Enhancing bioconversion process of lignocellulosis biomass through ionic liquid pretreatment and application of pervaporation for separation of produced bioethanol

2 Bioethanol can be produced from lignocellulosic biomass via the following three steps: 1 pretreatment of lignocellulose to enhance the biomass digestibility; 2 hydrolysis of cellulose

Doctoral Dissertation

Enhancing bioconversion process of lignocellulosic biomass through ionic liquid pretreatment and application

of pervaporation for separation

of produced bioethanol

Department of Bioenergy Science and Technology

Graduate School, Chonnam National University

Trinh, Thi Phi Ly

August 2016

Doctoral Dissertation

Enhancing bioconversion process of lignocellulosic biomass through ionic liquid pretreatment and application

of pervaporation for separation

of produced bioethanol

Department of Bioenergy Science and Technology

Graduate School, Chonnam National University

Trinh, Thi Phi Ly

August 2016

So Yeon, Gyeong Jin and Om Min who assist me in the experiments

Finally, I express my deepest sense to my entire family for their love, inspiration and support me Thank my husband and my daughter for giving me a happy and awesome time in Korea

Trinh Thi Phi Ly Chonnam National University August 2016

II

TABLE OF CONTENTS

Acknowledgement I Table of contents II List of Figures VI List of Table VIII

General introduction 1

References 4

Chapter 1 Characterization of [Bmim]Cl pretreatment of mixed softwoods 8

Abstract 9

1 Introduction 10

2 Materials and Methods 12

2.1 Materials 12

2.2 Pretreatment of softwood by ionic liquid 12

2.3 Characterization of native and pretreated biomass 12

2.4 Analysis of carbohydrates and lignin 13

2.5 Enzymatic hydrolysis 14

3 Results and Discussion 15

3.1 Characterizations of ionic liquid pretreated softwoods 15

3.2 Effect of the pretreatment variables on the compositions and recovery of softwood 22

3.3 Effect of the pretreatment variables on enzymatic hydrolysis 25

4 Conclusions 29

5 References 30

III

Chapter 2 An efficient approach to [Bmim]Ac pretreatment of mixed

softwoods using optimization of process and recycling of ionic liquid 35

Abstract 36

1 Introduction 37

2 Materials and Methods 39

2.1 Materials 39

2.2 Pretreatment of softwood with ionic liquid 39

2.3 Separate hydrolysis and fermentation (SHF) 40

2.4 Characterization of native and pretreated biomass 40

2.5 Analysis of fermentable sugars and bioethanol 41

2.6 Experimental design and process analysis with RSM 42

3 Results and Discussion 43

3.1 Effects of pretreatment variables on the recovery and composition of biomass 43

3.2 Effects of pretreatment variables on enzymatic hydrolysis 48

3.3 Recycling and reuse of ionic liquid 53

3.4 Cellulose crystallinity of pretreated softwoods 57

3.5 Fermentation of hydrolysates 60

4 Conclusions 62

5 References 63

Chapter 3 Acidified ionic liquid pretreatment of various feedstocks for bioethanol production 68

Abstract 69

1 Introduction 70

2 Materials and Methods 72

2.1 Materials 72

IV

2.2 Acidified IL pretreatment of different biomass 72

2.3 Separate hydrolysis and fermentation 73

2.4 Characterization of untreated and pretreated biomass 73

2.5 Analysis of fermentable sugars and bioethanol 73

3 Results and discussion 75

3.1 Effect of acid type, acid concentration, and reaction time 75

3.2 Effect of water content and biomass loading 78

3.3 Acidified IL pretreatment of different feedstocks 81

3.4 XRD analysis of acidified IL pretreated feedstocks 85

3.5 Separate hydrolysis and fermentation of pretreated feedstocks 87

4 Conclusions 90

5 References 91

Chapter 4 Separation and concentration of bioethanol produced from mixed softwood by pervaporation 96

Abstract 97

1 Introduction 98

2 Materials and Methods 100

2.1 Experimental setup for pervaporation 100

2.2 Bioethanol production from mixed softwood 102

2.3 Analysis 102

3 Results and discussion 104

3.1 Influence of initial ethanol concentration on the pervaporation performance 104

3.2 Influence of operating temperature on pervaporation performance 107

3.3 Pervaporation of bioethanol produced by fermentation 110

4 Conclusions 115

V

5 References 116

List of Publications 120 Abstract in Korean 121

Fig 4 SEM images of untreated and pretreatedsamples (800×magnification):

(a) untreated mixed softwood; (b) pretreated at 70 oC for 18 h, (c) pretreated at

120 oC for 18 h, and (d) pretreated at 130 oC for 18 h 21

Fig 5 Effect of pretreatment temperatures on biomass digestibility and

glucose yield (pretreatment time of 18 h) 27

Chapter 2

Fig 1 Response surface plots of the effect of pretreatment conditions on the

recovery of solid and carbohydrates: (a) solid recovery, (b) glucan recovery,

and (c) xylan recovery 45

Fig 2 Response surface plots of the effect of pretreatment conditions

on enzymatic saccharification performance: (a) cellulose digestibility,

(b) xylan digestibility, and (c) fermentable sugar yield 50

Fig 3 Enzymatic saccharification of original and recycled [Bmim]Ac

pretreated softwoods 55

Fig 4 XRD patterns of original and recycled [Bmim]Ac pretreated softwoods (1):

untreated; (2): original IL pretreatment; (3) – (8): 1st- 6th recycled IL pretreatment 58

Fig 5 Bioethanol production by the fermentation of [Bmim]Ac

pretreated softwood hydrolysates 61

VII

Chapter 3

Fig 1 Cellulose digestibility of untreated and pretreated biomass 84 Fig 2 Crystallinity index of untreated and pretreated biomass 86 Fig 3 Ethanol fermentation of pretreated biomass 88

Chapter 4

Fig 1 Pervaporation performance with initial ethanol concentration in the

Synthetic solutions 105

Fig 2 Permeate ethanol concentration with initial ethanol concentration

in the synthetic solutions 106

Fig 3 Effect of temperature on pervaporation performance in the synthetic

solutions 108

VIII

LIST OF TABLES

Chapter 1

Table 1 Effect of pretreatment temperatures on the compositions and the recovery

of biomass components 23

Table 2 Effect of pretreatment times on the compositions and the recovery of biomass components 24

Table 3 Effect of pretreatment times on the biomass digestibility and the glucose yield 28

Chapter 2 Table 1 Central composite design of two variables with compositions for ionic liquid pretreatment of mixed softwoods 46

Table 2 Main compositions of untreated and pretreated mixed softwoods 47

Table 3 Central composite design of two variables with bioconversion efficiency for ionic liquid pretreatment of mixed softwoods 51

Table 4 Regression coefficients and ANOVA of the response surfaces 52

Table 5 Solid recovery and main compositions in the recycling and reuse of [Bmim]Ac pretreated mixed softwoods 56

Table 6 Crystallinity index of [Bmim]Ac pretreated softwoods 59

Chapter 3 Table 1 Effect of acid types and reaction time on glucose and xylose yield 76

Table 2 Effect of acid concentration on glucose and xylose yield 77

Table 3 Effect of biomass loading on glucose and xylose yield 79

Table 4 Effect of water content on glucose and xylose yield 80

Table 5 Chemical compositions of untreated and pretreated biomass 82

estimated from experiments with ethanol (6.4 wt%) and water mixtures 109

Table 3 The consecutive pervaporative separation of fermentation broth 112 Table 4 Pervaporation performance of fermentation broth and synthetic

solution 113

Table 5 Pervaporation of synthetic solutions 114

1

Enhancing bioconversion process of lignocellulosic biomass through ionic liquid pretreatment and application of pervaporation for separation of produced bioethanol

Trinh, Thi Phi Ly

Department of Bioenergy Science and Technology

Chonnam National University (Supervised by Professor Lee Won Heong)

General introduction

Bioethanol is one of the promising alternative fuels that can be produced from biomass

by microbial fermentation The conversion of carbohydrates to ethanol has recently been more considered to the production of biofuels Until now, most bioethanol has been produced from starch and sucrose-containing feedstocks such as corn, sugarcane, cassava and potato However, concerns have been appeared that the production of bioethanol from edible biomass directly affects food production and security worldwide Lignocellulosic biomass, the most abundant bio-material on Earth, comes from various sources including agricultural and forest residues, and municipal waste An interest in the utilization of lignocellulosic biomass for the bioethanol production has been increased due to larger quantity and lower cost compared to starch and sucrose based materials (Brandt et al., 2013)

2

Bioethanol can be produced from lignocellulosic biomass via the following three steps: (1) pretreatment of lignocellulose to enhance the biomass digestibility; (2) hydrolysis of cellulose and hemicellulose into fermentable sugars; and (3) microbial fermentation of the sugars to bioethanol (Zhang et al., 2007)

Lignocellulosic biomass consists of cellulose, hemicellulose, and lignin, which are highly recalcitrant to chemicals or enzymes due to their complex arrangement and high degree of polymerization (Himmel et al., 2007) Many pretreatment technologies have been developed to overcome the recalcitrance of lignocellulosic biomass and subsequently to improve biomass digestibility including acid pretreatment, alkali pretreatment, steam explosion, organosolv, biological pretreatment, and others

Ionic liquids (ILs) are defined as molten salts that exist as liquids at relatively low temperature (Pinkert et al., 2009) ILs appear to be a green alternative to traditional organic solvents because of their interesting chemical and physical properties such as low vapor pressure, chemical and thermal stability, non-flammability, and the ability to dissolve a wide range of organic and inorganic compounds (Liu et al., 2012; Wang et al., 2012) ILs are capable of disrupting the hydrogen bonding network in cellulose due to the association of anions and cations in the IL with the hydroxyl protons and oxygen in cellulose, respectively, thereby resulting in the dissolution of cellulose (Cheng et al., 2011; Li et al., 2010; Tan & Lee, 2012) Advantages of pretreating lignocellulosic biomass with ILs over conventional pretreatment methods include low toxicity, no inhibitor formation, easy processing, and recyclability of ILs (Ha et al., 2011) (Nguyen et al., 2010; Zhu et al., 2012) ILs have been observed in the pretreatment of a variety of feedstocks such as woody biomass (Sun et al., 2009; Sun et al., 2013b; Torr et al., 2012; Trinh et al., 2015; Wu et al., 2013), and agricultural residues (Ang et al., 2012; Perez-Pimienta et al., 2013; Qiu & Aita, 2013; Sun et al., 2013a; Xu et al., 2012) In fact, IL pretreatment significantly improves biomass properties and enhances the production of fermentable sugars and fermentation efficiency by

a removal of lignin, modification of crystalline structure and depolymerization of

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hemicelluloses (Cox & Ekerdt, 2013; Perez-Pimienta et al., 2013; Weerachanchai et al.,

2012; Xu et al., 2012) However, high cost and viscous behavior of ILs limit their potential

applications for biomass processing Many studies have approached an effective and

economical pretreatment by optimizing the processing conditions, adding water to IL

solution and recycling ILs (Auxenfans et al., 2012; Fu & Mazza, 2011; Lee et al., 2013; Qiu

& Aita, 2013; Qiu et al., 2014)

Following fermentation process, produced bioethanol must be separated and

concentrated using separation and purification technique as its concentration is very low in

fermentation broth Currently, distillation has been used for the separation and purification of

bioethanol, but it has high energy requirements and low efficiency particularly at low ethanol

concentrations Pervaporation is one of the most promising approaches to recover alcohols

produced from fermentation (Beltran et al., 2013; Shao & Huang, 2007) The mass transport

across pervaporation membranes is governed by three processes: (1) sorption of selective

molecules in the surface of the membrane; (2) diffusion of the dissolved molecules across the

membrane; (3) desorption of the dissolved molecules into the vapor phase on the permeate

side (Shao & Huang, 2007) In the pervaporation process, components having higher affinity

and diffusivity with active layer in the membrane are preferentially removed from a liquid

mixture (Mohammadi et al., 2005; Wu et al., 2006)

In this work, we present four topics: (1) Characterization of [Bmim]Cl pretreatment of

mixed softwoods; (2) An efficient approach to [Bmim]Ac pretreatment of mixed softwoods

using optimization of process conditions and recycling of ionic liquid; (3) Acidified ionic

liquid pretreatment of various feedstocks for bioethanol production; (4) Separation and

concentration of bioethanol produced from mixed softwood by pervaporation

4

References for General introduction

1 Ang, T.N., Ngoh, G.C., Chua, A.S., Lee, M.G 2012 Elucidation of the effect of ionic liquid pretreatment on rice husk via structural analyses Biotechnol Biofuels, 5(1), 67

2 Auxenfans, T., Buchoux, S., Djellab, K., Avondo, C., Husson, E., Sarazin, C 2012 Mild pretreatment and enzymatic saccharification of cellulose with recycled ionic liquids towards one-batch process Carbohydrate Polymers, 90(2), 805-813

3 Beltran, A.B., Nisola, G.M., Vivas, E.L., Cho, W., Chung, W.J 2013 Poly(octylmethylsiloxane)/oleyl alcohol supported liquid membrane for the pervaporative recovery of 1-butanol from aqueous and ABE model solutions Journal of Industrial and Engineering Chemistry, 19(1), 182-189

4 Brandt, A., Grasvik, J., Hallett, J.P., Welton, T 2013 Deconstruction of lignocellulosic biomass with ionic liquids Green Chemistry, 15(3), 550-583

5 Cheng, G., Varanasi, P., Li, C., Liu, H., Melnichenko, Y.B., Simmons, B.A., Kent, M.S., Singh, S 2011 Transition of cellulose crystalline structure and surface morphology of biomass as a function of ionic liquid pretreatment and its relation to enzymatic hydrolysis Biomacromolecules, 12(4), 933-41

6 Cox, B.J., Ekerdt, J.G 2013 Pretreatment of yellow pine in an acidic ionic liquid: extraction of hemicellulose and lignin to facilitate enzymatic digestion Bioresour Technol, 134, 59-65

7 Fu, D., Mazza, G 2011 Optimization of processing conditions for the pretreatment of wheat straw using aqueous ionic liquid Bioresour Technol, 102(17), 8003-10

8 Ha, S.H., Mai, N.L., An, G., Koo, Y.M 2011 Microwave-assisted pretreatment of cellulose in ionic liquid for accelerated enzymatic hydrolysis Bioresour Technol, 102(2), 1214-9

9 Himmel, M.E., Ding, S.Y., Johnson, D.K., Adney, W.S., Nimlos, M.R., Brady, J.W., Foust, T.D 2007 Biomass recalcitrance: Engineering plants and enzymes for biofuels production Science, 315(5813), 804-807

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10 Lee, K.M., Ngoh, G.C., Chua, A.S.M 2013 Process optimization and performance evaluation on sequential ionic liquid dissolution-solid acid saccharification of sago waste Bioresource Technology, 130, 1-7

11 Li, C.L., Knierim, B., Manisseri, C., Arora, R., Scheller, H.V., Auer, M., Vogel, K.P., Simmons, B.A., Singh, S 2010 Comparison of dilute acid and ionic liquid pretreatment

of switchgrass: Biomass recalcitrance, delignification and enzymatic saccharification Bioresource Technology, 101(13), 4900-4906

12 Liu, C.Z., Wang, F., Stiles, A.R., Guo, C 2012 Ionic liquids for biofuel production: Opportunities and challenges Applied Energy, 92, 406-414

13 Mohammadi, T., Aroujalian, A., Bakhshi, A 2005 Pervaporation of dilute alcoholic mixtures using PDMS membrane Chemical Engineering Science, 60(7), 1875-1880

14 Nguyen, T.A.D., Kim, K.R., Han, S.J., Cho, H.Y., Kim, J.W., Park, S.M., Park, J.C., Sim, S.J 2010 Pretreatment of rice straw with ammonia and ionic liquid for lignocellulose conversion to fermentable sugars Bioresource Technology, 101(19), 7432-7438

15 Perez-Pimienta, J.A., Lopez-Ortega, M.G., Varanasi, P., Stavila, V., Cheng, G., Singh, S., Simmons, B.A 2013 Comparison of the impact of ionic liquid pretreatment on recalcitrance of agave bagasse and switchgrass Bioresour Technol, 127, 18-24

16 Pinkert, A., Marsh, K.N., Pang, S.S., Staiger, M.P 2009 Ionic Liquids and Their Interaction with Cellulose Chemical Reviews, 109(12), 6712-6728

17 Qiu, Z., Aita, G.M 2013 Pretreatment of energy cane bagasse with recycled ionic liquid for enzymatic hydrolysis Bioresour Technol, 129, 532-7

18 Qiu, Z.H., Aita, G.M., Mahalaxmi, S 2014 Optimization by response surface methodology of processing conditions for the ionic liquid pretreatment of energy cane bagasse Journal of Chemical Technology and Biotechnology, 89(5), 682-689

19 Shao, P., Huang, R.Y.M 2007 Polymeric membrane pervaporation Journal of Membrane Science, 287(2), 162-179

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20 Sun, N., Liu, H.B., Sathitsuksanoh, N., Stavila, V., Sawant, M., Bonito, A., Tran, K., George, A., Sale, K.L., Singh, S., Simmons, B.A., Holmes, B.M 2013a Production and extraction of sugars from switchgrass hydrolyzed in ionic liquids Biotechnology for Biofuels, 6

21 Sun, N., Rahman, M., Qin, Y., Maxim, M.L., Rodriguez, H., Rogers, R.D 2009 Complete dissolution and partial delignification of wood in the ionic liquid 1-ethyl-3-methylimidazolium acetate Green Chemistry, 11(5), 646-655

22 Sun, Y.C., Xu, J.K., Xu, F., Sun, R.C 2013b Structural comparison and enhanced enzymatic hydrolysis of eucalyptus cellulose via pretreatment with different ionic liquids and catalysts Process Biochemistry, 48(5-6), 844-852

23 Tan, H.T., Lee, K.T 2012 Understanding the impact of ionic liquid pretreatment on biomass and enzymatic hydrolysis Chemical Engineering Journal, 183, 448-458

24 Torr, K.M., Love, K.T., Cetinkol, O.P., Donaldson, L.A., George, A., Holmes, B.M., Simmons, B.A 2012 The impact of ionic liquid pretreatment on the chemistry and enzymatic digestibility of Pinus radiata compression wood Green Chemistry, 14(3), 778-787

25 Trinh, L.T.P., Lee, Y.J., Lee, J.W., Lee, H.J 2015 Characterization of ionic liquid pretreatment and the bioconversion of pretreated mixed softwood biomass Biomass & Bioenergy, 81, 1-8

26 Wang, H., Gurau, G., Rogers, R.D 2012 Ionic liquid processing of cellulose Chemical Society Reviews, 41(4), 1519-1537

27 Weerachanchai, P., Leong, S.S.J., Chang, M.W., Ching, C.B., Lee, J.M 2012 Improvement of biomass properties by pretreatment with ionic liquids for bioconversion process Bioresource Technology, 111, 453-459

28 Wu, H., Liu, L., Pan, F.S., Hu, C.L., Jiang, Z.Y 2006 Pervaporative removal of benzene from aqueous solution through supramolecule calixarene filled PDMS composite membranes Separation and Purification Technology, 51(3), 352-358

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29 Wu, L., Lee, S.H., Endo, T 2013 Effect of dimethyl sulfoxide on ionic liquid methylimidazolium acetate pretreatment of eucalyptus wood for enzymatic hydrolysis Bioresource Technology, 140, 90-96

1-ethyl-3-30 Xu, F., Shi, Y.C., Wang, D 2012 Enhanced production of glucose and xylose with partial dissolution of corn stover in ionic liquid, 1-Ethyl-3-methylimidazolium acetate Bioresour Technol, 114, 720-4

31 Zhang, Y.H.P., Ding, S.Y., Mielenz, J.R., Cui, J.B., Elander, R.T., Laser, M., Himmel, M.E., McMillan, J.R., Lynd, L.R 2007 Fractionating recalcitrant lignocellulose at modest reaction conditions Biotechnology and Bioengineering, 97(2), 214-223

32 Zhu, Z., Zhu, M., Wu, Z 2012 Pretreatment of sugarcane bagasse with NH4OH-H2O2 and ionic liquid for efficient hydrolysis and bioethanol production Bioresour Technol,

119, 199-207

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Chapter 1

Characterization of [Bmim]Cl pretreatment of mixed

softwoods

accessibility

Many pretreatment technologies have been developed to overcome the recalcitrance of woody biomass and subsequently to improve biomass digestibility including acid pretreatment, alkali pretreatment, steam explosion, organosolv, biological pretreatment, and others Recently, ionic liquids (ILs), which are regarded as green solvents, have attracted much attention due to their interesting properties such as low vapor pressure, chemical and thermal stability, non-flammability, and phase behavior (Liu et al., 2012; Wang et al., 2012) Furthermore, ILs can be easily prepared using different cations and anions, resulting in hydrophobic or hydrophilic types (Fukaya & Ohno, 2013; Trinh et al., 2013; Weingartner, 2008) Ionic liquids have been utilized in the dissolution of both softwood and hardwood (Sun et al., 2009), depolymerization of hemicelluloses for yellow pine (Cox & Ekerdt, 2013), and also delignification of eucalyptus, switchgrass and bagasse (Perez-Pimienta et al., 2013; Varanasi et al., 2012) ILs have been reported as promising solvents capable of disrupting the inter- and intra-molecular hydrogen bonds in the native cellulose crystalline structure and breaking the major chemical linkages in matrix polymers of various biomass feedstocks

11

(Cheng et al., 2011; Li et al., 2010; Tan & Lee, 2012)

IL pretreatment typically decreases cellulose crystallinity, partial removal of lignin and hemicellulose or increase in specific surface area, etc, thus enhancing the digestibility and the fermentability of sugars (Perez-Pimienta et al., 2013; Qiu et al., 2012; Torr et al., 2012; Weerachanchai et al., 2012) The previous works showed that pretreatment temperature and time are crucial factors, which intensively affect saccharification performance (Li et al., 2009; Shill et al., 2012; Uju et al., 2012) Therefore, choosing the suitable operation conditions in the biomass pretreatment process is necessary to increase the pretreatment efficiency and minimize the energy consumption (Li et al., 2011; Uju et al., 2012; Yoon et al., 2012)

As far as we know, only a few studies were reported for the ionic liquid pretreatment of woody biomass and characteristic properties of pretreated biomass Indeed, woody biomass

is always hard to process due to the tough and strong physical structure and high degree of lignifications (Zhu & Pan, 2010) In this study, mixed softwoods were pretreated with an ionic liquid, 1-butyl-3-methylimidazolium chloride ([Bmim]Cl), under a wide range of temperatures and duration times The changes in the crystalline cellulose, surface morphology, structural and chemical features of pretreated biomass were characterized by XRD, FT-IR and SEM Moreover, the enzymatic hydrolysis experiments were performed to estimate the effect of ionic liquid pretreatment on the bioconversion efficiency

12

2 Materials and methods

2.1 Materials

Mixed softwood (Pinus rigida and Pinus densiflora) chips were purchased from Poong

Lim Inc (Daejeon, Korea) and ground into powder (40 – 60 mesh) Ionic liquid, methylimidazolium chloride, [Bmim]Cl) (> 95% purity), was purchased from Sigma-Aldrich For enzyme hydrolysis, cellulase (Celluclast 1.5L), -glucosidase (NS 50013) and xylanase (NS 22036) were kindly provided by Novozyme

(1-butyl-3-2.2 Pretreatment of softwood by ionic liquid

Pretreatment was conducted by dissolution of about 0.2 g of mixed softwood (dry weight basis) with 4 g [Bmim]Cl in a glass tube placed into an oil bath (RCX – 1000S, EYELA) with magnetic stirring at 500 rpm Pretreatment reactions were conducted at temperatures of 70 – 150 oC and the duration time of 5 – 24 h For biomass recovery, 50 ml

of distilled water was added to the pretreatment solution and the solution was mixed with magnetic stirring at 500 rpm for at least 1 h at room temperature

Regenerated biomass was separated by vacuum filtration using a ceramic funnel with

a mixed cellulose ester membrane of 0.45 m; the biomass was then washed three times with distilled water (3 × 50 ml) to remove any residual ionic liquid Wet regenerated biomass was directly used for enzymatic saccharification and the other fraction of solid residue was dried

in an oven at 75 oC for 24 h for compositional analysis and characterizations The recovery

of solid and sugars were calculated according to the following equations:

Solid recovery (%) =Mass of raw biomass as dry basisMass of regenerated biomass × 100 (1)

Sugar recovery (%) =Sugar in pretreated biomass (%)× solid recovery (%)

Sugar in native biomass (%) (2)

2.3 Characterization of native and pretreated biomass

The crystallinity of the cellulose in native and pretreated biomass was analyzed using an

13

X-ray diffractometer (PANalytical X'Pert PRO) equipped with Cu radiation ( = 1.5406 Å ;

40 kV; 30 mA) The samples were scanned within 5 – 30o with a step size of 0.013o The crystallinity index (CrI) was calculated according to the XRD peak height method using the following equation (Park et al., 2010):

𝐶𝑟𝐼 =𝐼002 −𝐼 𝑎𝑚

where I 002 is the scattered intensity at the main peak and I am the scattered intensity due to the amorphous portion evaluated as the minimum intensity between the main and secondary peaks

Changes in the surface morphology of biomass after pretreatment were analyzed using scanning electron microscopy (SEM) (Hitachi S-4700) at an accelerating voltage of 5 kV Chemical structures of untreated and pretreated biomass were characterized by FT-IR (Bruker Optik GmbH VERTEX 70) The spectra were recorded within the range of 4000–

380 cm-1 with 4 scans at a resolution of 4 cm-1

2.4 Analysis of carbohydrates and lignin

Structural carbohydrates of untreated and pretreated softwood were determined through the two-step acid hydrolysis procedure of the National Renewable Energy Laboratory TP-510-42618 (Sluiter et al., 2012) Each sample was analyzed in triplicate Concentration of fermentable sugar was measured by HPLC (Waters 2695 system, MA, USA) equipped with

an Aminex HPX-87P column (300×7.8 mm, Bio-Rad, Hercules, CA, USA) and a refractive index detector (Waters 2414 system) The analysis was performed with 5 mM H2SO4 as the mobile phase at an isocratic flow rate of 0.6 mL/min

For lignin analysis in biomass, the acid insoluble lignin was estimated by gravimetric analysis of the solid residue on the glass filter after drying at 105 oC for 24 h In addition, the acid soluble lignin was measured using UV-Vis spectrophotometer at 205 nm, with the extinction coefficient of 110 L g-1 cm-1 (Lee et al., 2009)

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2.5 Enzymatic hydrolysis

Enzymatic hydrolysis of the wet pretreated samples was conducted at 50 oC for 96 h and

5% (w/w) of the softwood (dry weight basis) in a shaking incubator at 150 rpm and the

results were compared to those of untreated biomass The mixture containing biomass and

0.1 M sodium citrate buffer (pH 4.8) with 2% sodium azide was prepared prior to the

enzyme cocktail loading of cellulase 17.5 FPU/g, -glucosidase 6.25 CBU/g and xylanase 25

FXU/g After saccharification, the liquid stream separated by centrifugation from the solid

fraction was applied for the fermentable sugar analysis Concentrations of fermentable

sugars were determined using high performance liquid chromatography (HPLC) as

mentioned below All enzymatic hydrolysis experiments were conducted in duplicate The

biomass digestibility and glucose yield were calculated based on the following equations:

Glucan digestibility (%) =Glucose produced via enzymatic hydrolysis × 0.9Glucan in pretreated biomass × 100 (3) Xylan digestibility (%) =Xylose produced via enzymatic hydrolysis × 0.88

Xylan in pretreated biomass × 100 (4) Glucose yield (%) =Glucose produced via enzymatic hydrolysis

Glucan in native biomass × 1.11 × 100 (5)

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3 Results and discussion

3.1 Characterizations of ionic liquid pretreated softwoods

After [Bmim]Cl pretreatment, the changes in the crystalline cellulose of pretreated biomass were observed through XRD analyses and the results were shown in Fig 1 In the untreated softwood, two sharp peaks were found at angles of 15o and 22o, corresponding to (101) and (002) lattice planes of crystalline cellulose I polymorph, respectively (Moniruzzaman & Ono, 2013; Qiu et al., 2012) As shown in the figure, XRD patterns of softwood pretreated at 70 – 100 oC resemble those of the untreated sample but a decrease in intensity of two main peaks was clearly observed It is thought that cellulose crystals are partially swollen with [Bmim]Cl due to the structural heterogeneity and complexity of cell-wall constituents at mild pretreatment conditions (Zhang et al., 2014) For the pretreatmented biomass at high temperatures (120 and 130 oC), the main peak at 22o split into two peaks with lower intensities and the peak at around 15o disappeared, suggesting that cellulose I was strongly modified to a low order structure (Socha et al., 2013; Uju et al., 2012; Zhang et al., 2014) The structure with a low degree of order, which is recognized as amorphous region, is less recalcitrant and more readily digestible compared to cellulose structure in untreated biomass (Cheng et al., 2011; Li et al., 2010) In addition, a new peak appeared at 12oindicates the characteristic peak of cellulose II lattice (Cheng et al., 2011) It is assumed that the van der Waals interaction between hydrogen-bonded layers of cellulose II is weaker than that of cellulose I (Wada et al., 2010) Therefore, cellulose II is more efficiently hydrolyzed

by cellulase enzymes than cellulose I (Cheng et al., 2011; Wada et al., 2010) Softwood pretreated by [Bmim]Cl at the temperature pretreatment of 120 – 130 oC caused transformation of crystalline cellulose into amorphous structure and formation of cellulose II, thus facilitating the subsequent bioconversion process

The crystallinity index (CrI) refers to the relative amount of crystalline material in cellulose (Cheng et al., 2011; Park et al., 2010) The CrI of untreated softwood was estimated

to 39.5% After [Bmim]Cl pretreatment, the decrease CrI of regenerated softwoods was

16

identified as shown in Fig 2 Particularly, decrease in the CrI values was observed from 24%

to 16% when the pretreatment temperature increased from 70 oC to 130 oC (see Fig 2a) The fraction of cellulose swollen in IL increased with elevated temperature transformed into disordered structures during the regeneration process, leading to decrease in the crystallinity

of biomass (Zhang et al., 2014) Similarly, the decreasing trend of the CrI with extended pretreatment time was observed as illustrated in Fig 2b

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Fig 3 shows the FT-IR spectra of untreated and pretreated softwoods with different pretreatment temperatures The absorbance bands at 1155 cm-1 and 2882 cm-1 disappeared in the softwood pretreated at 130 oC The peak changes are related to a C–O–C asymmetric bridge stretching vibration in hemicellulose and a C-H stretch of cellulose (Karatzos et al., 2012; Labbe et al., 2005; Li et al., 2011) In addition, the peak at 1725 cm-1 (C=O stretching vibration in acetyl groups of hemicelluloses) showed decreased intensity in the softwood pretreated at 130 oC, indicating that [Bmim]Cl pretreatment caused the dissolution of carbohydrates at a high temperature The lignin peaks at 1599 and 1509 cm-1 (C=C stretching vibration in aromatic ring), and 1263 cm-1 (guaiacyl ring and C-O stretching vibration) maintaining their intensity were found in all pretreated materials Through the FT-IR spectra regarding the softwood pretreatment with different temperatures, a significant change in the carbohydrates was observed, but lignin has remained throughout the pretreated softwoods The surface morphology of pretreated and untreated softwoods was examined by SEM Softwood pretreatment at 70 – 100 oC showed a corrugated surface while untreated softwood exhibited a compact and rigid surface Especially, more porous structures were observed in samples pretreated at 120 – 130 oC, revealing the disruption of tissue network and a loss of high order structure which is consistent with the results obtained by XRD (see Fig 4)

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Fig 3 FTIR spectra of untreated and IL pretreated softwoods for 18 h: (a) 2885 cm-1; (b)

1725 cm-1; (c) 1599 cm-1; (d) 1509 cm-1; (e) 1263 cm-1; (f) 1155 cm-1

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3.2 Effect of the pretreatment variables on the compositions and recovery of softwood

Firstly, the influence of the pretreatment temperatures on the chemical compositions and the recovery of biomass components were investigated and the results were shown in Table 1 The solid recovery of regenerated softwood significantly decreased from 91% to 73% as the pretreatment temperature increased from 70 oC to 130 oC at the 18 h reaction Moreover, the recovery of carbohydrates (glucan, xylan and mannan) in the pretreated biomass decreased with increasing temperatures It is noted that the recovery of xylan and mannan was lower than that of glucan at 130 oC, indicating that [Bmim]Cl at high temperatures could dissolve hemicellulose more effectively than cellulose (Karatzos et al., 2012) The removal of hemicelluloses is believed to expose the crystalline cellulose core of cell-wall microfibrils, which may enhance the cellulose degradation through enzymatic hydrolysis (Himmel et al., 2007; Karatzos et al., 2012) The lignin content slightly increased as the pretreatment temperature increased from 70 oC to 130 oC The results were consistent with the evidence representing in FT-IR spectra as discussed earlier

Table 2 shows the influence of the reaction times on the composition and the recovery

of biomass components A decrease in the recovery of solid residues and sugars was observed with increasing pretreatment time from 5 h to 24 h for the pretreatment at 120 oC and 130 oC The softwood pretreatment at 120 oC showed the negligible change in the carbohydrates and lignin content compared to the untreated sample However, pretreatment

at 130 oC resulted in considerable decrease in the solid residues and sugars recovery The recovery of solid residues and glucan was measured to 53% and 49%, respectively, in the pretreated biomass at 130 oC for 24 h The lower recovery of carbohydrates at higher temperature and longer time is attributed to the solubilization of fermentable sugars into the liquid fraction (Li et al., 2011; Qiu et al., 2012; Sun et al., 2009) It is interesting that the [Bmim]Cl pretreatment at given conditions showed a little effect on delignification of mixed softwood (Zhu & Pan, 2010)

Acid insoluble lignin (%)

Total lignin (%)

Glucan (%)

Xylan (%)

Mannan (%)

Solid recovery (%)

Glucan recovery (%)

Xylan recovery (%)

Mannan recovery (%) Untreated 1.0  0.0 32.0  0.4 33.0  0.4 41.2  0.7 5.4  0.5 9.8  0.8 100 100 100 100

Acid soluble lignin (%)

Acid insoluble lignin (%)

Total lignin (%)

Glucan (%)

Xylan (%)

Mannan (%)

Solid recovery (%)

Glucan recovery (%)

Xylan recovery (%)

Mannan recovery (%)

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3.3 Effect of the pretreatment variables on enzymatic hydrolysis

Enzymatic hydrolysis of the pretreated and untreated softwoods was conducted with

three different enzymes, i.e., cellulase, -glucosidase, and xylanase; the results are illustrated

in Fig 5 and Table 3 The enzymatic hydrolysis of untreated softwood released only 5% of initial glucan as glucose and 4% of initial xylan as xylose monomer as shown in the table When pretreatment temperature increased from 70 to 130 oC, the cellulose and xylan digestibility notably increased from 29% to 97% and 29% to 46%, respectively (see Fig 5) Significant enhancements on the enzymatic degradation were found especially in the pretreatment at 120 and 130 oC (see Table 3) The results were in good agreement with the literature report in which pretreatment at higher temperatures resulted in higher sugar conversion (Weerachanchai et al., 2012) Moreover, the transformation of crystalline cellulose into amorphous structure and the formation of more hydrolysable cellulose II through XRD analysis were observed at the pretreatment of 120 – 130 oC

As shown in Fig 5, the maximum digestion of cellulose was measured to 97% at the pretreatment of 130 oC and 18 h It is assumed that high temperature reaction caused less crystalline cellulose structure having fewer and weaker inter-chain hydrogen bonds that substantially promote cellulose accessible to hydrolyzing enzymes (Bergenstrahle et al., 2007)

In this study, the effect of pretreatment times on the saccharification performance was investigated in the pretreatment at 120 oC and 130 oC Table 3 shows increasing biomass digestibility with extending time When the reaction time increased from 5 h to 24 h at 120

o

C, cellulose and xylan digestibility increased from 51% to 91% and from 28% to 59%, respectively Increasing digestibility of fermentable sugars at high pretreatment temperatures could be understood by the decrease in CrI with increasing pretreatment time, as mentioned previously

Softwood pretreatment at 130 oC showed the similar tendency to the cellulose digestion

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under different pretreatment times As given in Table 2 and Table 3, softwoods pretreated at

130 oC resulted in much higher biomass digestibility even though they possess higher lignin content (Torr et al., 2012) In addition, the preferential removal of hemicelluloses essentially contributes to the improvement of enzymatic degradation as discussed above (Cox & Ekerdt, 2013; Himmel et al., 2007)

In the present work, the glucose yield was estimated to elucidate the overall to-glucose conversion According to Fig 5, glucose yield significantly increased with increasing temperature from 70 oC to 130 oC Increasing pretreatment temperatures facilitated the biomass digestion but lowered the recovery of glucan as shown in Table 2 and Table 3 At 130 oC, glucose yield reached 78% in the pretreatment for 15 h but decreased as reaction extended longer than 15 h (see Table 3) After pretreatment at 130 oC for 24 h, about 96% of cellulose was converted into glucose, overall glucose yield being only 47% based on glucan fraction of the raw biomass The maximum glucose yield at the pretreatment condition of 130 oC for 15 h was about 78%, which can be comparable to other types of ionic liquid pretreatments (Bahcegul et al., 2012; Doherty et al., 2010)

Glucan digestibility (%)

Xylan digestibility (%)

Glucose yield (%)