Study on the Structure and Properties of Xylan Extracted from Eucalyptus, Sugarcane Bagasse and Sugarcane Straw

LICENTIATE THESIS IN FIBRE AND POLYMER SCIENCE STOCKHOLM, SWEDEN 2015

Study on the structure and properties of xylan extracted from eucalyptus, sugarcane bagasse and sugarcane straw

DANILA MORAIS DE CARVALHO

Eucalyptus

xylan

Sugarcane bagasse xylan

Sugarcane straw xylan

KTH ROYAL INSTITUTE OF TECHNOLOGY SCHOOL OF CHEMICAL SCIENCE AND ENGINEERING

Study on the structure and properties of xylan extracted from eucalyptus, sugarcane bagasse and sugarcane straw

DANILA MORAIS DE CARVALHO

Licentiate Thesis No. 59, 2015 KTH Royal Institute of Technology Fibre and Polymer Science Department of Fibre and Polymer Technology SE-100 44 Stockholm, Sweden

TRITA-CHE Report 2015:59 ISSN 1654-1081 ISBN 978-91-7595-718-0

Akademisk avhandling som med tillstånd av KTH i Stockholm framlägges till offentlig granskning för avläggande av teknisk licentiatexamen fredagen den 13 november kl. 10:00 i Rånbyrummet, Teknikringen 56, Stockholm.

The paper was reprinted with permission of © Elsevier 2015

Abstract

Lignocellulosic biomasses are an important source of chemical components such as cellulose, lignin and hemicelluloses, and can be used for a variety of purposes in both the pulp and paper and chemical conversion industries. Xylan, the main hemicellulose found in hardwood and grass plants, plays an important role during the pulping/pretreatment process reactions, including those used in 2nd generation bioethanol production. It may also play an important role in the production of certain novel materials.

This thesis evaluates the composition of eucalyptus (Eucalyptus urophylla x Eucalyptus grandis), sugarcane bagasse and sugarcane straw, with a specific focus on the structure and properties of xylan. The chemical characterization of biomasses showed that sugarcane bagasse and straw contain larger amounts of extractives, ash and silica than eucalyptus. The large amount of silica leads to an overestimation of the Klason lignin content, if not corrected. By using a complete mass balance approach, sugarcane bagasse and straw were shown to contain smaller amounts of lignin (18.0% and 13.9%, respectively) than previously reported for these raw materials, and certainly a much smaller amount of lignin than was found in eucalyptus (27.4%). The hemicellulose content in sugarcane bagasse (28.7%) and straw (29.8%) was much higher than that in eucalyptus (20.3%).

In order to investigate the structure of the xylan in greater detail, it was extracted with dimethyl sulfoxide from holocellulose, obtained by either peracetic acid or sodium chlorite delignification. The structure of the isolated xylans was confirmed by FTIR and 1H NMR analysis. In eucalyptus, the O-acetyl-(4-O-methylglucurono)xylan (MGX) was identified. This had a molar ratio of xylose units to branches of 4-O-methylglucuronic acid of 10:1.1 and a degree of acetylation of 0.39. All 4-O- methylglucuronic acid groups were attached to position O-2 of the xylose units, which had an acetyl group in position O-3. The acetyl groups were distributed in positions O-3 (64%), O-2 (26%) and O-2,3 (10%). The MGX had a molecular weight (Mw) of about 42 kDa.

In bagasse and straw, arabinoxylan (AX) was identified. This had a molar ratio of xylose units to arabinosyl substitutions of 10:0.5 for bagasse and 10:0.6 for straw. A degree of acetylation was 0.29 and 0.08 for bagasse and straw, respectively. The arabinose units were attached preferentially to position O-3 in AX. In the xylan from bagasse, the acetyl groups were found in positions O-3 (60%), O-2 (13%) and O-2,3 (27%), while in the xylan from straw, the acetyl groups were distributed between positions O-3 (67%) and O-2 (33%).

The AX had a molecular weight (Mw) of about 38 kDa and 30 kDa for bagasse and straw, respectively.

The differences in the structure of xylan present in the various biomasses played an important role during hydrothermal pretreatment, which is often used as the first step in 2nd generation ethanol production. The varying amounts of uronic acid and acetyl groups resulted in different starting pH levels of liquor and, thus, affected the chemical transformation in the biomasses in different ways. The hydrothermal pretreatment resulted mostly in the removal and/or transformation of hemicelluloses, but also in the formation of a significant number of pseudo-lignin structures. In addition, in eucalyptus, pseudo-extractives structures were generated. The sugarcane straw showed the highest mass loss during the investigated pretreatment.

Sammanfattning

Lignocellulosa i biomassa är en viktig källa till kemiska komponenter som cellulosa, lignin, och hemicellulosa och har stora användningsområden inom både kemikalie- samt massa och papperindustrin. Xylan, den huvudsakliga hemicellulosan i lövträd och gräsväxter, spelar en viktig roll under kok- och förbehandlingsprocesser, samt de processer som används vid produktion av 2:a generationens bioetanol. Xylan kan också spela en viktig roll för produktionen av nya material.

I denna avhandling utvärderas sammansättningen av eukalyptus (Eucalyptus urophylla x Eucalyptus grandis), sockerrörsbagass, och sockerrörsstrå med fokus på xylans struktur och egenskaper. Den kemiska karaktäriseringen av biomassan visade att sockerrörs bagass och strå innehåller större mängder extraktivämnen, aska och kisel än eukalyptus. Om inte hänsyn tas till kisel leder den högre halten till att mängden klasonlignin överskattas. Genom att använda en fullständig massbalans visades att sockerrörs-bagass och -strå innehåller lägre halter av lignin (18,0% för bagass och 13,9% för strå) än vad tidigare rapporterats för dessa råmaterial, och definitivt en mycket lägre halt lignin än vad som förekommer i eukalyptus (27,4%). Andelen hemicellulosa i sockerrörs bagass och (28,7%) och strå (29,8%) var mycket högre än i eukalyptus (20,3%).

För att mer detaljerat undersöka strukturen hos xylan extraherades xylanet ur holocellulosa med dimetylsulfoxid. Holocellulosan hade erhållits genom delignifiering med antingen perättiksyra eller natriumklorit. De isolerade xylanes struktur bekräftades med FTIR och 1H NMR. I eukalyptus identifierades O-acetyl-(4-O-metylglukurono)xylan (MGX). Molförhållandet av xylosenheter till 4-O-metylglukuronsyra var 10:1,1 och acetyleringsgraden var 0,39. Alla 4-O-metylglukuronsyra grupper var bundna till position O-2 i xylosenheterna, som hade en acetylgrupp på position O-3. Acetylgrupperna var fördelade på position O-3 (64%), O-2 (26%) och O-2,3 (10%). MGX hade en molekylvikt

(Mw) på ca 42 kDa.

I bagass och strå identifierades arabinoxylan (AX). Molförhållande av xylosenheter till arabinosyl substitutioner var 10:0,5 för bagass och 10:0,6 för strå. Acetyleringsgraden var 0,29 för bagass och 0,08 för strå. I AX var arabinosenheterna främst bundna till position O-3. I xylan från bagass hittades acetylgrupper på positionerna O-3 (60%), O-2 (13%) och O-2,3 (27%). I strå var acetylgrupperna fördelade på O-3 (67%) och O-2 (33%). AX hade en molekylvikt (Mw) på ca 38 kDa för bagass och 30 kDa för strå.

Skillnaderna i de identifierade xylanstrukturerna från de olika biomassor spelade en betydande roll för den hydrotermiska förbehandlingen som ofta används som ett första steg vid produktion av 2:a generationens bioetanol. De olika halterna uronsyra- och acetylgrupper gjorde att det ursprungliga pH värdet i processvätskan varierade vilket, sin tur påverkade den kemiska omvandlingen av biomassan på olika sätt. Det var främst hemicellulosor som frigjordes och förändrades under den hydrotermiska förbehandlingen, vilket följdes av att en betydande mängd pseudo-lignin strukturer bildades. Dessutom bildades pseudo-strukturer av extraktivämnen för eukalyptus. Under den studerade förbehandlingen var massförlusten högst för sockerrörsstrå.

List of appended papers

Paper I

Danila Morais de Carvalho, Olena Sevastyanova, Lais Souza Penna, Brunela Pereira da Silva, Mikael E. Lindström and Jorge Luiz Colodette (2015). Assessment of chemical transformations in eucalyptus, sugarcane bagasse and straw during hydrothermal, dilute acid, and alkaline pretreatments, Industrial Crops and Products, 73, 118-126.

Paper II

Danila Morais de Carvalho, Antonio Martinez Abad, Jorge Luiz Colodette, Mikael E. Lindström, Francisco Vilaplana and Olena Sevastyanova (2015). Comparative characterization of acetylated heteroxylan from eucalyptus, sugarcane bagasse and sugarcane straw (Manuscript).

The results of this work were also presented at the following scientific meetings:

1. Isolation and characterization of acetylated xylan from sugarcane bagasse. Danila Morais de Carvalho, Antonio Martinez Abad, Francisco Vilaplana, Mikael E. Lindström, Jorge Luiz Colodette, Olena Sevastyanova. Cost Action FP1105 (WoodCellNet) workshop, 2015, San Sebastian, Spain (Poster presentation).

2. Chemical characterization and evaluation of various pretreatments methods to the bioconversion of eucalyptus into bioethanol. Danila Morais de Carvalho, Olena Sevastyanova, Mikael E. Lindström, Jorge Luiz Colodette. 7th International Colloquium on Eucalyptus Pulp (ICEP), 2015, Vitória, Brazil (Poster presentation).

3. Chemical transformations in eucalyptus, sugarcane bagasse and sugarcane straw during hydrothermal, acid and alkaline pretreatments. Danila Morais de Carvalho, Olena Sevastyanova, Lais Souza Penna, Brunela Pereira da Silva, Mikael E. Lindström, Jorge Luiz Colodette. 18th International Symposium on Wood, Fibre and Pulping Chemistry (ISWFPC), 2015, Vienna, Austria (Oral presentation).

4. Comparative study on the structure of acetylated xylans from eucalyptus and sugarcane bagasse and straw. Danila Morais de Carvalho, Francisco Vilaplana, Antonio Martinez Abad, Jorge Luiz Colodette, Mikael E. Lindström, Olena Sevastyanova. 18th International Symposium on Wood, Fibre and Pulping Chemistry (ISWFPC), 2015, Vienna, Austria (Poster presentation).

List of Abbreviations

1H NMR Proton nuclear magnetic resonance spectroscopy Ara or Araf Arabinose or arabinofuranose ATR Attenuated total reflectance AX Arabinoxylan D Polydispersity index

D2O Deuterium oxide or heavy water DA Degree of acetylation DMSO Dimethyl sulfoxide EDTA Ethylenediamine tetracetic acid FTIR Fourier transform infrared spectrometry G (lignin) Guaiacyl lignin GalA Galacturonic acid Gal or Galp Galactose or galactopyranose GC-MS Gas chromatography-mass spectrometry Glc or Glcp Glucose or glucopyranose H (lignin) p-hydroxyphenyl lignin HDO Heavy water HMF Hydroxymethyl furfural HPLC High-performance liquid chromatography IC Ion chromatography LiBr/DMSO Solution of lithium bromide in dimethyl sulfoxide Man or Manp Mannose or mannopyranose MeGlcA 4-O-methylglucuronic acid

Mw Molecular weight MGX O-acetyl-(4-O-methylglucurono)xylan

Mn Molecular number

NaClO2 Sodium chlorite NaOH Sodium hydroxide

NaClO2/DMSO Delignification with NaClO2 followed by xylan isolation with DMSO PAA Peracetic acid PAA/DMSO Delignification with PAA followed by xylan isolation with DMSO PMAAs Permethylated alditol acetates Rha or Rhaf Rhamnose or rhamnofuranose S (lignin) Syringyl lignin SEC Size-exclusion chromatography SEM Scanning electron microscopy S/G ratio Syringyl/guaiacyl ratio of lignin UV (spectroscopy) Ultraviolet (spectroscopy) Xyl or Xylp Xylopyranose

List of Technical Terms

Bagasse (sugarcane) Lignocellulosic biomass remained after juice removal from sugarcane stalks. Pseudo-extractives Structures formed mainly from degradation products from xylan during acidic and alkaline pretreatments and quantified together with extractives due to similar solubility in neutral organic solvents. Pseudo-lignin Structures formed mainly from degradation products from xylan during acidic pretreatments and quantified together with Klason lignin. Straw (sugarcane) The term straw for sugarcane describes a different tissue in the plant from that usually used for other lignocellulosic biomasses, such as corn, rice or wheat. Sugarcane straw is the lignocellulosic biomass formed by sugarcane leaves and stalk tips.

Contents

1. Introduction ...... 1 1.1 Objectives ...... 7 2. Experimental ...... 8 2.1 Working plan ...... 8 2.2 Raw materials and chemicals ...... 9 2.3 Isolation of acetylated xylan samples ...... 9 2.3.1 PAA delignification ...... 9

2.3.2 NaClO2 delignification ...... 10 2.3.3 DMSO extraction ...... 10 2.4 Hydrothermal pretreatment ...... 10 2.5 Chemical characterization of biomasses ...... 11 2.5.1 Ash, silica and total extractives contents ...... 11 2.5.2 Lignin and sugar content ...... 11 2.5.3 Mass balance calculation for biomasses ...... 12 2.6 Chemical and structural characterization of xylan samples ...... 12 2.6.1 Delignification yields and xylan isolation steps ...... 12 2.6.2 Chemical composition of xylan samples ...... 12 2.6.3 Linkage analysis ...... 12 2.6.4 Size-exclusion chromatography (SEC) ...... 13 2.6.5 Acetyl content and degree of acetylation ...... 13 2.6.6 1H nuclear magnetic resonance spectroscopy (1H NMR) ...... 14 2.6.7 Fourier transform infrared spectrometry (FTIR) ...... 14 2.6.8 Scanning electron microscopy (SEM) ...... 14 3. Results and Discussion ...... 15 3.1 Chemical characterization of biomasses (Paper I) ...... 15 3.1.1 Eucalyptus ...... 15 3.1.2 Sugarcane bagasse and straw ...... 16 3.1.3 The influence of silica content on Klason lignin quantification ...... 17 3.1.4 Complete mass balance: from extractives-free sawdust to whole biomass ...... 18 3.2 Chemical and structural characterization of acetylated xylan (Paper II) ...... 20 3.2.1 Preparation of holocellulose samples ...... 20 3.2.2 Extraction of xylan ...... 25 3.2.3 Chemical and structural characterization of xylan samples ...... 27

3.2.3.1 Structure and composition of xylan ...... 27 3.2.3.2 Molecular weight of xylan samples ...... 30 3.2.3.3 Acetyl groups in xylan ...... 31 3.2.3.4 Linkage analysis after acid hydrolysis of xylan samples ...... 32 3.2.3.5 Characterization of xylan structure by 1H NMR method ...... 34 3.2.3.6 Empirical structure of xylan from eucalyptus, bagasse and straw ...... 37 3.3 Role of xylan during the hydrothermal pretreatment (Paper I) ...... 39 4. Conclusions ...... 44 5. Appendix ...... 45 6. Acknowledgments ...... 46 7. References ...... 47

1. Introduction

The development of new technologies for the production of more advanced materials, fuels and chemicals from renewable resources, such as wood, agricultural crops and residues, has increased substantially throughout the world over the past several years (Alekhina et al., 2014; Egüés et al., 2014; Fall et al., 2014; Cardoso et al., 2013; Lundberg et al., 2013; Zhang et al., 2013; Çetinkol et al., 2012; Zhang et al., 2012; Prakash et al., 2011). Over the next few years, it is expected that plant biomasses will be used strategically as renewable resources in various novel and integrated industries. According to Ragauskas et al. (2006), political support for investments in these new industries is as important as the development of methods to increase efficiency and sustainability in chemical conversion processes. Sugarcane is one of Brazil’s main agricultural crops. Juice (rich in sucrose) is extracted from the sugarcane and used to produce sugar, technical ethanol and, to a lesser extent, beverages and candies. During the 2014/15 harvest in Brazil, an estimated 659 million tons of sugarcane was cultivated over 9.1 million ha of land, with an average productivity of 72 tons/ha (Conab, 2014). In addition to food and fuel production, a large amount of lignocellulosic residues are inevitably generated by the sugarcane industry - mainly bagasse (sugarcane stalks remaining after juice extraction) and straw (sugarcane leaves and stalk tips) (Figure 1). After being processed, approximately 140 kg of bagasse and 140 kg of straw is generated (on a dry weight basis) from each ton of sugarcane (Oliveira et al., 2013). Based on the above estimates, this means that approximately 92 million tons of bagasse and 92 million tons of straw were generated during the 2014/15 harvest. Eucalyptus is an important hardwood specie and is cultivated around the world. Indeed, it is the most cultivated wood specie in Brazil, where it is used for diverse industrial purposes (Gonzáles-García et al., 2012). The large number of lignocellulosic biomasses, both from the main culture and its residues, opens up new possibilities for the biofuel and the advanced materials industries. However, a correct assessment of their chemical composition is crucial. In lignocellulosic biomasses, the main components are ash, extractives, cellulose, hemicelluloses and lignin (Figure 2).

Figure 1. Schematic representation of a sugarcane plant and the main lignocellulosic residues generated from it: bagasse and straw.

The ash components present in lignocellulosic biomasses are all inorganic compounds that are usually absorbed directly from the soil. Calcium, potassium, magnesium, manganese, iron and copper are the main inorganic compounds found in biomasses. In addition, for some specific biomasses, such as grass plants, the silica components have important physiological functions as a growth promoter and as self-protection against herbivory. Silica might also replace carbon-based support in the intercellular spaces of grass stalks (McNaughton et al., 1985). Extractives are a generic name for a large group of organic components, normally of relatively low molecular weight, that assumes different functions in the plant, such as colour, smell, chemical protection, nutritive reserve and taste, among others. Cellulose, hemicelluloses and lignin, which are all cell wall components, are the main components from lignocellulosic biomasses for chemical conversion purposes. Cellulose is formed by a linear chain of β-(1→4)-D-glucopyranose units. Although the cellulose differs in various biomasses in terms of their degree of polymerization and percentage of crystalline and amorphous parts, from a chemical point of view it is quite similar in all higher plants. The hemicelluloses are polysaccharide compounds which chain contains different sugars: D-glucose, D-xylose, L-arabinose, D-galactose, D-mannose, D-glucuronic acid, 4-O- methyl-α-D-glucuronic acid, D-galacturonic acid, traces of L-rhamnose, L-fucose and some O-methyled neutral sugars. Acetyl groups can also be found (Bian et al., 2010). The main

2 hemicellulose in grasses, such as sugarcane, and in hardwood, such as eucalyptus, is xylan, which is formed by a backbone of β-(1→4)-D-xylopyranose units, with the possibility of substitution in C-2 and/or C-3, in addition to acetyl groups. Normally, the substitutions in xylans are arabinofuranose (Araf) and 4-O-methylglucuronic acid (MeGlcA) in grasses and hardwood, respectively. Other substitutions in xylan have also been detected in some cases, but more rarely (Magaton et al., 2008; Ebringerová et al., 2005; Evtuguin et al., 2003).

Figure 2. Chemical components in lignocellulosic biomasses.

The exact structure of lignin is still under debate, but, in general, lignin is formed by p- hydroxyphenyl (H) (non-methylated phenolic ring), guaiacyl (G) (methyl in position 3 in phenolic ring) and syringyl (S) (methyl in positions 3 and 5 in phenolic ring) units. One positive effect of a higher S/G lignin ratio is that it prevents the formation of condensed structures of lignin during chemical processing (Brandt et al., 2013; Santos et al., 2011). Also, a higher S/G ratio has proven to be beneficial during the various pretreatments used for the chemical conversion of biomasses. The chemical composition and percentage of each component present in biomasses varies significantly between plants (eucalyptus vs sugarcane), between parts of the same plant (leaves vs stalks) and even between parts of the same tissue (sapwood vs heartwood).

Sugarcane bagasse typically contains 5-7% extractives, 1-3% ash, 39-45% cellulose, 23- 27% hemicelluloses and 19-32% lignin (Canilha et al., 2011; Rabelo et al., 2011; Rocha et al., 2011; da Silva et al., 2010). In straw, the typical chemical composition is: 5-17% extractives, 2-12% ash, 33-45% cellulose, 18-30% hemicelluloses and 17-41% lignin (Santos et al., 2014; Costa et al., 2013, da Silva et al, 2010; Saad et al., 2008). The main xylan found in bagasse and straw is the arabinoxylan (AX), with substitutions of arabinose and slightly acetylation in the backbone of xylose (Ebringerová et al., 2005). Lignin in bagasse and straw differs from those in hardwood due the presence of H lignin in addition to G and S (Brandt et al., 2013). In general, eucalyptus from tropical areas contains small amounts of extractives (2-5%) and ash (0.1-0.2%), which results in more than 90% of its chemical composition being formed by cellulose (46-49%), hemicelluloses (18-32%) and lignin (29-33%) (Pereira et al., 2013; Zanuncio et al., 2013). The xylan in eucalyptus has branches of methylglucurono acids in addition to acetyl groups in the backbone of xylose (Magaton et al., 2008; Ebringerová et al., 2005; Evtuguin et al., 2003). In eucalyptus, the lignin is formed by G and S lignin units (Brandt et al., 2013). The use of lignocellulosic biomasses in the production of biofuels and advanced materials requires conversion processes during which the biomass is first chemically treated to remove undesirable chemical components and decrease its recalcitrance in the pretreatments stages. Diluted acid pretreatment is considered the most promising process for the treatment of biomass, especially when the goal is the release of sugars for enzymatic hydrolysis (Pu et al., 2013; Yang and Wyman, 2008; Chandra et al., 2007). During diluted acid pretreatment, chemical transformations in the biomass occur due to a combined action of acid pH, pressure, residence time (1 min to 1 h) and temperature (150-200ºC) (Pu et al., 2013; Saha et al., 2005), and a combination of high temperature and short residence time or low temperature and long residence time is usually used (Alvira et al., 2010). Sulfuric acid is typically used in acid pretreatments. Hydrothermal pretreatment, which is also called autohydrolysis or hot water pretreatment, is a variation of the diluted acid treatment in which the only acid source is the organic acids from xylans. This process is milder than acid pretreatment, during which an external acid source is provided. Chemical reactions during hydrothermal pretreatment are catalyzed by the hydronium ion that is released by water, as well as by the acetyl and uronic acid groups released from xylan (Vegas et al., 2008; Garrote et al., 2007). From a chemical point of view, the reactions that occur during hydrothermal pretreatment are similar to those that occur in dilute acid pretreatment, but they proceed to a lesser extent. Although milder than acid pretreatment, the hydrothermal process has been considered to be a feasible pretreatment process for

4 lignocellulosic biomasses, as it promotes adequate chemical transformations in biomass, with low-cost reactor construction and low operational costs for both the consumption of chemicals and maintenance (due to acid corrosion). Moreover, it is an environmentally friendly process (Alvira et al., 2010; Garrote et al., 2007). The main effects of hydrothermal pretreatment are the increase in cellulose accessibility, which is achieved through the removal of hemicelluloses, and the swelling of the entire structure. In addition, degradation products from xylan can be formed and modified during the pretreatment, thereby generating condensed structures that are usually referred to as pseudo-lignin (Hu et al., 2012; Sannigrahi et al., 2011; Alvira et al., 2010; Lora and Wayman et al., 1978). The formation of pseudo-lignin from polysaccharides degradation has also been demonstrated recently, especially at a combination of high temperature, long residence time and high acid concentration (Sannigrahi et al., 2011). The presence of pseudo-lignin in pretreated biomasses inhibits the enzymes in enzymatic hydrolysis, for which milder hydrothermal conditions are recommended (Hu et al., 2012). In addition to avoiding pseudo-lignin formation, milder hydrothermal conditions also decrease the risk of generating degradation products from xylan, such as furfural and hydroxymethyl furfural (HMF), which are generated primarily in high severity conditions and which inhibit fermentative yeasts (Pu et al., 2013; Saha et al., 2005). The hemicelluloses play a crucial role during the hydrothermal process because of:

1) they are present in lignocellulosic biomasses in large amounts; 2) they are the chemical components that are removed from the biomass most substantially during hydrothermal pretreatment; 3) they contain organic acids that are released during pretreatment and that catalyze the chemical reactions during the hydrothermal process (since no additional chemical is provided); and 4) they are involved in the formation of inhibitors (pseudo-lignin, furfural and HMF).

In addition to their importance during the pretreatment process, hemicelluloses have a promising future themselves. Over the coming years, it is anticipated that there will be a decrease in the use of petroleum-based products, with renewable sources replacing fossil sources. The use of polysaccharides, including hemicelluloses, are considered versatile alternatives for the production of more environmentally friendly advanced materials, fuels and chemicals, and even foodstuffs (Svärd et al., 2015; Zhang et al., 2013; Ebringerová et al., 2005).

Assuming that different biomasses contain hemicelluloses that are chemically different from one another (i.e., different acid groups content, side groups, chain size, etc.), it is important to assess the chemical structure of these hemicelluloses correctly, considering their sugar composition, as well as their content of acetyl and uronic acid groups. This can provide useful guidance for researchers when determining the appropriate conditions for biomass pretreatments, especially the hydrothermal process. The biggest problem regarding the chemical characterization of biomasses is probably the use of different methodologies (and, therefore, different reporting methods) for the same biomass. There is, thus, an obvious need for the standardization of the methodologies employed for both the analysis and the reporting of results (Canilha et al., 2012). A more accurate protocol for the assessment of the chemical composition of biomasses could also provide a better evaluation of the chemical transformation of the biomasses during the pretreatments, by presenting the results of chemical composition using the same basis for both the raw materials and the pretreated biomasses. In addition, a more detailed evaluation of the chemical and structural features of hemicelluloses could provide useful information for researchers on their more feasible industrial uses. The main challenge, especially in the study of hemicelluloses, is to isolate the hemicelluloses in large quantities and intact (Evtuguin et al., 2003). For structural studies, the isolation is usually performed in holocellulose produced from extractive-free sawdust, for which the mixture ethanol:toluene 1:2 (v/v) is generally used (Sun et al., 2004; Shalatov et al., 1999). Holocellulose can be obtained through different delignification processes, although the most frequently used processes use peracetic acid (PAA) (Marques et al., 2010; Magaton, 2008; Evtuguin et al., 2003) and sodium chlorite (Bian et al., 2010; Magaton, 2008; Evtuguin et al., 2003; Shatalov et al., 1999; Hägglund et al, 1956). Dimethyl sulfoxide (DMSO) is typically used to isolate acetylated hemicelluloses from holocellulose, which has the advantage of preserving the acetyl ester compounds and glycosidic linkages, which is desirable for structural studies (Peng et al., 2012). However, DMSO is also suitable for isolating xylose and non-xylan polysaccharides (Hägglund et al, 1956). In xylan isolated by DMSO, no significant difference in the position of acetyl groups in the eucalyptus xylan backbone occurred during the delignification process, regardless of whether PAA or sodium chlorite was used (Evtuguin et al., 2003), which indicates that both chemicals are feasible for delignification. The growing interest in the use of renewable resources for the production of biofuels and advanced materials, together with the importance of hemicelluloses in the conversion processes of biomasses, were the chief motivations for this thesis.

1.1 Objectives

The main goals of this study were:

1) to propose a more accurate characterization protocol for the assessment of the chemical composition of eucalyptus, sugarcane bagasse and sugarcane straw biomasses, taking into consideration each biomass constituent, including the extractives and silica contents; 2) to determine the chemical structure of xylan extracted from eucalyptus, sugarcane bagasse and sugarcane straw; and 3) to assess the effect of the different chemical compositions of the biomasses, and especially the effects of xylan structure, on hydrothermal pretreatment.

It is hoped that the knowledge achieved in the present study can contribute to an improvement in chemical conversion technologies and to the development of advanced materials from lignocellulosic biomasses.

2. Experimental

2.1 Working plan

Figure 3 depicts the working plan. The chemical composition of eucalyptus, bagasse and straw (ash, extractives, lignin and carbohydrates) were investigated using classical methodologies. A new approach for the reporting of chemical composition, using the complete mass balance, was adopted (Carvalho et al., 2015). Xylan samples were obtained from eucalyptus, bagasse and straw and chemically and structurally characterized using the following methodologies: carbohydrates, methylation linkage analysis, size-exclusion chromatography (SEC), Fourier transform infrared spectrometry (FTIR), 1H nuclear magnetic resonance spectroscopy (1H NMR) and scanning electron microscopy (SEM). Hydrothermal pretreatment was performed on the biomasses, and the chemical composition of the pretreated eucalyptus, bagasse and straw were investigated using complete mass balance approach that combine the classical methodologies and the actual yield.

Figure 3. Working plan for chemical characterization of untreated and pretreated biomasses (Paper I) and chemical and structural characterization of acetylated xylan samples (Paper II).

2.2 Raw materials and chemicals

The raw materials used were eucalyptus, sugarcane bagasse and sugarcane straw. Chips of a 7-year old clonal hybrid of eucalyptus (Eucalyptus urophylla x Eucalyptus grandis) were supplied by a pulp mill whose eucalyptus plantation is located in Brazil. Small pieces (10 mm diameter) of 5-month old sugarcane bagasse (stalks after juice removal) and straw (leaves and tips) from the cultivar RB867515 were supplied by Ridesa Experimental Station (Viçosa, Minas Gerais state, Brazil). The materials were dried and then stored in airtight plastic bags at room temperature prior to use. All chemicals used were of analytical grades.

2.3 Isolation of acetylated xylan samples

The biomasses were converted into sawdust and submitted to extractives removal using ethanol/toluene 1:2 (v/v) for 12 h in a Soxhlet extractor (Sun et al., 2004; Shalatov et al., 1999). The moisture of the extractives-free sawdust was determined according to TAPPI T 264 cm-07.

2.3.1 PAA delignification

An EDTA pretreatment was performed on the bagasse and straw prior to PAA delignification in order to remove metal ions that can cause decomposition of PAA. Approximately 500 mL of 0.2% (w/v) EDTA was added to the extractives-free sawdust of bagasse and straw in an erlenmeyer flask and shaken at 90ºC for 1 h (Brienzo et al., 2009). The suspension was filtered through a porous glass filter P2 (porosity 100), washed with distillated water and used in the delignification. For the delignification, 500 mL of 10% PAA at pH 3.5 (adjusted with sodium hydroxide solution) was added to 10 g of extractives- free sawdust of bagasse and straw (pretreated with EDTA) and of eucalyptus in a erlenmeyer flask, covered with an inverted erlenmeyer flask and then shaken at 85ºC for 30 minutes with constant stirring in a well-ventilated fume hood. A reaction time of 40 minutes was also tested during the PAA process. After PAA delignification, the suspension was cooled in an ice bath and diluted twice with water. The delignified material was filtered, washed with 5 L of warm distilled water and with 50 mL of acetone/ethanol 1:1 (v/v) (adapted from Evtuguin et al., 2003). The holocellulose was dried at room temperature (24ºC) and stored in airtight containers.

2.3.2 NaClO2 delignification

A sample of 10 g of extractives-free sawdust was treated with 530 mL of buffered sodium chlorite solution: 388 mL water, 15 mL acetic acid 100%, 72 mL sodium acetate

30% (w/v) and 55 mL sodium chlorite 30% (w/v). NaClO2 delignification was performed in erlenmeyer flask, covered with an inverted erlenmeyer flask, at 75ºC for 30 minutes, with constant stirring in a well-ventilated fume hood. The addition of chemical reagents in three steps, with 30 min intervals between additions, was also tested during the NaClO2 process.

After NaClO2 delignification, the suspension was filtered through a polystyrene membrane (porosity 60 µm), washed with 5 L of distilled water and, soon after, with 100 mL of acetone (adapted from Magaton, 2008). The holocellulose was dried at room temperature (24ºC) and stored in airtight containers.

2.3.3 DMSO extraction

130 mL of DMSO was added to 6 g of holocellulose (PAA-holocellulose or NaClO2- holocellulose) in a plastic (polyethylene) bottle and shaken at 24ºC for 24 hours under nitrogen atmosphere (adapted from Hägglund et al., 1956). The solution was filtered through a polystyrene membrane (porosity 60 µm) with ~20 mL of distilled water. The supernatant was added to 600 mL of ethanol at pH 3.5 (adjusted with formic acid) (adapted from Magaton et al., 2008) in a plastic (polyethylene) bottle and the complete xylan precipitation occurred after 12 hours at 4ºC. The xylan samples were isolated by centrifugation (10 min at 45000 rpm) and then washed 5 times with methanol (adapted from Evtuguin et al., 2003). The xylan samples were dried in a desiccator at room temperature (24ºC) and then in a vacuum drier (5 h, 50ºC).

2.4 Hydrothermal pretreatment

Samples of eucalyptus, bagasse and straw (100 g each) were subjected to hydrothermal pretreatment (with water). The liquor:biomass ratio used was 2:1 for eucalyptus and 7:1 for bagasse and straw (on a dry weight basis). Hydrothermal pretreatment was performed in a Regmed reactor (2 L capacity) with constant agitation for 90 min until the set temperature (175ºC) was reached, and an additional 15 min for the pretreatment itself. Afterwards, the reactor was cooled and the pretreated biomass was washed with an excess of water and centrifuged at 800 rpm for 4 minutes. The pretreated biomass was dried for 24 h at 23 ± 1

ºC and 50 ± 2% relative humidity and then stored at room temperature in airtight containers.

2.5 Chemical characterization of biomasses

2.5.1 Ash, silica and total extractives contents

The biomasses were ground into 40/60 mesh sawdust using a Wiley mill bench model, dried (23 ± 1 ºC and 50 ± 2% relative humidity) and then used for chemical analysis. The moisture was determined according to TAPPI T 264 cm-07. The ash content of the biomasses was measured according to TAPPI 211 om-02. The silica content in the ash was measured according to TAPPI 244 cm-11 using hydrochloric acid. The total extractives content was measured according to TAPPI T 264 cm-07 using a sequential treatment with 1:2 ethanol/toluene (5 h), 95% ethanol (4 h) and hot water (1 h). The total extractives content was determined gravimetrically based on the dry matter loss during analysis.

2.5.2 Lignin and sugar content

The lignin content was determined as a sum of acid insoluble lignin (Klason lignin) and acid soluble lignin. The Klason lignin was determined according to Gomide and Demuner, (1986). The acid soluble lignin was determined by UV-spectroscopy at 215 and 280 nm wavelengths (Goldschimid, 1971). Neutral sugars were analyzed in acid hydrolysate from the Klason lignin analysis according to Wallis et al. (1996) by ion chromatography (IC). The neutral sugar content was reported as anhydrosugar. For uronic acid determination, 0.3 mL of acid hydrolysate from the Klason lignin analysis was treated with 0.3 mL of sodium chlorite/boric acid solution and then hydrolyzed with sulfuric acid. The 5-formyl-2-furoic acid generated was determined by UV spectroscopy at a 450 nm wavelength (Scott, 1979). Acetyl groups content was determined in 300 mg of extractives-free biomass after hydrolysis with oxalic acid at 120ºC for 80 minutes (Solar et al., 1987) by high performance liquid chromatography (HPLC).

2.5.3 Mass balance calculation for biomasses

A complete mass balance calculation was developed in order to report the chemical composition of the biomasses correctly. This new approach takes in into consideration the silica co-precipitation in the Klason lignin (acid-insoluble lignin) analysis, as well as the extractives content influence on the total mass balance. The detailed procedure can be found in Carvalho et al. 2015 (Paper I). The chemical composition of the pretreated biomasses was obtained by combining the complete mass balance and the actual yield after pretreatment (see Carvalho et al., 2015).

2.6 Chemical and structural characterization of xylan samples

2.6.1 Delignification yields and xylan isolation steps

The delignification yield (PAA and NaClO2) was measured gravimetrically based on the extractives-free biomass (on a dry basis) and the DMSO extraction yield was measured gravimetrically based on the PAA-holocellulose or NaClO2-holocellulose (on a dry basis). The xylan yield (estimated as xylose content) after the delignification step was calculated taking into consideration the amount of xylose in the extractives-free biomasses and in the holocellulose, combined with the delignification yield. The xylan yield (estimated as xylose content) after DMSO extraction was estimated based on the xylose content in the extractives-free biomasses and in xylan samples, combined with the DMSO isolation yield.

2.6.2 Chemical composition of xylan samples

The sugar content (neutral sugars) of the xylan samples was determined by sulfuric acid (72%) hydrolysis and subsequent high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), in triplicate. The content of uronic acids (glucuronic and galacturonic acid) was determined by methanolysis according to Appeldoorn et al. (2010) and Bertaud et al. (2002).

2.6.3 Linkage analysis

Xylan samples were activated and reduced according to Kim and Carpita (1992). Then the samples were further swelled according to Ciucianu and Kerek (1984) and hydrolyzed,

12 reduced and acetylated according to Albersheim et al. (1967). The obtained permethylated alditol acetates (PMAAs) were separated and analyzed by gas chromatography. The mass spectra of the fragments obtained from the permethylated alditol acetates were compared to those of the reference polysaccharide derivatives and to available data (Carpita and Shea, 1989). For eucalyptus samples the reduction of uronic acids groups was performed before activation. The quantification was based on the carbohydrate composition and effective carbon response of each compound, as detected by GC-MS.

2.6.4 Size-exclusion chromatography (SEC)

2 mg of the samples were dissolved in 0.5% LiBr/DMSO at 60ºC overnight. The solution was filtered and the molar mass distributions of the xylan extracts from eucalyptus, sugarcane bagasse and straw were analyzed by size-exclusion chromatography coupled to a refractive index detector. Pullulan standards of known molar masses were used for calibration.

2.6.5 Acetyl content and degree of acetylation

9 mg of the xylan samples were hydrolyzed with NaOH at 70ºC overnight. The acetyl groups content was determined from extracts using high-performance liquid chromatography (HPLC) with UV detection. The degree of acetylation (DA) was determined according to Eq. 1 (Xu et al., 2010) from the acetyl content in the xylan samples.

132 % (1) 100 1 %

where: DA is the degree of acetylation, % acetyl is the acetyl content determined by

-1 -1 analysis, Macetyl is the acetyl groups molecular weight (43 g mol ) and 132 g mol is xylose molecular weight.

2.6.6 1H nuclear magnetic resonance spectroscopy (1H NMR)

The xylan samples were dissolved in D2O and treated with xylanase in order to improve the dissolution. The 1H nuclear magnetic resonance (NMR) spectra (400 Hz) were recorded at room temperature on a Bruker Advance 400 Hz instrument by using the standard Bruker pulse program. The software MestReNova was used for the spectra evaluation.

2.6.7 Fourier transform infrared spectrometry (FTIR)

The holocellulose and xylan samples were studied by Fourier transform infrared spectrometry (FTIR). The spectra (wavelength 4000-600 cm-1) were recorded by a Perkin- Elmer Spectrum 2000 FTIR spectrometer (Waltham, MA, USA) equipped with an ATR system, Spectac MKII Golden Gate (Creecstone Ridge, GA, USA). The spectra were obtained from dry samples, which were subjected to 16 scans at a resolution of 4 cm-1 and an interval of 1 cm-1 at room temperature. The software Origin 9.1 was used for the spectra evaluation.

2.6.8 Scanning electron microscopy (SEM)

Scanning electron microscopy (SEM) images were obtained from holocellulose samples by using a JEOL JSM-5400 scanning microscope (JEOL Ltd., Japan).

3. Results and Discussion

3.1 Chemical characterization of biomasses (Paper I)

3.1.1 Eucalyptus

The chemical composition of eucalyptus, based on the complete mass balance, is set out in Figure 4. The results observed for ash and extractives confirmed those found in the literature: ash in the range of 0.1-0.3% and extractives in the range of 2.1-5% (using the same experimental methodology) (Pereira, et al., 2013; Zanuncio, et al., 2013; Alves et al., 2010). Together, ash and extractives accounted for 2.5% of dry eucalyptus, a relatively low value that has little influence on the mass balance.

Figure 4. Chemical composition of eucalyptus taking into consideration the complete mass balance.

Lignin (27.4%) and sugars (70.1%) accounted for the other 97.5% of chemical components in eucalyptus. In percentage, the glucose was the main chemical component in eucalyptus, representing almost 50% of dry eucalyptus and more than 70% of the total sugars content. The significant content of xylose, uronic acids and acetyl in the sugars content confirmed that xylan is the main hemicellulose in eucalyptus (Evtuguin et al., 2003; Shatalov et al., 1999).

3.1.2 Sugarcane bagasse and straw

Not surprisingly, high ash and extractives contents were observed in bagasse (Figure 5), similar to that reported in the literature (Andrade and Colodette, 2014; Alves et al., 2010), and significant higher than the values observed in eucalyptus wood. In addition, results indicated that silica accounted for 62% of the total ash content, in accordance with the results obtained by McNaughton et al. (1985), which suggested that the silica acts as a growth promotor in grasses and, moreover, may replace the carbon-based support in the intercellular spaces of grass stalks. Ash and extractives together accounted for 17.3% of dry bagasse.

Figure 5. Chemical composition of bagasse taking into consideration the complete mass balance.

The other 82.7% of the components present in bagasse corresponded to 18.0% lignin and 64.7% sugars. The results demonstrated that the cell wall in bagasse was less lignified than in eucalyptus, but with presence of G and H lignin units, which can increase the possibility of condensation reactions during chemical processes (Brandt et al., 2013). Glucose in bagasse accounted for only 36% of dry bagasse and 56% of the total sugars content. As is the case in eucalyptus, the results confirmed that xylan is the main hemicellulose in bagasse (Brienzo et al., 2009). However, the amount of xylan present in bagasse was almost twice the amount present in eucalyptus. Although chemically different from bagasse, the chemical composition of straw was certainly more similar to that of bagasse than to eucalyptus. Discrepancies in the chemical

16 composition between the two parts of the same plant are most likely due the different physiological function of each tissue. For example, the high silica content in grasses, which has been reported as functioning as self-protection against herbivory (McNaughton et al., 1985), differs from the physiological function of silica in stalks. The silica represented 73% of the total ash content in straw, and both ash and silica were found in significantly higher quantities in straw than in bagasse (Figure 6). Ash and extractives together accounted for 20.1% of dry straw. In the other 79.9% of the straw components, the total lignin accounted for only 13.9% of dry straw, with a probable presence of S, G and H lignin units (Brandt et al., 2013). The total sugars content accounted for 66% in straw (on a dry basis), of which 55% was glucose (36.3% on a dry straw basis).

Figure 6. Chemical composition of straw taking into consideration the complete mass balance.

3.1.3 The influence of silica content on Klason lignin quantification

Results presented in Figures 4, 5 and 6 indicated a higher content of silica in bagasse and, especially, in straw, than in eucalyptus. The large amounts of silica present in bagasse and straw are not soluble in acid and, as a result, can lead to the overestimation of Klason lignin by co-precipitating with acid insoluble lignin and the results of this process are presented in Figure 7. For both bagasse and straw, the correction of the overestimation in Klason lignin was very important. The Klason lignin contents of bagasse and straw were 19.5% and 14.0%,

17 respectively, which values were substantially lower than those found in the literature (Costa et al., 2013; Pitarelo, 2007), although in that case the silica content was not removed from the Klason lignin. This correction is also recommended for other biomasses, especially those rich in silica content, such as grass plants. In addition, recognizing the presence of silica in the acid- insoluble residue is important for estimating the correct mass balance. In eucalyptus, the overestimation in Klason lignin due to silica co-precipitation was negligible. The Klason lignin content of eucalyptus was 24.0%, which was similar to that found in the literature (Zanuncio et al., 2013; Santos et al., 2011).

Figure 7. The influence of silica content on Klason lignin quantification. n.d. non- determined, value under the limit of detection.

3.1.4 Complete mass balance: from extractives-free sawdust to whole biomass

The content of ash and extractives are usually obtained directly from experimental data, unlike the content of carbohydrates and lignin, which are determined from extractives-free sawdust. The results revealed a substantially higher content of ash and extractives in the chemical composition of bagasse (17.3%) and straw (20.1%) than for eucalyptus (2.5%) (Figure 8). As a result, the interpretation error due to assessing the chemical composition based on extractives-free sawdust is very small for eucalyptus, unlike that for bagasse and straw.

Figure 8. General chemical profile of eucalyptus, bagasse and straw.

Table 1 sets out the results of the chemical composition, determined according to the approach described in the Experimental section.

Table 1. Results of chemical composition determined on extractives-free sawdust before and after (boxes) the accounting for losses of sugars % Eucalyptus Bagasse Straw Silicaa n.d. 1.7 6.6 Klason ligninb 24.0 (0.1) 19.5 (0.1) 14.0 (0.3) Acid-soluble lignin 4.0 (0.02) 1.9 (0.06) 2.2 (0.08) Glucose 49.4 (0.7) 51.1 41.8 (0.8) 42.8 41.4 (1.2) 42.4 Xylose 12.0 (0.2) 12.4 24.8 (0.2) 25.4 26.0 (0.2) 26.6 Galactose 1.2 (0.1) 1.2 0.9 (0.1) 0.9 0.9 (0.06) 1.0 Mannose 0.9 (0.05) 0.9 0.9 (0.1) 1.0 0.3 (0.1) 0.3 Arabinose 0.3 (0.1) 0.3 2.3 (0.2) 2.3 3.9 (0.05) 4.0 Uronic acids 4.0 (0.05) 1.5 (0.05) 1.3 (0.02) Acetyl groups 1.9 (0.04) 3.0 (0.02) 1.7 (0.01) Totalc 97.7 100.0 98.3 100.0 98.2 100.0 Standard deviations (…) aDetermined in sawdust with extractives and converted to extractives-free sawdust. n.d. non- determined, value under the limit of detection. bSilica co-precipitation deducted. cSum of the average of components in extractives-free sawdust (silicaa, lignin and sugars).

For all biomasses, the sum of the sugars (after redistribution of losses of sugars) and lignin content in the extractives-free sawdust was higher than that for the carbohydrates and lignin in the whole biomass (blue bars in Figure 8). Accounting for the content of

19 extractives and ash thus the correct values for each constituent in eucalyptus, bagasse and straw are set out in Figures 4, 5 and 6. Chemical characterization of biomasses before chemical processing is widely used by researchers as a way to obtain information that is useful for evaluating biomass features, defining parameters for chemical processing and anticipating the biomass's behavior and yield during chemical processing (Carvalho et al., 2014; Pereira et al., 2013; Ramos e Paula et al., 2011; Santos et al., 2011). The use of the complete mass balance can help to achieve a more accurate chemical evaluation of biomasses, even after chemical processing.

3.2 Chemical and structural characterization of acetylated xylan (Paper II)

3.2.1 Preparation of holocellulose samples

Especially in the case of bagasse and straw, a substantial higher amount of xylan was observed, with a different chemical structure and different number and type of side groups and acetyl groups content. A more detailed study of the xylan from these biomasses was performed in order to identify their different chemical and structural features.

Two delignification methods - PAA and NaClO2 - were employed prior to the extraction of xylan using DMSO. Isolating xylan in a high yield and without affecting its structure is a challenge (Evtuguin et al., 2003) due to the interlinkages between various polymers in the cell wall. In the present study, the biomasses were delignified prior to the DMSO extraction in order to remove the lignin and, therefore, to obtain more pure xylan samples. Two delignification conditions were used for each process: PAA for 30 or 40 minutes and

NaClO2 with one or three additions of chemical reagents. The FTIR spectra for the holocellulose samples produced through the delignification processes are set out in Figure 9. Slight differences were observed in the FTIR spectra of holocelluloses obtained under different delignification conditions and delignification processes. The presence of acetylated xylan in the holocellulose samples was confirmed by absorption at 1734 cm-1 in both the PAA-holocellulose samples and the NaClO2-holocellulose samples (Bian et al., 2010). In addition, results indicated that, even when using the PAA-delignification process for 40 min and the NaClO2-delignification process with three additions of chemical reagents, the holocellulose samples contained small amounts of lignin, which was evidenced by the presence of a slight band at 1510 cm-1 due to the aromatic skeletal vibration (Sun et al., 2004).

A B

Figure 9. FTIR spectra of holocellulose samples delignified by PAA process (A) during 30 min for eucalyptus (spectrum a), bagasse (spectrum c) and straw (spectrum e) and during 40 min for eucalyptus (spectrum b), bagasse (spectrum d) and straw

(spectrum f), and FTIR spectra of holocellulose samples delignified by NaClO2 process (B) with one addition of chemical reagents for eucalyptus (spectrum g), bagasse (spectrum i) and straw (spectrum k) and with three additions of chemical reagents for eucalyptus (spectrum h), bagasse (spectrum j) and straw (spectrum l).

The holocellulose samples obtained from eucalyptus using the PAA-delignification process exhibited the lowest Klason lignin content among all of the biomasses. In addition, a reduction in the Klason lignin content was observed in the holocellulose obtained from eucalyptus due to the reaction time during the PAA-delignification process and the additions of chemical reagents in the NaClO2-delignification process (Figure 10). The increasing of the reaction time during the PAA-delignification process did not improve delignification in holocelluloses obtained from bagasse and straw. This was in contrast to the effect of adding chemical reagents three times instead of only once for the same biomasses during the NaClO2-delignification process. In general, the PAA- delignification process was more effective for lignin removal than the NaClO2- delignification methods used.

B A

Figure 10. Klason lignin content in holocellulose samples from eucalyptus (A), bagasse

(B) and straw (C) obtained by PAA (30 or 40 minutes) and NaClO2 (one and three additions of chemical reagents). Klason lignin was not corrected due to silica co- precipitation.

The delignification processes changed the morphology of the raw materials, as shown in Figures 11, 12 and 13, with the eucalyptus showing the fewest changes. Rezende et al. (2011) observed that delignification increased the fragility of and holes in the cell wall structure of sugarcane bagasse. In the present study, similar results were observed for all biomasses. The organization of the fibres was more open in the holocellulose samples than in the biomass raw materials, but no difference was observed in holocellulose produced by different methods from the same biomass. These results suggest that the delignification processes (PAA or NaClO2) and the different conditions employed during the delignification processes had similar effects on the fibre surface. Fibres (indicated by arrow F) were the only structural element observed in the SEM images for eucalyptus (Figure 11). In addition to fibres, the pith (indicated by arrow P) has also been observed as a morphological feature in bagasse (Cruz et al., 2013; Driemeier et al., 2011; Rezende et al., 2011) (Figure 12). Pith is a cell formed, basically, by polysaccharides. Fibres and pith are probably present in both the bagasse raw materials and the holocellulose samples obtained from bagasse, although in the present study fibres were not observed in the SEM images for

22 certain holocellulose samples. This was most likely due the intense surface changes that occurred during the delignification process, which resulted in the pith completely covering the fibres in the images. During the delignification process, the lignin from both the lamella and the lignified cells was removed, which can indirectly increase the area of the pith due to cell wall disruption.

F F

A F B C

F F D E

Figure 11. SEM images of eucalyptus raw material (A), eucalyptus PAA-holocellulose (30 min) (B), eucalyptus PAA-holocellulose (40 min) (C), eucalyptus NaClO2-holocellulose

(one addition of chemical reagents) (D) and eucalyptus NaClO2-holocellulose (three additions of chemical reagents) (E).

F F P P P A B C

P D E

Figure 12. SEM images of bagasse raw material (A), bagasse PAA-holocellulose (30 min)

(B), bagasse PAA-holocellulose (40 min) (C), bagasse NaClO2-holocellulose (one addition of chemical reagents) (D) and bagasse NaClO2-holocellulose (three additions of chemical reagents) (E).

Figure 13 shows changes in the fibre surface of straw due to delignification. Compared to the raw material, the holocellulose samples showed a more open structure. In the raw material, only fibres were observed in the SEM images, whereas in the holocellulose samples obtained from straw piths were also observed, most likely due to lignin removal from the lamella and the adjacent lignified cells. The SEM images provided evidence that the delignification processes, in addition to causing changes in the chemical composition (mostly lignin removal), also changed the fibre surface of the eucalyptus, bagasse and straw. For bagasse and straw, even milder delignification conditions resulted in a more open structure, and the use of more severe delignification conditions had no additional effect on the surface features.

F P F F A B C

P F P F D E

Figure 13. SEM images of straw raw material (A), straw PAA-holocellulose (30 min) (B), straw PAA-holocellulose (40 min) (C), straw NaClO2-holocellulose (one addition of chemical reagents) and (D) straw NaClO2-holocellulose (three additions of chemical reagents) (E).

3.2.2 Extraction of xylan

Xylan samples were extracted from the corresponding holocelluloses using DMSO. For eucalyptus and bagasse, the holocellulose yield from the NaClO2 process was higher than from the PAA process (Table 2). For straw, the PAA process resulted in slightly higher yield values than the NaClO2 process. In addition, results indicated that the delignification yield decreased with increases in reaction time (PAA-delignification process) and additions of chemical reagents (NaClO2-delignification process) by up to 7% and 9%, respectively, which suggests that it is likely that, under more severe conditions, carbohydrates (together with lignin) were removed during delignification. The main goal of the present study was to prepare xylan samples with the least chemical modification possible. Despite the presence of small amounts of lignin in the holocellulose samples (indicated by FTIR spectra in Figure 9 and Klason lignin analysis in

Figure 10), milder conditions for the PAA (30 min) and NaClO2 (one addition of chemical reagents) delignification processes were used for the holocellulose preparation in order to minimize the loss of xylan during delignification (bold values in Table 2).

Table 2. Delignification yield (in %) based on extractives-free biomasses (dry basis)

PAA delignification NaClO2 delignification Biomass 1 addition of 3 additions of 30 min 40 min chemical chemicals Eucalyptus 71.0 65.4 85.0 77.9 Bagasse 75.2 71.5 84.6 75.6 Straw 74.3 69.7 72.3 64.4

The xylan isolation yield was measured gravimetrically and calculated based on the weight of the holocellulose used in the isolation process (on a dry basis). The PAA/DMSO process was more efficient for xylan isolation, resulting in a significantly higher isolation yield for the PAA/DMSO process than for the NaClO2/DMSO process, irrespective of the biomass (Table 3). The present results confirmed those found in the literature for eucalyptus, in which a higher xylan yield was also observed for the PAA/DMSO process than for the NaClO2/DMSO process (Evtuguin et al, 2003).

Table 3. Xylan isolation yield (in %) based on holocellulose (dry basis)

Biomass PAA/DMSO NaClO2/DMSO Eucalyptus 6.93 0.23 Bagasse 5.70 0.85 Straw 7.69 1.53

The xylan yields (estimated as xylose content) after delignification and DMSO extraction are set out in Table 4. During the delignification step, the main chemical component removed was the lignin, although a certain amount of xylan was also removed. A large amount of xylose was lost from the starting materials during the xylan isolation steps, irrespective of the holocellulose (PAA or NaClO2) and biomass (eucalyptus, bagasse or straw). A significantly higher xylan yield was obtained when using the PAA/DMSO process. For NaClO2-holocelluloses samples a substantial presence of lignin was observed (Figure 10).

Table 4. Xylan yield (in %) after delignification and xylan isolation step Delignification step Xylan isolation step Biomass PAA NaClO2 PAA/DMSO NaClO2/DMSO Eucalyptus 80.4 100.0 29.1 0.2 Bagasse 58.0 68.4 10.2 1.1 Straw 81.8 81.1 12.6 1.7

3.2.3 Chemical and structural characterization of xylan samples

3.2.3.1 Structure and composition of xylan

The structure of the xylan samples isolated through the PAA/DMSO and

NaClO2/DMSO processes was confirmed by FTIR analysis (Figure 14). In all spectra, a typical signal for xylan was observed: a sharp band at 1039 cm-1, which is due to the C-O, C- C stretching or C-OH bending in the sugar units (Chaikumpollert et al., 2004). In addition, the bands between 1175 and 1000 cm-1, also typical of xylans, were observed (Sun et al., 2004). The dominance of β-glycosidic linkages between the xylose units were evidenced by the presence of a sharp band at 897 cm-1 (Gupta et al., 1987).

Figure 14. FTIR spectra of xylan samples of eucalyptus PAA/DMSO (a), eucalyptus

NaClO2/DMSO (b), bagasse PAA/DMSO (c), bagasse NaClO2/DMSO (d), straw

PAA/DMSO (e) and straw NaClO2/DMSO (f).

The hydroxyl stretching vibrations of xylans (3350-3330 cm-1), as well as the C-H stretching vibrations caused by the water involved in the hydrogen bonding (2920 cm-1) (Sun et al., 2004), were observed in the spectra. The band at 1241 cm-1, which is due to the

-1 C-O stretching, and at 1370 cm , which is due to the C-CH3 stretching, were also detected (Xu et al., 2010). The absorption at 1628 cm-1 corresponded principally to the water absorbed by xylans (Kačuráková et al., 1998). A low amount of lignin was observed in the xylan spectra, evidenced by the presence of a slight band at 1510 cm-1 due to the aromatic skeletal vibration (Sun et al., 2004). The isolation of the acetylated xylan samples from the biomasses was confirmed by FTIR analysis. The presence of acetyl groups in the xylan samples was confirmed by the absorption at 1734 cm-1, which is due to the C=O stretching (Bian et al., 2010). The band at 1160 cm-1, observed especially in bagasse and straw, evidenced the presence of arabinose substitution in the xylan samples (Egüés et al., 2014; Brienzo et al., 2009). This was also confirmed by sugar analysis and linkage analysis (Figure 15 and Table 7, respectively). The absence of bands around 1720 cm-1, which appear due to ketone carbonyls stretching that can be generated from xylans through oxidation, suggested that the glycosidic linkages and hydroxyl groups of xylans were not attacked or oxidized during the isolation processes (Magaton et al., 2008; Sun and Tomkinson, 2002). Appendix A presents a detailed interpretation of the main band seen in Figure 14. The changes in the composition of the biomasses due to the various treatments were monitored by sugar analysis and the data therefrom is set out in Figure 15. During the delignification processes, the main chemical component removed was the lignin. A similar sugar composition was observed between PAA-holocellulose and NaClO2-holocellulose for all of the biomasses, with only slight differences due to the varying delignification conditions. The glucose content in the holocellulose samples was marginally greater (0.22%

- 3.5%) with the PAA-40 min and NaClO2-3 chemical reagents additions processes than with PAA-30 min and NaClO2-1 chemical reagents addition processes, respectively. A longer reaction time in PAA-delignification and higher chemical charge in NaClO2- delignification both resulted in the loss of xylan together with lignin during the delignification process. Thus, the use of milder conditions in the delignification step probably contributed to a decrease in the loss of xylan from raw materials.

Figure 15. Sugar composition of eucalyptus (A), bagasse (B) and straw (C) for extractives- free biomass, PAA-holocellulose (30 and 40 minutes), NaClO2-holocellulose (1 and 3 additions of chemical reagents) and xylan samples isolated by PAA/DMSO and

NaClO2/DMSO. The delignification processes used for xylan preparation are underlined.

Xylose was the second most abundant sugar present in both the raw materials and the holocellulose samples. In bagasse and straw (extractives-free biomasses), the xylose contents were 2.2 and 2.4 times higher than in eucalyptus, respectively. Especially in

29 bagasse and straw, arabinose was observed to be an important sugar in xylans, which was also confirmed in the FTIR spectra (band at 1160 cm-1) (Figure 14). In extractives-free biomasses and holocellulose samples, the glucose, xylose and arabinose yielded more than 92% of the total sugars. The PAA/DMSO process produced the purest xylan samples, with 91.0%, 81.4% and

80.0% xylose for eucalyptus, bagasse and straw, respectively, while the NaClO2/DMSO process resulted in hemicelluloses samples with a higher glucose content. Magaton et al. (2008) found a similar sugar composition of xylan from eucalyptus isolated by the PAA/DMSO process.

3.2.3.2 Molecular weight of xylan samples

The xylan samples for each biomass had different molecular weights (Table 5) and weight distributions (Table 16). In general, the molecular weight distribution for the xylan samples followed monomodal size distributions. Only the xylan sample extracted from eucalyptus with NaClO2/DMSO showed a broader and multimodal distribution (Figure 16), which could be linked to the presence of a heterogeneous population of polysaccharides, as suggested by the monosaccharide composition (Figure 15) and linkage analysis (Table 7).

Table 5. SEC results – – Samples Extraction process Mn, kDa Mw, kDa D PAA/DMSO 30.8 (0.3) 42.2 (0.3) 1.4 Eucalyptus NaClO2/DMSO 9.7 (0.3) 55.2 (2.4) 5.7 PAA/DMSO 25.2 (0.0) 37.6 (0.3) 1.5 Bagasse NaClO2/DMSO 23.7 (0.5) 40.8 (2.9) 1.7 PAA/DMSO 17.9 (0.5) 29.5 (0.5) 1.7 Straw NaClO2/DMSO 18.2 (1.1) 34.2 (3.2) 1.9 Standard deviation (…)

For PAA/DMSO xylan samples from eucalyptus, the molecular weight value was 42 kDa, a value lower than that observed for NaClO2/DMSO xylan samples (55 kDa). The polydispersity indexes for eucalyptus were 1.4 and 5.7 for PAA/DMSO xylan samples and

NaClO2/DMSO xylan samples, respectively. These results reinforced the observation that the NaClO2/DMSO extraction process did not succeed in isolating relevant xylan fractions from eucalyptus, but, rather, it isolated a mixture of shorter polysaccharide chains with a high polydispersity index. In the present study, the molecular weight value for PAA/DMSO

30 xylan samples was slightly higher than the value of 36 kDa reported for the Eucalyptus globulus (Evtuguin et al., 2003). The average molar mass values for sugarcane bagasse and straw were in the range of 41-30 kDa, which were similar to the values previously obtained for sugarcane bagasse xylans (Peng et al., 2009; Sun et al., 2004) and for straw xylans from other grasses (e.g. wheat straw) (Persson et al., 2009). The polydispersity indexes of the bagasse and straw xylan samples were in the range of 1.5-1.9, which was lower than the values previously obtained for sugarcane bagasse xylans (Peng et al., 2009; Sun et al., 2004).

Figure 16. Molecular weight distribution of xylan from eucalyptus, bagasse and straw.

3.2.3.3 Acetyl groups in xylan

Each biomass presented a different degree of acetylation. The PAA/DMSO process resulted in the isolation of xylan samples with the highest acetyl content and degree of acetylation for all biomasses (Table 6), with eucalyptus xylan having the highest degree of acetylation. In NaClO2/DMSO xylan samples, bagasse had the highest degree of acetylation. Straw had the lowest degree of acetylation, irrespective of the xylan isolation process used, and only a slight difference was observed between the two methods. Based on the chemical characterization of the starting biomasses, approximately 9.1%, 8.9% and 4.7% of the acetyl groups were observed in hemicelluloses for eucalyptus, bagasse and straw raw materials, respectively (Figures 4-6). In the present study, the acetyl content was lower than that observed by Evtuguin et al. (2003) for Eucalyptus globulus (19.6%), but there was a similar degree of acetylation between the eucalyptus xylan isolated by the PAA/DMSO process and the xylan samples from beech and birch studied by Teleman et al. (2002). Magaton et al. (2008) observed a

31 substantially higher degree of acetylation for Eucalyptus urophylla × Eucalyptus grandis (0.55) than was observed in the present study for eucalyptus. Acetyl groups play an important role during the chemical processing of biomasses, and knowledge of the differences in the degree of acetylation of xylan in various biomasses can help to optimize chemical conversion processes.

Table 6. Acetyl content and degree of acetylation in xylans Acetyl content (%) DAa Sample PAA/DMSO NaClO2/DMSO PAA/DMSO NaClO2/DMSO Eucalyptus 11.3 (0.17) 3.3 (0.07) 0.39 (0.01) 0.10 (0.00) Bagasse 8.7 (0.37) 5.4 (0.22) 0.29 (0.01) 0.17 (0.01) Straw 2.4 (0.45) 1.5 (0.00) 0.08 (0.01) 0.05 (0.00) Standard deviation (…) aDegree of acetylation.

3.2.3.4 Linkage analysis after acid hydrolysis of xylan samples

Xylan has been reported as being the main hemicellulose in both hardwood and grass plants, but the exact structure and pattern of the branching differs for different plants (Ebringerová et al., 2005). Many studies had shown that in eucalyptus the empirical structure of the xylan is the O-acetyl-(4-O-methylglucurono) xylan, with branches of 4-O- methylglucurono acids at position 2 and acetyl groups at positions 2 and/or 3 (Magaton et al., 2008; Evtuguin et al., 2003). In addition, residues of glucan and ramnoarabinogalactan can be found chemically linked to the 4-O-methylglucuronic acids, as described by Evtuguin et al. (2003). In grasses, such as sugarcane, the arabinoxylan (AX) has been identified as the main hemicellulose. The backbone is formed by β-(1→4)-D-xylopyranose, with possibile substitutions of α-L-arabinofuranose at positions 2 and/or 3. Arabinoxylan can also be slightly acetylated (Ebringerová et al., 2005). Results of the linkage analysis revealed Xylp as the main unit in the backbone of xylan in eucalyptus, bagasse and straw based on the substantially higher molar proportion of 4- Xylp (1 →4)-linked units (Table 7). In addition, an evident presence other polysaccharides in the NaClO2/DMSO xylan samples was observed in all biomasses, in consistency with the sugar composition results. In bagasse and straw, these other polysaccharides most likely came from arabinan and arabinogalactan, in addition to glucans and cellulose. In eucalyptus, the presence of these other polysaccharides is most likely due the presence of cellulose and other additional pectins, such as rhamnogalacturonan.

In xylan samples from eucalyptus, the branches of terminal MeGlcA (GlcpA-(1→), in addition to 4-O-methylglucuronic acid (→2)-GlcpA-(1→) units, occurred preferentially in position O-2 of the xylose. The molar ratio between the xylose units and MeGlcA branches was 10:1.1. The number of branches in the xylose chain (2,4-Xylp, 3,4-Xylp and 2,3,4- Xylp) accounted for a larger molar amount than MeGlcA units, most likely due to incomplete MeGlcA quantification. The presence of substituted glucuronic acid moieties has been reported before in PAA-extracted eucalyptus xylans (Pinto et al., 2005; Evtuguin et al., 2003). In this study, the results suggested the substitution of MeGlcA with mainly terminal galactosyl units, which are consistent with the results for PAA (Table 7). Branches in 3,4-Xylp and 2,3,4- Xylp were observed in small amounts and might arise from the incomplete saponification of the acetyl groups during the methylation process (Pinto et al., 2005). Magaton et al. (2008) suggested that the chemical linkage between glucose and galactose to the xylan chain via uronic acids could have been the reason for the presence of glucose and galactose in the xylan samples from eucalyptus. Evtuguin et al. (2003) supported those findings, obtaining remarkable amounts of glucose, arabinose and rhamnose in the xylan isolated from eucalyptus through the PAA/DMSO process. They also suggested that these sugars most likely came from short polysaccharide fragments of different origins, chemically linked to the backbone of the xylans and not simply adsorbed during the xylan isolation process. Based on the linkage analysis results obtained by the present study for eucalyptus, the linkage of xylan to glucose and pectins was not proved. In xylan samples from bagasse and straw, a substantially higher molar proportion was observed for 2,4-Xylp and 3,4-Xylp than for 2,3,4-Xylp, which confirmed the observations of Ebringerová et al. (2005). However, this was in contrast to the observations made by Heikkinen et al. (2013), who detected relatively abundant double substitutions in xylan from wheat. The chemical structure of xylan samples from bagasse and straw were similar to each other. Xylan from straw was substituted slightly more by arabinosyl units (10 xylose units: 0.6 arabinosyl units) than was xylose from bagasse (10 xylose units: 0.5 arabinosyl units). In xylan extracted from bagasse and straw through the PAA/DMSO process, the molar ratio between terminal residues (t-Araf) and branched xylose units (2,4-Xylp, 3,4- Xylp and 2,3,4-Xylp) were 0.94 and 1.00, respectively. This indicated that no undermethylation occurred during the analysis. On the other hand, for xylans isolated by the NaClO2/DMSO process, the molar ratios between terminal residues and branched xylose units for bagasse and straw, were only 0.65 and 0.69, respectively. For these samples lower total xylose content was also obtained (Figure 15), most likely due to the

33 presence of other types of polysaccharides. As a result, the NaClO2/DMSO process was not efficient for xylan preparation, resulting in a xylan which exhibited different chemical behavior, such as having a higher resistance towards methylation. Table 7. Linkage analysis Relative abundance (% mol) Structural units Linkage Eucalyptus Bagasse Straw deduced PAA NaClO2 PAA NaClO2 PAA NaClO2 t-Araf Araf-(1 → <0,1 0,8 (0,0)1 4,6 (0,1) 3,6 (0,3) 5,3 (0,4) 6,0 (0,7) 2-Araf → 2) Araf-(1 → <0,1 n.d n.d 0,2 (0,2) 0,5 (0,1) 0,8 (0,0) 3-Arap → 3) Arap-(1 → n.d n.d n.d 0,1 (0,0) <0,1 0,3 (0,1) 5-Araf → 5) Araf-(1 → <0,1 0,4 (0,1) 0,2 (0,0) 0,3 (0,1) 0,5 (0,3) 0,7 (0,3) 2,5-Araf → 2,5) Araf-(1 → <0,1 1,1 (0,6) n.d <0,1 <0,1 <0,1 Total Ara1 0,2 (0,0) 2,4 (0,8) 4,8 (0,1) 4,1 (0,5) 6,4 (0,9) 7,6 (1,0) t-Xylp Xylp-(1 → 1,6 (0,2)* 2,0 (0,1) 2,5 (0,2) 2,8 (0,2) 2,5 (0,4) 2,5 (0,5) 2-Xylp → 2) Xylp-(1 → n.d n.d n.d 0,2 (0,1) 0,1 (0,1) 0,4 (0,1) 4-Xylp → 4)-Xylp-(1 → 74,4 (2,3) 30,9 (3,5) 81,8 (0,7) 66,3 (2,1) 75,3 (2,4) 56,6 (0,3) 2,4-Xylp → 2,4)-Xylp-(1 → 9,5 (1,0) 3,2 (0,2) 1,2 (0,1) 1,1 (0,1) 1,0 (0,2) 1,3 (0,0) 3,4-Xylp → 3,4)-Xylp-(1 → 0,6 (0,1) 0,8 (0,0) 3,6 (0,1) 4,3 (0,3) 4,2 (0,8) 7,2 (0,7) 2,3,4-Xylp → 2,3,4) Xylp-(1 → 0,3 (0,1) 1,2 (0,5) <0,1 <0,1 <0,1 0,2 (0,0) Total Xyl1 86,4 (3,6) 38,1 (4,3) 89,1 (1,2) 74,9 (2,8) 83,4 (3,9) 68,5 (1,7) t-Glcp Glcp(1 → n.d n.d 0,2 (0,0) 0,8 (0,3) 0,2 (0,0) 0,3 (0,0) 3-Glcp → 3)- Glcp A-(1 → 0,3 (0,2) 2,0 (0,5) 2,6 (0,0) 6,7 (0,7) 3,4 (1,2) 5,0 (0,8) 4-Glcp → 4) Glcp-(1 → 0,4 (0,1) 29,4 (7,4) 2,2 (0,0) 9,9 (1,9) 4,2 (0,0) 15,3 (0,6) 4,6-Glcp → 4,6)-Glcp-(1 → <0,1 2,6 (0,2) n.d n.d n.d n.d 3,4-Glcp → 3,4)-Glcp-(1 → n.d 1,6 (0,3) n.d n.d n.d n.d Total Glc1 0,7 (0,3) 34,6 (9,4) 5,0 (0,1) 17,3 (2,8) 7,9 (1,2) 20,6 (1,5) 4-Manp → 4) Manp-(1 → 0,1 (0,0) 8,4 (0,1) n.d. n.d. n.d. n.d. 4,6-Manp → 4,6) Manp-(1 → n.d. 0,4 (0,1) n.d. n.d. n.d. n.d. Total Man1 0,1 (0,0) 8,8 (0,2) <0,1 0,3 (0,0) <0,1 <0,1 t-Galp Galp-(1 → 1,8 (0,2) 1,3 (0,9) 0,5 (0,2) 1,3 (0,3) 0,8 (0,0) 1,6 (0,1) 6-Galp → 6) Galp-(1 → n.d n.d <0,1 0,1 (0,1) 0,2 (0,1) 0,1 (0,0) 3-Galp → 3) Galp-(1 → n.d n.d <0,1 0,5 (0,1) 0,3 (0,2) 0,1 (0,0) 3,4-Galp → 3,4) Galp-(1 → n.d 0,6 (0,1) n.d n.d n.d n.d 2,3-Galp → 2,3) Galp-(1 → n.d 0,8 (0,3) n.d n.d n.d n.d 2,4-Galp → 2,4) Galp-(1 → n.d 1,3 (0,2) n.d n.d n.d n.d 3,6-Galp → 3,6) Galp-(1 → 0,2 (0,0) 1,6 (0,1) <0,1 0,2 (0,1) <0,1 0,1 (0,1) 4,6-Galp → 4,6) Galp-(1 → 0,1 (0,0) 2,7 (0,4) 0,1 (0,0) 0,7 (0,3) 0,5 (0,2) 0,6 (0,0) Total Gal1 2,2 (0,3) 8,2 (2,0) 0,9 (0,1) 2,8 (1,0) 1,8 (0,6) 2,6 (0,3) 3-Rhaf → 3) Rhaf-(1 → 0,6 (0,1) n.d. n.d. n.d. n.d. n.d. 2,4-Rhaf → 2,4) Rhaf-(1 → n.d. 1,6 (1,1) n.d. n.d. n.d. n.d. Total Rha1 0,6 (0,1) 1,6 (1,1) n.d. <0,1 n.d <0,1 2-GalpA → 2) GalpA-(1 → 1,4 (0,5) 1,1 (0,2) n.e. n.e. n.e. n.e. 4-GalpA → 4) GalpA-(1 → 1,2 (0,2) 2,4 (1,9) n.e. n.e. n.e. n.e. Total GalA1 2,6 (0,6) 2,9 (2,0) 0,2 (0,0) 0,2 (0,0) 0,5 (0,0) 0,2 (0,0) t-GlcpA GlcpA(1 → 6,3 (1,1) 3,0( 0,5) n.e. n.e. n.e. n.e. 2-GlcpA → 2) GlcpA-(1 → 0,8 (0,1) 0,4 (0,1) n.e. n.e. n.e. n.e. Total GlcA1 7,2 (0,2) 3,4 (0,6) <0,1 0,5 (0,0) <0,1 0,2 (0,0) Standard deviation (…) 1Monosaccharide composition as calculated by acid methanolysis n.d: not detected n.e: not evaluated

3.2.3.5 Characterization of xylan structure by 1H NMR method

The structure of the acetylated xylan, including the position of the acetyl groups in the xylan molecules, was investigated using the 1H NMR method (Figure 17). PAA/DMSO xylan extracted from eucalyptus, bagasse and straw, were chosen for this study. In the spectra, the hydrogen from the acetyl groups (CH3-CO-) (δH 2.01-2.11), the D2O (HDO) (δH 4.71) and the solvent impurities (δH 1.94, 2.00, 2.16, 2.47, 2.62, 2.82, 3.07 and 3.25) were also assigned, in addition to the main signals, in accordance with data found in the literature (Sun et al., 2011; Marques et al., 2010; Magaton et al., 2008; Evtuguin et al., 2003; Hoffmann et al., 1992; Izydorczyk and Biliaderis, 1992; Cavagna et al., 1984).

The anomeric hydrogens in the region between δH 4.4-5.5 were identified as following patterns in the xylan chain: a non-acetylated xylose unit in the backbone isolated from other acetylated xylose units; a non-acetylated xylose unit neighboring an acetylated xylose unit; a xylose unit acetylated in oxygen 3; a xylose unit acetylated in oxygen 2; and a xylose unit di-acetylated in oxygens 2 and 3. For eucalyptus, in addition to assigning the positions of the acetyl groups, the branches of 4-O-methylglucuronic acid in the acetylated xylan (Xyl-3Ac-2MeGlcA), the 4-O-methylglucuronic acid (MeGlcA) and the 4-O- methylglucuronic acid linked to the galactosyl unit (MeGlcA-2Gal) were also assigned. For bagasse and straw, arabinose linked to oxygen 3 in monosubstituted xylan (δH 5.39) and arabinose linked to oxygen 3 (δH 5.29) and 2 (δH 5.23) in disubstituted xylan (Izydorczyk and Biliaderis, 1992) were assigned (Table 8). The 1H NMR spectrum for xylan from eucalyptus is set out in Figure 17A. Some signals of anomeric protons related to acetyl groups in the xylose units and the position of the 4-O- methylglucuronic acid units, which appear in the region between δH 4.6-4.9, were not completely identified due to an overlap with the solvent signal (HDO). This was mainly the case for the proton chemical shift at H-1 and H-2 in the xylose unit. The other proton chemical shifts in the same xylose unit (H-3, H-4 and H-5) suggested the presence of acetyl groups in O-2, O-3 and O-2,3, in addition to O-3 of xylose substituted by MeGlcA. In the xylan samples from eucalyptus, the 4-O-methylglucuronic acid attached via α-(1→2) linkage to xylose, which was observed in the results of the linkage analysis, was confirmed

1 in the H NMR spectra by the presence of an anomeric proton at δH 5.20 and of a singlet at

δH 3.38 (Teleman et al., 2000; Cavagna et al., 1984). 4-O-methylglucuronic acid can be present in deacetylated xylose units (Shatalov et al., 1999) or in acetylated xylose units (Xyl-3Ac-2MeGlcA) (Magaton et al., 2008; Evtuguin et al., 2003). In the present study, the integration of the proton at H-3 (δH 4.99) from the structural element Xyl-3Ac-2GlcA and the proton H-1 (δH 5.20) from the 4-O-methylglucuronic acid resulted in a ratio of 1:1. This proved that all 4-O-methylglucuronic acid units are present in acetylated xylose. This observation is consistent with those found by Magaton et al. (2008) for the same eucalyptus hybrid and by Evtuguin et al. (2003) for Eucalyptus globulus.

Figure 17. 1H NMR spectrum of acetylated xylan from eucalyptus (A), bagasse (B) and straw (C) extracted by PAA/DMSO process. Designation of the structural fragments is presented in Table 8. *solvent impurities.

The 1H NMR spectra for xylan from sugarcane bagasse and straw are set out in Figure 17B and 17C, respectively. Acetyl groups in xylan from bagasse were assigned at positions O-2, O-3 and O-2,3. In xylan from straw, acetyl groups were assigned at positions O-2 and O-3 only. In xylan samples from both bagasse and straw, the terminal arabinose attached

via α-(1→) linkage to xylose units at positions O-2 and O-3, as observed earlier in the linkage analysis, was confirmed in the 1H NMR spectra. The 1H NMR spectra also suggested the presence of disubstituted xylose (Xyl-2,3Ara) units in the xylan samples from bagasse and straw, but it was not confirmed by linkage analysis in representative molar abundance.

Table 8. Proton NMR chemical shifts (δH, ppm) for structural units of acetylated xylan extracted by PAA/DMSO process from eucalyptus, bagasse and straw. H5 Biomass Structural units H1 H2 H3 H4 ax eq Xyl (isol.) 4.49 3.30 3.56 3.79 3.42 n.d. Xyl (Xyl-Ac) 4.37 3.19 3.54 3.69 n.d. n.d. Xyl-3Ac 4.60 3.47 4.98 3.94 3.47 n.d. Xyl-2Ac n.d. n.d. 3.79 3.85 3.45 n.d. Eucalyptus Xyl-2,3Ac n.d. n.d. 5.09 4.03 3.54 n.d. Xyl-3Ac-2MeGlcA n.d. 3.69 4.99 3.97 3.47 n.d. MeGlcA 5.20 3.56 3.82 3.17 n.d. n.d. MeGlcA-2Gal 5.45 3.76 3.82 3.19 n.d. n.d. Xyl (isol.) 4.46 3.30 3.55 3.79 3.37 n.d. Xyl (Xyl-Ac) 4.42 3.22 3.54 3.71 3.36 n.d. Xyl-3Ac 4.58 3.47 4.92 3.91 3.47 4.15 Xyl-2Ac 4.64 n.d. 3.79 3.85 3.43 4.15 Bagasse Xyl-2,3Ac 4.84 4.82 5.19 4.05 3.51 4.20 Xyl-3Ara 5.39 4.18 3.91 4.18 3.75 3.77 Xyl-2,3Ara 5.29 4.18 3.91 4.18 3.75 3.77 Xyl-2,3Ara 5.23 4.16 3.94 4.14 3.75 3.77 Xyl (isol.) 4.50 3.30 3.58 3.72 3.39 4.09 Xyl (Xyl-Ac) 4.38 3.20 3.54 3.70 3.35 n.d. Xyl-3Ac 4.52 3.46 4.91 3.92 3.46 n.d. Xyl-2Ac 4.61 4.63 3.81 3.84 3.46 4.19 Straw Xyl-2,3Ac n.d. n.d. n.d. n.d. n.d. n.d. Xyl-3Ara 5.32 4.20 3.92 4.20 3.74 3.76 Xyl-2,3Ara 5.29 4.20 3.92 4.20 3.74 3.76 Xyl-2,3Ara 5.26 4.19 3.93 4.11 3.74 3.76 n.d.non determined or inexistent. The following designations used were: Xyl (isol.) → non-acetylated Xylp in the backbone isolated from other acetylated Xylp units; Xyl (Xyl-Ac) → non-acetylated Xylp linked with neighboring acetylated Xylp; Xyl-3Ac - 3-O-acetylated Xylp; Xyl-2Ac → 2-O-acetylated Xylp; Xyl-2,3Ac → 2,3- di-O-acetylated Xylp; Xyl-3Ac-2MeGlcA → MeGlcA 2-O-linked and 3-O-acetylated Xylp; MeGlcA → MeGlcA unit; MeGlcA-2Gal → Galp 2-O-linked MeGlcA; Xyl-3Ara – terminal arabinose linked to O-3 of monosubstituted xylose; Ara Xyl-2,3Ara – terminal arabinose linked to O-3 in disubstituted xylose; and Xyl-2,3 - terminal arabinose linked to O-2 in disubstituted xylose.

3.2.3.6 Empirical structure of xylan from eucalyptus, bagasse and straw

Based on a combination of the experimental results for degree of acetylation (Table 6), methylation linkage analysis (Table 7) and 1H NMR spectroscopy (Table 8 and Figure 17), a designation for the structural fragments in xylans extracted from eucalyptus, bagasse and straw (Table 9) and the empirical structures of xylan (Figure 18) were obtained.

In xylan extracted from eucalyptus, a molar ratio between the xylose units and the branches of 4-O-methylglucuronic acid units of 10:1.1 was revealed by methylation linkage analysis. A degree of acetylation of 0.39 acetyl groups/xylose units resulted in the followed distribution of acetyl groups: 36% at Xyl-3Ac, 26% at Xyl-2Ac and 10% at Xyl-2,3Ac. In addition, 28% of the acetyl groups were located at position O-3 of xylose substituted at O-2 by 4-O-methylglucuronic acid (Xyl-3Ac-2MeGlcA). All 4-O-methylglucuronic acids were linked to the acetylated xylose unit, as determined by 1H NMR spectroscopy. In addition, the presence of 4-O-methylglucuronic acid linked to terminal galactosyl unit, in low molar abundance, was also shown by 1H NMR spectroscopy and confirmed by linkage analysis. The assignment for the position of acetyl groups in the xylan was obtained by the integration of the H-3 proton chemical shift in the 1H NMR spectrum, since for H-1 and H- 2 some signals were overlapped by the solvent signal (HDO).

Table 9. Empirical structure of xylan from eucalyptus, bagasse and straw Relative abundance (per 100 Xylp units) Structural fragments and short designation Eucalyptus Bagasse Straw →4)-β-D-Xylp-(1 → (Xyl) 63 70 86 →4)[3-O-Ac]-β-D-Xylp-(1 → (Xyl-3Ac) 14 17 5 →4)[2-O-Ac]-β-D-Xylp-(1 → (Xyl-2Ac) 10 4 3 →4)[3-O-Ac] [2-O-Ac]-β-D-Xylp-(1 → (Xyl-2,3Ac) 2 4 - →4)[4-O-Me-α-D-GlcpA(1→2)] [3-O-Ac] -β-D-Xylp-(1 → (Xyl-3Ac-2MeGlcA) 11 - - -4- O-Me-α-D-GlcpA-(1 → (MeGlcA) 7* - - → 2)-4- O-Me-α-D-GlcpA-(1 → (MeGlcA-2Gal) 1* - - →4)[α-L-Arabf(1→3)]-β-D-Xylp-(1 → (Xyl-3Ara) - 4 5 →4) [α-L-Arabf(1→2)]-β-D-Xylp-(1 → (Xyl-2Ara) - 1 1 α-L-Araf-(1 → (Ara) - 5 6 *Value for MeGlcA units underestimated by methanolysis.

In xylan extracted from sugarcane bagasse, a molar ratio between the xylose units and the arabinosyl substitutions of 10:0.5 was revealed by methylation linkage analysis. A significantly greater quantity of arabinose was observed by methylation linkage analysis at position O-3 than at position O-2, in addition to a negligible molar quantity of arabinose in positions O-2 and O-3 in the same xylose unit (Xyl-2,3Ara). A degree of acetylation of 0.29 acetyl groups/xylose units resulted in the following distribution of acetyl groups: 60% at Xyl-3Ac, 13% at Xyl-2Ac and 27% at Xyl-2,3Ac. In xylan extracted from sugarcane straw, a molar ratio between the xylose units and the arabinosyl substitutions of 10:0.6 was revealed by methylation linkage analysis, with greater quantity of arabinose at position O-3 than at position O-2 and a negligible molar quantity of arabinose in positions O-2 and O-3 in the same xylose unit (Xyl-2,3Ara). A degree of acetylation of 0.08 acetyl groups/xylose units resulted in the following distribution of acetyl groups: 67% at Xyl-3Ac and 33% at Xyl-2Ac.

Figure 18. Empirical structure of xylan from eucalyptus (A), bagasse (B) and straw (C).

3.3 Role of xylan during the hydrothermal pretreatment (Paper I)

During hydrothermal pretreatment, the only chemical used is water, and the hydrolysis is catalyzed by organic acids released from the biomass xylan itself (Pu et al.., 2013; Vegas et al., 2008; Garrote et al., 1999). Hydrothermal pretreatment, which is performed in mild acid conditions, leads to hemicellulose hydrolysis and removal of the hemicellulose from the biomass, thereby increasing cellulose accessibility (Alvira et al., 2010). The pH levels that were observed after hydrothermal pretreatment are set out in Table 10. The values obtained for eucalyptus, bagasse and straw were consistent with the content of acetyl acid and uronic acid found in xylan from these biomasses (see Figures 4-6 and Table 6).

Table 10. Final pH after hydrothermal pretreatment Eucalyptus Bagasse Straw pH 3.4 (0.07) 4.5 (0.02) 5.3 (0.01) Standard deviations (…)

The total content of sugars (glucose, xylose, galactose, mannose and arabinose) in the biomasses after hydrothermal pretreatment was 62.2%, 68.0% and 68.8% (Table 11) for eucalyptus, bagasse and straw, respectively. The C6 sugars (glucose, galactose and mannose), which are converted to bioethanol more efficiently by using traditional yeasts (Baeyens et al., 2015), corresponded to 55.4%, 43.0% and 41.3% for eucalyptus, bagasse and straw, respectively. These results indicate that eucalyptus should present greater advantages for bioethanol production if the content of sugars is completely accessible. However, the glucose, especially from cellulose, is not easily accessible due to the cellulose recalcitrance. The recalcitrance has been described as a protection mechanism, of lignocellulosic biomass, that hinder the degradation of cellulose by enzymes and, thus, creates a challenge for enzymatic hydrolysis (Foston and Ragauskas, 2012; Zhao et al. 2012; Pu et al., 2013; Chandra et al., 2007). This necessitates the use of various pretreatment processes in order to improve cellulose accessibility.

Table 11. Total chemical composition of the pretreated biomasses (%)*

Pseudo- Klason Soluble Biomasses Ash Glucose Xylose Galactose Mannose Arabinose extractives** Lignin Lignin

Eucalyptus 0.2 12.4 22.1 3.1 54.0 6.9 0.5 0.9 - Bagasse 1.0 5.6 24.4 1.1 42.6 24.3 0.2 0.2 0.7 Straw 5.2 3.8 21.2 1.0 40.4 25.5 0.3 0.6 2.1 *Calculated from average of chemical components. **Structures which were removed by extraction with toluene, ethanol and hot water according to TAPPI T 264 cm-07 method (see experimental).

When performed at a lower pH, the hydrothermal pretreatment for eucalyptus resulted in the removal of fewer chemical components (i.e., resulting in the highest yield) (Figure 19). The inverse correlation between pretreatment pH and yield suggested that bagasse and, especially, straw were more suited to hydrothermal pretreatment than eucalyptus. A significant level of hemicellulose removal (xylose, galactose, mannose and arabinose) was observed during hydrothermal pretreatment, irrespective of the biomass. Hemicellulose removal of up to 63%, 25% and 23 % was observed in eucalyptus, bagasse and straw, respectively. The higher level of hemicellulose removal in eucalyptus was most likely due to a lower pH level during the pretreatment, resulting from the larger content of

40 acetyl groups in eucalyptus xylan (compared to the bagasse or straw xylans). Glucose was slightly removed through the hydrothermal pretreatment. In addition to chemical transformations in the polysaccharides, the results indicated an increase in the relative percentages of extractives (in eucalyptus) and lignin (in bagasse and straw) after hydrothermal pretreatment. The quantity of extractives increased 4.9 times for eucalyptus, which indicated that other chemical components were most likely transformed during the hydrothermal pretreatment into structures that had similar solubility to extractives. The structures formed during the hydrothermal pretreatment, named “pseudo-extractives”, were quantified together with extractives in accordance with the standard method (see Experimental 2.5.1). In eucalyptus, the significant level of hemicellulose removal, combined with the formation of pseudo-extractives, suggests that the degradation products from xylan reacted with fragments of lignin or themselves to form pseudo-extractives. In addition, the amount of hemicellulose in eucalyptus removed through the hydrothermal pretreatment (12.8 g) was higher than the total loss of mass during the pretreatment (8.5 g), which further strengthens the hypothesis that degradation products from xylan contribute to the formation of pseudo-extractives. Some formation of pseudo-extractives could also occur in the sugarcane bagasse and straw. The total amount of lignin in the pretreated eucalyptus decreased 15.7% compared to the amount of lignin in the raw material, and it is reasonable to assume the possible presence of lignin fragments, together with hemicellulose fragments, in the pseudo- extractives structures. The total amount of lignin in the pretreated bagasse and straw increased by up to 1.2 and 1.3 times, respectively, compared to the amount of lignin in their respective raw materials. In addition, the results indicated a significant increase in Klason lignin during the pretreatment process (see Table 11). This phenomenon has been observed previously, and was described to the formation of pseudo-lignin (Hu et al., 2012; Sannigrahi et al., 2011; Li et al., 2007; Li et al., 2005). The present results expand current knowledge and show that in grass plants that are subjected to acid hydrolysis, pseudo- lignin is also generated, even in mild conditions. A recent study demonstrated that pseudo- lignin was generated to a larger extent from polysaccharide degradation than from lignin during acid pretreatments of the biomass, especially under high severity conditions (temperature, reaction time and acid concentration) (Sannigrahi et al., 2011). A practical implication of the presence of pseudo-lignin in pretreated biomasses is the inhibition of enzymes during enzymatic hydrolysis of biomasses (Hu et al., 2012). To avoid this phenomenon, milder pretreatment conditions are recommended.

Figure 19. Chemical composition of pretreated eucalyptus, bagasse and straw (obtained by the combination of mass balance and actual yield for each biomass).

The different chemical compositions of xylan in the various biomasses proved to be very important during hydrothermal pretreatment. The results showed that the xylan has at least three important roles during hydrothermal pretreatment:

1) Catalyst: the reactions that occurred during the hydrothermal pretreatment were promoted by the acid groups (uronic acids and acetyl groups) that were released from the biomasses. The eucalyptus, in addition to having a larger number of uronic groups and acetyl groups in the xylan (Figures 4-6), also showed a higher degree of acetylation (Table 6) in the xylan than did the bagasse and straw. This was consistent with the pH levels after pretreatment (Table 10);

2) Matter removal: during the hydrothermal pretreatment, the xylan was removed more extensively than any other chemical component in the biomasses, which improved cellulose accessibility without a significant loss of glucose; and

3) Formation of modified structures: degradation products from xylan (furan type structures), which formed during the pretreatment process, reacted to form pseudo- extractives (in eucalyptus) and pseudo-lignin (in bagasse and straw).

In summary, the comparative study of the structure of xylans from various biomasses contributes to the current knowledge of the structural features of xylan in different biomasses, such as O-acetyl-4-O-methylglucuronoxylan in eucalyptus and arabinoxylan in

42 sugarcane bagasse and straw, and provides a better understanding of the role of these polysaccharides during chemical pretreatments, especially hydrothermal pretreatment.

4. Conclusions

 The suggested novel approach, based on the complete mass balance, proved to be a useful tool for the comparison of the chemical composition of various biomasses. The calculations, which took into consideration the content of ash and extractives, allowed for a more accurate assessment of the chemical composition of bagasse and straw, which both contain large amounts of those chemical components. It was shown that sugarcane bagasse and straw contain larger quantities of ash (2.3% and 7.9%, respectively) and extractives (15.0% and 12.2%, respectively) than eucalyptus (0.16% ash and 2.3% extractives).  The large quantity of silica in sugarcane bagasse and straw leads to an overestimation of the Klason lignin content, which needs to be corrected. Based on the corrected values, it was shown that bagasse and straw had a smaller total content of lignin (18.0% and 13.9%, respectively) than previously reported, and certainly a smaller amount than that found in eucalyptus (27.4%).  Xylan from eucalyptus, sugarcane bagasse and sugarcane straw differ from each other in terms of their structure. In eucalyptus, the xylan was the O-acetyl-4-O- methylglucuronoxylan with a molar ratio of xylose units to 4-O-methylglucuronic acid branches of 10:1.1, degree of acetylation of 0.39 and molecular weight of 42 kDa. Galactosyl unit linked to 4-O-methylglucuronic acid in eucalyptus xylan was also observed. In sugarcane bagasse and straw, the xylan was the arabinoxylan. In bagasse, the xylan had a molar ratio of xylose units to arabinosyl substitutions of 10:0.5, degree of acetylation of 0.29 and molecular weight of 38 kDa, while in straw, the xylan had a molar ratio of xylose units to arabinosyl substitutions of 10:0.6, degree of acetylation of 0.08 and molecular weight of 30 kDa. Arabinose units in xylan from bagasse and straw were observed in positions O-2 and O-3.  It was shown that, during hydrothermal pretreatment, the structure of the xylan (i.e., the number of acetyl groups) affected the process conditions (pH of the liquor) and may have contributed to the formation of a significant amount of pseudo- extractives in eucalyptus and pseudo-lignin in bagasse and straw.

5. Appendix

Appendix A: FTIR interpretation Wavenumber (cm-1) Functional groups Interpretation 3400 – 3200 -OH stretching H bond hydroxyl groups stretching 2970 – 2860 C-Hn stretching Symmetric vibration 1765 – 1715 C=O stretchin Ketone and ester carbonyl groups 1628 Water absorption 1560-1510 C=O stretchin Ketone and carbonyl 1510 Lignin 1472 C-H or OH Deformation 1470-1430 0-CH3 Methoxyl -O-CH3 1467, 1414 C-H, OH and CH2 C-H, OH and CH2 bending 1402 CH bending 1381 C-H Deformation 1370 C-CH3 1270 C-O-C 1252 C-H or OH Deformation 1241 C-O stretching 1232 C-O-C stretching Aryl-alkyl ether 1170, 1082 C-O-C stretching vibration Pyranose ring skeletal 1175 Arabinose chain 1169 CH3-O-C CH3 stretchin, deformation and stretching vibration 1160 Arabinose residues 1108 C-OH OH association 1060 C-OH C-O stretching and C-O 1051 C-O Stretching 1043 C-O-(H) COH bending modes 1039 Xylan 990 Arabinosyl units 950 - 700 C-H deformation C-H deformation 900 - 700 C-H Aromatic hydrogen 897 ß-glucosidic linkages Signal typical of acetylated xylan and signals typical of lignin.

Sources: Egües et al. (2014), Bian et al. (2010), Xu et al. (2010), Brienzo et al. (2009), Magaton et al. (2008), Yang et al. (2007), Chaikumpollert et al. (2004), Sun et al. (2004), Fang et al. (2002), Kačuráková et al. (1998) and Gupta et al. (1987).

6. Acknowledgments

To God, the GREATER LOVE, that keeps me and leads me. Thanks also for make this beautiful journey possible. To Professor Mikael Lindström, my supervisor, and the Dr. Olena Sevastyanova, my co- supervisor, for all the help, patience and supervision gently offered during this entire project. Your dedication, knowledge and commitment certainly are the reasons for the existence of this thesis. Thank you! To Antonio and Francisco, for the friendship and the productive cooperation. Thanks for help me to make this project possible. To my advisor in Brazil (UFV) the Professor Jorge Luiz Colodette, for the guidance, understanding and teaching and all the contribution you offered that are helping to become the professional I want to be. To Capes and CNPq foundations, for the scholarship in Brazil and Sweden (Sandwich program), respectively. To my parents, José Helvécio and Mariínha and my brother Diego, for the unconditional love, for all the support and for understand my absence. To my other relatives, for all motivation and confidence and for share with me my fears and achievements. To my colleagues at the Fiber and Polymer Technology Department and Wallenberg Wood Science Center and all the amazing people that I have met during the last two years, especially to Verónica, the most “chévere” friend and to Rafa, the most amazing gift the Northern lands could give me. To my other friends from LCP (Viçosa), Coronel Fabriciano, Stockholm or even other places around the world, for all the encouragement and friendship. For all of you my sincere gratitude,

THANKS!

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