figure 5-14: Xylose content of sugar maple extracts with increasing and decreasing non- volatile solids contents............................................................................................103 Figure 5-15: Extract pH from hot water extraction of treated and untreated maple wood. .................................................................................................................................105 Figure 5-16: Titration of 8:1 (L:W) extraction of 50g OD maple wood with acetic acid. .................................................................................................................................106 Figure 5-17: pH of 160ºC hot water extracts from untreated and biotreated maple chips and 160ºC weak acid extract from untreated maple chips .....................................107 Figure 5-18: Non-volatile solids buildup in extract from 140ºC hot water and 140ºC weak acid hydrolysis (1% acetic acid by mass of liquor) of untreated and biotreated maple chips........................................................................................................................108 Figure 5-19: Non-volatile solids buildup in 160°C weak acid hydrolysis and 160ºC hot water extract of sugar maple...................................................................................109 Figure 8-1: 1 H NMR spectra of model sugars in acidic D 2 O: anomeric region (courtesy Dave Kiemle ).........................................................................................................117

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Equations Equation 3-1: Calculation of xylan content from NMR data............................................46 Equation 3-2: Calculation of void volume in chips..........................................................47 Equation 4-1: Equation used to calculate concentration based on refractive index signals. ...................................................................................................................................68 Equation 4-2: Modified Rayleigh equation relating M w to the signal from the light scattering detector.....................................................................................................69

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Barber, Vincent A. Extraction of Hemicelluloses from Sugar Maple Chips after Biotreatment with Ceriporiopsis subvermispora. Word processed and bound thesis, 146 pages, 56 figures, 34 tables, 2007

ABSTRACT

The effect that biotreatment with Ceriporiopsis subvermispora has on sugar maple (Acer saccharum) chips, and its ability to enhance the extraction of hemicellulose was investigated. The effect of the biotreatment on the chip was analyzed to assess selectivity towards the removal of the non-carbohydrate mass as well as the physical and chemical changes in the chips, which can affect the removal of hemicelluloses. The loss of lignin from the chips increased with the loss of mass during biotreatment and represented the majority of the mass loss. Klason lignin losses were 12%, 29.8%, and 41.1% in biotreated chips with total mass losses of 3.8%, 10.6%, and 18.6%. Images taken with a scanning electron microscope show degradation and erosion of the cell walls, which result in a more open structure. The opening of the structure was confirmed by analysis of the mass of water in a given volume of saturated biotreated chips. The pH of water extract of the chips was also seen to decrease after the biotreatment process dropping from 5.29 for untreated wood to 3.26, and 3.23 for biotreated chips with mass losses of 10.6% and 18.6% respectively. The benefits of biotreatment on the alkali extraction or hydrolysis of hemicellulose was then evaluated. The hemicelluloses that can be extracted with 25% KOH from the maple wood chips increased with even moderate biotreatment. Xylan is the primary hemicellulose found in hardwood and was measured as 19.4% of the mass of the untreated wood. The mass of xylan that was extracted increased from 2.9% for untreated chips to 6.7%, 7.1%, and 7.6% of the raw wood mass, for biotreated wood with mass losses of 3.8%, 10.6% and 18.6%, respectively. Autohydrolysis of the maple chips with temperatures in the range of 140ºC to 180ºC was used to extract hemicellulose from the maple chips. The total sugars and the xylose in the extract correlated well to the total non-volatile solids content of the extract for both biotreated and untreated chips at all temperatures and times used for the experiments. From these correlations a predicted maximum xylose content equivalent to 10.6% of the mass of the raw wood can be removed and retained in the extract. The rate of removal from the biotreated chips was found to be twice as fast as from the untreated chips at each of the temperatures used.

Key Words: Ceriporiopsis subvermispora, white-rot fungi, bio-delignification, sugar maple, hemicellulose, xylan, carbohydrate, alkali extraction, autohydrolysis

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Author’s name in full: Vincent Albert Barber Candidate for the degree of: Doctor of Philosophy Date: February 2007 Major Professor: Dr. Gary M. Scott Faculty: Paper and Bioprocess Engineering

Division of Environmental and Resource Engineering State University of New York, College of Environmental Science and Forestry, Syracuse, New York

Signature of Major Professor ________________________________________ Dr. Gary M. Scott

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ACKNOWLEDGEMENTS

I express my deepest gratitude to my steering committee: Dr. Gary M. Scott, Dr. Arthur Stipanovic, and Dr. Thomas E. Amidon. Their knowledge and guidance has been invaluable throughout the course of this work. I also thank Mr. David Kiemle for his assistance with NMR sugar analysis, Dr Iris Vazquez-Cooz for her assistance with scanning electron microscopic analysis, William Burring for his help with pH titrations along with many other experiments, Alton Brown for his assistance with hydrolysis experiments and Jeremy Bartholomew for his assistance with biotreatment. I thank the faculty, staff, and students of Walters Hall for all their help and encouragement throughout this research, especially Ray Appleby, Linda Fagan, Lynn Mickinkle, Ashutosh Mittal, and Todd Bolton. Last, but not least, I would like to thank my family; my parents Harold and Paula Barber, who always stressed the value of knowledge, putting my education first, and taught me the value of hard work, and my wife Katie, whose love and support has helped me through this journey. I would like to dedicate this thesis to my daughter, Haylie Elizabeth Barber, every day you teach me as much as I teach you. I hope that someday you will find the love of learning I have found, and you will be lucky enough to surround yourself with people willing to give of themselves, like the many that have made this research such a rewarding experience.

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Chapter 1: Introduction

The improved use of natural resources plays an important part in today’s society with both ecological and economical implications. Woody materials are primarily made up of three fractions: cellulose, hemicellulose, and lignin. To effectively utilize these materials it is important to develop methods that effectively and efficiently separate the fractions for their particular end uses. The hemicellulose fraction represents a large, and mostly untapped, source of polymers and sugars, especially in the paper industry where they are mostly a waste product. Today 70% of the pulp produced is produced by chemical pulping methods,

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most commonly the kraft process. The kraft process utilizes the cellulose for pulp production and is able the use the lignin fraction as a heat source, but the hemicellulose fraction is not efficiently utilized. Hemicelluloses retained in the pulp help to increase the strength properties, but during the kraft process 50% of the hemicelluloses are removed with the black liquor. To remove the hemicelluloses from the black liquor would be energy intensive, so they are burned in recovery boilers where they produce lower heat values compared to lignin. Extraction of the hemicelluloses prior to the pulping process would draw a whole new line of products from a source that is currently a waste stream. The pre-extraction of the hemicellulose would not only represent a better use of our natural resources, but also could help to reduce the chemicals required for further processing. Both the sugars and the polymers from lignocellulosic materials can be used to reduce the global consumption of petroleum, which is depleting crude oil reserves at an ever increasing rate. Global increases in energy and chemical consumption require sources that are renewable and will not increase the amount of atmospheric CO 2 , commonly believed to be the main cause of global warming. The sugars derived from hemicelluloses can be fermented into fuel products, such as ethanol, which can decrease the consumption of petroleum-based gasoline and other transportation fuels. Hemicelluloses used as a polymer, have the potential to replace some polymers from petroleum sources for use in biodegradable plastics. Woody materials utilize carbon dioxide from the atmosphere as a carbon source. As a whole, the use of these materials for the production of plastics and fuels will result in no net gain in the atmospheric carbon dioxide, as it is recycled from plants to products to atmosphere back to plants.

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In this research, the use of a biotreatment with Ceriporiopsis subvermispora prior to the extraction process is utilized as a method to partially delignify the wood and open the pore structure. The ability of the biotreatment to facilitate the removal of high- molecular-weight (HMW) xylan and enhance the removal of low molecular weight (LMW) xylan or xylose from sugar maple (Acer saccharum), one of the most abundant wood species in the northeast United States is evaluated. 1.1 Outline of study

Chapter 2:

Literature Review . In this chapter, the state of the science is reviewed by looking at previous research relevant to this work. The review is divided into two sections. The first section is devoted to wood chemistry. This section focuses on xylan, the primary hemicellulose in hardwoods, and its interaction with the other wood constituents, along with extraction methods. The second section of this chapter is devoted to biotreatment. This section describes the known effects of biotreatment on wood, and also discusses some of the ways it can be utilized in the paper industry.

Chapter 3:

Biotreatment of maple wood . In this chapter, the focus is on the biotreatment of the wood chips with C. subvermispora, a white-rot fungus that preferentially degrades lignin. The chips are biotreated for various durations and the effects of the biotreatment on the chips are measured by measuring composition changes during the biotreatment. The changes that are expected to have an effect on the extraction of hemicellulose are the focus of this section. The mass loss during the biotreatment is established as a method to evaluate the biotreatment effectiveness. The lignin and sugar contents of the biotreated wood is evaluated to illustrate the selectivity of

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the white-rot fungus towards lignin, as well as to establish pre-extraction concentrations. The pH of the biotreated chips is measured through the titration of a cold-water extract. The pH plays an important role in the autohydrolysis process and could also effect alkali extractions. Decreased pH may indicate that there will be a increased consumption of alkali used in alkali extraction of hemicelluloses.

Chapter 4:

Enhancement of KOH extraction of xylan through the bio-treatment of

wood chips . This section utilized biotreatment prior to an alkali extraction of maple wood as a method to increase the extraction of polymeric hemicelluloses from the wood chips. Most previous work on the extraction of hemicelluloses focused on extraction from the holocellulose (composed of cellulose and hemicellulose) or milled wood, which required additional pretreatment steps that were chemical or energy intensive and degraded the structure of the fiber. Extractions of the hemicelluloses from chips with various degrees of biotreatment were performed. The lignin remaining in the chips was analyzed to determine how much was being removed in the extraction process. The hemicellulose from the extract was precipitated and then analyzed for sugar content to determine the amount of hemicelluloses in the extract. Size exclusion chromatography (SEC) was then utilized to determine the molecular weight of the precipitated hemicelluloses. The molecular weight of the hemicellulose that was extracted from biotreated wood was compared to that obtained from the untreated wood to determine the extent of polymer degradation.

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Chapter 5:

Effects of biotreatment on the hot water extraction of Hemicellulose .

In this chapter, the extracts that were obtained from the hot water extractions of the biotreated and untreated chips were analyzed to determine if the biotreated chips were more susceptible to the hydrolysis process. The mass of the non-volatile solids in the extract was measured along with a sugars analysis of the extract. The pH of the extract was also monitored to determine if there was a difference between the extracts of the biotreated and untreated chips. For the hydrolysis process, it becomes important to consider both the role played by the increase in the openness of the structure allowing for diffusion, and the role played by the increase in the pH of the biotreated chips in the hydrolysis of intermolecular bonds.

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Chapter 2: Literature Review

The structure of wood is primarily composed of elongated cells mostly aligned in the direction parallel to the stem. Trees grow in height through the buds at the ends of stems and branches; this growth is called primary growth. Secondary growth increases the girth or diameter of the stems and takes place in the cambial zone through the division of living cells. The new cells first enclose themselves in the primary wall composed of cellulose, hemicellulose, pectin, and protein. Once the cell has reached its full size, the formation of the secondary wall is initiated and it is composed of cellulose and hemicellulose. The lignin starts to form before the secondary wall is completed [1]. When done, the secondary wall will be composed of cellulose, hemicellulose, and lignin.

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The wood cells are commonly referred to as fibers. Figure 2-1 shows the components of the initial living cell and the locations of the layers within the fiber.

Figure 2-1: Wood fiber schematic. This schematic depicts the makeup of both the living and dead cell. [1]

Between the fibers is the middle lamella, a region mostly composed of lignin. This region serves to hold the fibers together and give strength to the wood structure. Figure 2-2 is a transmission electron micrograph of wood showing the layers of the fiber and the middle lamella that joins them.

Figure 2-2: Transmission electron micrograph of softwood tracheids showing: middle lamella (M), primary wall (P), and outer (S 1 ), middle (S 2 ), and inner (S 3 ) secondary wall layers [2].

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The general composition of wood is: cellulose (40-50%), hemicelluloses (25- 30%) and Lignin (20-30%). Sugar Maple (Acer saccharum) was chosen as the wood species of study for this report due to its high rate of occurrence in the forests of the Northeast United States, which are 77% hardwoods [3]. The envisioned biorefineries will be regional, relying on plant material that is local to minimize cost associated with transportation to meet efficiency requirements; therefore sugar maple is a logical choice. Sugar maple wood has a composition of 2.5% extractives, 25.2% lignin, 40.7% cellulose, 3.7% glucomannan, 23.6% glucuronoxylan, 3.5% other polysaccharides, and 0.8% residuals [1], though there can be variations in results due to compositional differences with a wood sample and different methods of analysis. 2.1 Cellulose Cellulose is a homogeneous linear polymer of glucose units, which are linked together by β (1-4) glycosidic bonds. The degree of polymerization (DP) is between 7,000 and 15,000 for plant cellulose [4]. Cellulose forms inter- and intra-molecular hydrogen bonds, leading to the assembly of molecular bundles called microfibrils. The microfibrils have both highly ordered crystalline domains and disordered amorphous regions and are insoluble in most solvents. 2.2 Lignin Lignin is a polymeric structure resulting from the dehydrogenative polymerization of three primary precursors: coniferyl alcohol, sinapyl alcohol, and coumaryl alcohol (see Figure 2-3 ). The lignin present in softwood is composed almost entirely of coniferyl alcohol, while that in hardwood is composed of both coniferyl, and sinapyl alcohols [2].

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Figure 2-3: Lignin precursors: (1) coumaryl alcohol, (2) coniferyl alcohol, (3) sinapyl alcohol [1].

The phenylpropane units that make up lignin can be combined by a variety of linkages (see Figure 2-4 and Table 2-1 ). The different linkages in the lignin result in a three-dimensional structure. The ether linkages are the most common in hardwood and softwood milled wood lignin (MWL) while softwoods also have a high amount of phenolic linkages.

Figure 2-4: Most common linkages between phenylpropane units in lignin [1].

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Table 2-1: Common lignin linkages and their occurrences in milled wood lignin (compiled from [1]). Spruce Birch A Arylglycerol- β -aryl ether 48 60 B Glyceraldehyde-2-aryl ether 2 2 C Noncyclic benzyl aryl ether 6-8 6-8 D Phenylcoumaran 9-12 6 E Structures condensed in 2- or 6- positions 2.5-3 1.5-2.5 F Biphenyl 9.5-11 4.5 G Diaryl ether 3.5-4 6.5 H 1,2-Diarylpropane 7 7 I β , β -linked structures 2 3 Bond Type Percentage

The difficulty in separation of lignin from the carbohydrates in wood has led to a heavy debate in the scientific community over the possibility of lignin-carbohydrate covalent bonding. One belief is that there is no bond between the lignin and the carbohydrate polymers, instead the polymers are immobilized within the three- dimensional structure of the lignin [5]. More current research has lead to the conclusion that the lignin is chemically bound to the carbohydrates in what are termed lignin- carbohydrate complexes (LCC) [5, 6]. Four structures have been proposed for the possible LCC linkages: benzyl ether ( Figure 2-5 ), benzyl ester ( Figure 2-6 ), glycoside ( Figure 2-7 ), and acetal ( Figure 2-8 ), with the first two being the most prevalent [5].

Figure 2-5: Proposed benzyl ether bond between lignin and hemicellulose ([5]).

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Figure 2-6: Proposed benzyl ester bond between lignin and hemicellulose ([5]).

Figure 2-7: Proposed glycoside bond between lignin and hemicellulose ([5]).

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Figure 2-8: Proposed acetal bond between lignin and hemicellulose ([5]).

2.3 Hemicelluloses 2.3.1 Properties Hemicelluloses are a group of branched polymers that are heterogeneous in nature and can be composed of multiple five carbon sugars (xylose, arabinose) and six-carbon sugars (glucose, mannose, rhamnose, and galactose). The hemicelluloses have a degree of polymerization of 100-200, which is much less than that of cellulose [7-10]. Hemicelluloses give strength to the wood structure by helping to bind cellulose, a linear polymer, to the lignin. It has also been found that there is a portion of the hemicelluloses that is in close association with the cellulose. This fraction makes up around 20% of the hemicellulose and can be found immobilized within or partially within the crystalline region of cellulose in the microfibrils [11].

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Glucuronoxylan, more simply referred to as xylan, is the predominate hemicellulose found in hardwoods. The xylan content of sugar maple has been determined by multiple authors with some variation in results. Each of the methods, first, hydrolyzes the hemicelluloses to sugars, then, determines the amount of xylan through quantification of xylose. Timell [12] reports the xylan content of extractive free wood as xylose 16.9%, methylglucuronoxylan (de-acetylated xylan) 18.2% and O-acetyl- methylglucuronoxylan (xylan) 21.1%. A xylose concentration of 17.4% of the raw wood was calculated based on the un-substituted xylan value reported by Kaar et al. [13]. The value of 23.6% for native xylan reported by Sjöström [1] corresponds to 18.9% xylose using the same ratio as the previously cited Timell data. In the northeast U.S., sugar maple is one of the dominant hardwood species, and it can seen from the above reported composition of sugar maple that xylan makes up the majority of the hemicellulose. Along with maple, beech and birch are the other significant species used for pulping in the region, and these species are also high in xylan content at 27- 29% xylan [1]. Because xylan makes up the overwhelming majority of the hemicellulose in hardwoods, the focus in this research will be on the xylan portion of the hemicellulose. The xylose molecules in the backbone of the xylan are linked by β (1-4) bonds. Residues on the backbone include acetyl groups at the C-2 or C-3 position with a substitution rate of 7 per 10 xylose units. Additionally, (1-2)-linked 4-O-methyl-α-D- glucuronic acid groups at the C-2 position occur at an average rate of 1 per 10 xylose units [1]. Figure 2-9 shows the structure of hardwood xylan with associated side groups.

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Figure 2-9: Structure of O-acetyl-4-O-methylglucuronoxylan (xylan) from hardwood [14]

The determination of the molecular weight of xylan is a complicated problem due to the different structures of the non-homogeneous polymer. Branching, different degrees of substitution of the possible side group, aggregation, and the alterations that occur during the extraction process affect the shape that the xylan will take in a solvent. The differences in shape changes the hydrodynamic volume of xylan of a given molecular weight, making molecular weight determination a complicated process when using SEC methods [15]. The apparent molecular weight of xylan will be extremely high when using pullulan as a standard [16] due in part to the branched nature of the xylan and higher flexibility of pullulan. There is also an observed tendency of the xylan to form aggregates [16, 17], which can easily give an erroneously high number for molecular weight. Table 2-2 gives the molecular weight of some common sources of xylan, which range from 31,500 to 76,000.

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Table 2-2: Reported molecular weights of xylan from various sources. Species Mw Mn [Dp] Analysis Method Source Sugar Maple (Acer saccharum)

(51,000)

(Osmometry) [9]

[190] [Osmometry] [7] White Birch (Betula papyrifera) 76,000 (51,000)

Light scattering (osmometry) [9] Yellow Birch 75,000 (46000)

Light scattering (osmometry) [9] White Elm 70,000 (46000)

Light scattering (osmometry) [9] Trembling Aspen (Populus tremuloides) 31,500

[200] Osmometry [10]

2.3.2 Uses The end uses of the xylan, along with the desired use of the remaining wood components, are important factors in determining the process that will be used during extraction of the hemicellulose. The HMW polymers show potential for use in green plastics [18] and hydrogels. Xylo-oligomers can be further degraded to xylose or used as a food source. The xylose sugars can be fermented to produce ethanol or xylitol and as a feed to produce other green plastics such as polyesters and polyhydroxyalkanoates [19- 24]. Ethanol is a product that has currently received renewed interest as a potential way of decreasing dependence on foreign oil and being more environmentally friendly, which drives much of the work towards hemicellulose saccarification through hydrolysis or enzyme systems. The sugars produced can then be fermented to ethanol using a variety of different bacteria, fungi, or yeasts [25, 26]. HMW polymers from hemicellulose can be blended with other polymers [27-29], which can be cellulosic or petroleum derived. The blended polymers can often retain much of the physical and mechanical properties of the pure polymer, and can reduce the use of petroleum based polymers while helping to produce biodegradable products.

Full document contains 147 pages
Abstract: The effect that biotreatment with Ceriporiopsis subvermispora has on sugar maple (Acer saccharum ) chips, and its ability to enhance the extraction of hemicellulose was investigated. The effect of the biotreatment on the chip was analyzed to assess selectivity towards the removal of the non-carbohydrate mass as well as the physical and chemical changes in the chips, which can affect the removal of hemicelluloses. The loss of lignin from the chips increased with the loss of mass during biotreatment and represented the majority of the mass loss. Klason lignin losses were 12%, 29.8%, and 41.1% in biotreated chips with total mass losses of 3.8%, 10.6%, and 18.6%. Images taken with a scanning electron microscope show degradation and erosion of the cell walls, which result in a more open structure. The opening of the structure was confirmed by analysis of the mass of water in a given volume of saturated biotreated chips. The pH of water extract of the chips was also seen to decrease after the biotreatment process dropping from 5.29 for untreated wood to 3.26, and 3.23 for biotreated chips with mass losses of 10.6% and 18.6% respectively. The benefits of biotreatment on the alkali extraction or hydrolysis of hemicellulose was then evaluated. The hemicelluloses that can be extracted with 25% KOH from the maple wood chips increased with even moderate biotreatment. Xylan is the primary hemicellulose found in hardwood and was measured as 19.4% of the mass of the untreated wood. The mass of xylan that was extracted increased from 2.9% for untreated chips to 6.7%, 7.1%, and 7.6% of the raw wood mass, for biotreated wood with mass losses of 3.8%, 10.6% and 18.6%, respectively. Autohydrolysis of the maple chips with temperatures in the range of 140ºC to 180ºC was used to extract hemicellulose from the maple chips. The total sugars and the xylose in the extract correlated well to the total non-volatile solids content of the extract for both biotreated and untreated chips at all temperatures and times used for the experiments. From these correlations a predicted maximum xylose content equivalent to 10.6% of the mass of the raw wood can be removed and retained in the extract. The rate of removal from the biotreated chips was found to be twice as fast as from the untreated chips at each of the temperatures used. Key words . Ceriporiopsis subvermispora , white-rot fungi, bio-delignification, sugar maple, hemicellulose, xylan, carbohydrate, alkali extraction, autohydrolysis