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S. M. MAHFUZUL ISLAM

PRETREATMENT, ENZYME PRODUCTION AND ENZYMATIC HYDROLYSIS

A Dissertation

Presented to

The Graduate Faculty of The University of Akron

In Partial Fulfillment

of the Requirements for the Degree

Doctor of Philosophy

S. M. Mahfuzul Islam

May, 2018 VALORIZATION OF GUAYULE AND SOY BIOMASS THROUGH

PRETREATMENT, ENZYME PRODUCTION AND ENZYMATIC HYDROLYSIS

S. M. Mahfuzul Islam

Dissertation

Approved: Accepted:

Advisor Department Chair Dr. Lu-Kwang Ju Dr. Michael Cheung

Committee Member Dean of the College Dr. J. Richard Elliott Dr. Donald P. Visco Jr.

Committee Member Executive Dean of the Graduate Dr. George Chase School Dr. Chand Midha

Committee Member Date Dr. Sadhan C. Jana

Committee Member Dr. Stephen Duirk

ABSTRACT

This research aims at developing guayule and soy material (soybean hull and meal) as biorefinery feedstock. Carbohydrate is the main component in guayule and soybean hull, with similar contents of cellulose (27-35%) and hemicellulose (19-22%) but different lignin contents (~36% in guayule and 1-4% in soybean hull). Carbohydrate

(30-35%) is the second largest component in soybean meal, next to protein (50%). The main objective is to increase value of these materials through enzymatic carbohydrate hydrolysis to fermentable monomeric sugars. For guayule and soybean hull, CO2-H2O based pretreatment conditions were studied for reducing generation of fermentation inhibitors while enabling high sugar conversion in subsequent enzymatic hydrolysis. The pretreatment condition for guayule was optimized at 1800 psi and 180 °C for 30 min, enabling 82% sugar conversion with low levels of hydroxymethylfurfural (0.07%), furfural (0.25%) and acetic acid (3.0%). The optimal condition for soybean hull was 1250 psi-130 °C-30 min, giving 90+% sugar conversion and negligible inhibitors generation.

The better outcomes with gentler pretreatment for soybean hull confirmed the hypothesis that lignin plays the critical role in required pretreatment severity. For soybean meal, additional objectives are to remove indigestible carbohydrates and increase protein content for food and feed uses. The main challenge lies in the complicated enzymes required, containing at least cellulase, xylanase, pectinase, α-galactosidase and sucrase activities, and their optimal composition for complete hydrolysis. A kinetic model was developed to describe the hydrolysis and help optimize the enzyme composition.

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Pectinase (half-maximum constant, Km = 6.8 U/g) and α-galactosidase (Km = 1.2 U/g) were found to be the most deficient activities produced by the chosen (and other common) enzyme producers. To improve their production, fed-batch Aspergillus niger fermentation conditions were studied and higher pectinase (19.1 ± 0.04 U/mL), α- galactosidase (15.7 ± 0.4 U/mL) and cellulase (0.88 ± 0.06 FPU/mL) activities were achieved. Soybean meal pretreatment and enzymatic hydrolysis was also studied to improve pectin hydrolysis by destabilizing Ca2+-bridged junctures with chelators and to decrease protein loss during the enzymatic hydrolysis. and protein recovery has been improved to 94.8% with only 5.2% protein loss in process. Overall this research has optimized pretreatment and enzymatic carbohydrate hydrolysis for guayule, soybean hull and meal, and increased value of these materials. New knowledge has also been gained in the effect of biomass composition on pretreatment requirement and associated inhibitor generation and in the interactions of mixed enzyme activities on complex carbohydrate structures. The work to further improve the model for enzyme hydrolysis kinetics of complex carbohydrates and the production of very high enzyme titers, along with reduction of protein loss, are the major steps forward for future soybean bioprocessing.

ACKNOWLEDGEMENTS

I would like to take this opportunity to extend my most profound gratitude to my advisor Dr. Lu-Kwang Ju for his constant support, motivation, and valuable guidance in helping me to develop my research abilities throughout this research. His scholarly way of advising with broad and deep scientific knowledge has been inspiring to me. I also would like to thank the rest of my committee members: Dr. J. Richard Elliott, Dr. George

Chase, Dr. Stephen Duirk and Dr. Sadhan C. Jana for their encouragement and insightful comments about my research. I would like to acknowledge the financial support from

United States Department of Agriculture (USDA), United Soybean Board (USB) and

Department of Chemical & Biomolecular Engineering. I am also indebted to my group members for their help and suggestions during this research. Special thanks go to Dr.

Abdullah Al Loman, Dr. Nicholas Callow, Dr. Qian Li, and Mr. Ashwin Sancheti whose work gave me valuable insight into this research project.

I would like to dedicate this dissertation to my daughter, my love, my heart-

Manha Mahfuz. She will always be the inspiration in every aspect of my life. I would especially like to thank my father Dr. Abdul Wazed; mother, Mrs. Helena Begum, brother S M Mazharul Islam, and all my family members for their sacrifice, love, and continuous support throughout my entire life and in all my pursuits. Last but not the least,

I would like to convey my sincere gratitude to my wife Salma Nazim for her encouragement, and love which helped in achieving my goal.

TABLE OF CONTENTS

Page

LIST OF TABLES ...... xiv

LIST OF FIGURES ...... xvi

CHAPTER

I. INTRODUCTION ...... 1

1.1 Background and Significance of the Study ...... 1

1.2 Objectives and Overview of the Study ...... 4

1.3 Hypothesis and Experimental Design ...... 7

II. BACKGROUND ...... 14

2.1 Guayule Biomass ...... 14

2.2 Soybean ...... 15

2.2.1 Soybean hull carbohydrate ...... 17

2.2.2 Soybean meal carbohydrate ...... 18

2.2.3 Soy protein ...... 20

2.3 Enzyme hydrolysis of different carbohydrate ...... 21

2.3.1 Cellulose hydrolysis ...... 21

2.3.2 Hemicellulose hydrolysis ...... 22

2.3.3 Pectin hydrolysis ...... 23

2.3.4 Galacto-Oligosaccharides hydrolysis ...... 23

2.4 Pretreatment ...... 24

2.4.1 CO2-H2O based pretreatment ...... 26

2.4.2 Fermentation Inhibitor generation ...... 28

2.4.3 Mechanism of Inhibition in fermentation ...... 30

2.5 Enzyme production ...... 31

III. MATERIALS AND METHODS ...... 33

3.1 Materials ...... 33

3.2 CO2-H2O based pretreatment ...... 34

3.3 Enzyme Hydrolysis ...... 35

3.4 Cultivation ...... 35

3.4.1 Cultivation of Kluyveromyces marxianus for guayule inhibition study ...... 35

3.4.2 Cultivation of Aspergillus niger for enzyme production ...... 36

3.5 Analytical Methods ...... 37

3.5.1 Carbohydrate composition analysis of soybean hull and soybean meal ...... 37

3.5.2 Total reducing sugar measurement ...... 37

3.5.3 High performance liquid chromatography (HPLC) ...... 38

3.5.4 Proteinaceous content determination ...... 38

3.5.5 Intracellular protein content measurement for cell growth ...... 39

3.5.6 Enzyme Assay ...... 40

3.5.6.1 Cellulase ...... 40

3.5.6.2 Xylanase ...... 41

3.5.6.3 Pectinase ...... 42

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3.5.6.4 Sucrase ...... 42

3.5.6.5 -Galactosidase ...... 43

IV. CO2-H2O BASED PRETREATMENT AND ENZYME HYDROLYSIS OF GUAYULE BIOMASS FOR HIGH SUGAR-LOW TOXIC HYDROLYSATE GENERATION ...... 44

4.1 Introduction ...... 44

4.2 Materials and Methods ...... 47

4.2.1 Materials...... 47

4.2.2 CO2-H2O based pretreatment ...... 48

4.2.3 Enzymatic hydrolysis ...... 48

4.2.4 Determination of acetic acid, HMF and furfural in pretreated Biomass ...... 49

4.2.5 K. marxianus cultivation for inhibition study ...... 49

4.2.6 Analyses ...... 50

4.3 Results and Discussion ...... 52

4.3.1 HMF, furfural and acetic acid formation at different CO2-H2O pretreatment conditions ...... 52

4.3.2 Effects of pretreatment conditions on sugar conversion from enzyme hydrolysis ...... 56

4.3.3 Explosion effect ...... 60

4.3.4 Inhibition study ...... 61

4.3.5 Compromise between inhibitor formation and sugar conversion 66

4.3.6 Comparison with other pretreatment/hydrolysis methods ...... 69

4.4 Conclusion ...... 71

V. CO2-H2O BASED PRETREATMENT AND ENZYMATIC HYDROLYSIS OF SOYBEAN HULLS ...... 72

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5.1 Introduction ...... 72

5.2 Materials and Methods ...... 75

5.2.1 Materials ...... 75

5.2.2 Pretreatment of soybean hull ...... 76

5.2.3 Enzymatic hydrolysis ...... 77

5.2.4 Evaluation of fermentation inhibitor production ...... 79

5.2.5 Analytical methods ...... 79

5.3 Results and Discussion ...... 80

5.3.1 Sugar composition in soybean hull ...... 80

5.3.2 Effect of grinding soybean hull on enzymatic hydrolysis ...... 81

5.3.3 Soybean hull hydrolysis by different loadings of A. niger and T. reesei enzyme broths ...... 82

5.3.4 Pretreatment effect ...... 87

5.3.4.1 Reducing sugar release ...... 87

5.3.4.2 Monomeric sugar release ...... 91

5.3.4.3 Acetic acid, HMF and furfural formation during pretreatment ...... 95

5.3.4.4 Optimal pretreatment condition and vessel cost analysis . . 97

5.3.5 Effect of physical explosion ...... 100

5.3.6 Enzymatic hydrolysis of pretreated hulls at higher enzyme Loading ...... 102

5.4 Conclusion ...... 102

VI. HIGH MONOMERIC SUGAR YIELDS FROM ENZYMATIC HYDROLYSIS OF SOYBEAN MEAL AND EFFECTS OF MILD HEAT PRETREATMENTS WITH CHELATORS ...... 105

6.1 Introduction ...... 105

6.2 Materials and Methods ...... 108

6.2.1 Materials ...... 108

6.2.2 Enzymatic hydrolysis ...... 108

6.2.3 Effect of soybean meal particle size on enzymatic hydrolysis . . . 109

6.2.4 Effects of heat pretreatment with chelating agents EDTA, HMP and citric acid on enzymatic hydrolysis ...... 109

6.2.5 Factorial design for effects of heat and citric acid pretreatments and SPEZYME CP supplementation on enzymatic hydrolysis . . . 110

6.2.6 Analytical methods ...... 111

6.2.6.1 Carbohydrate composition analysis of soybean meal . . . . 111

6.2.6.2 Sugar analysis ...... 112

6.3 Results and Discussion ...... 113

6.3.1 Sugar composition of soybean meal ...... 113

6.3.2 Effect of soybean meal particle size on enzymatic hydrolysis . . . 115

6.3.3 Effects of EDTA, HMP and citric acid ...... 116

6.3.4 Factorial design for effects of heat and citric acid pretreatments and SPEZYME CP supplementation ...... 119

6.3.4.1 Total reducing sugar yields ...... 119

6.3.4.2 Monomeric sugar yields ...... 121

6.4 Conclusion...... 127

VII. BETTER UNDERSTANDING OF ENZYMATIC PROCESSING OF SOYBEAN FLOUR CARBOHYDRATE THROUGH MODELING MONOMERIC SUGAR RELEASE ...... 130

7.1 Introduction ...... 130

7.2 Materials and Methods ...... 131

7.2.1 Materials ...... 131

7.2.2 Enzymatic hydrolysis of soybean flour ...... 132

7.2.3 Analytical methods ...... 132

7.2.3.1. Enzyme activities measurement ...... 132

7.2.3.2. Monomeric sugar analysis ...... 134

7.3. Results and discussion ...... 135

7.3.1 Effect of enzyme loading monomeric sugar yield in enzyme hydrolysis: Modeling approach ...... 135

7.3.1.1 Yield Model ...... 138

7.3.1.2. Best-fit model parameters for monomeric sugar yield at 48 h ...... 139

7.3.2 Kinetic profiles of monomeric sugar during enzyme hydrolysis . . 142

7.3.2.1 Kinetic Model ...... 145

7.3.2.2 Best-fit model parameters for kinetic performances . . . . . 147

7.4 Conclusion ...... 149

VIII. EFFECTS OF IONIC STRENGTH, PROTEASE AND HEAT TREATMENT ON PROTEINACEOUS RELEASE FROM SOY FLOUR DURING ENZYME PROCESSING ...... 150

8.1 Introduction ...... 150

8.2 Materials and Methods ...... 152

8.2.1. Materials ...... 152

8.2.2 Common methods used for enzymatic processing of soy flour . . . 154

8.2.3 Ionic strength effect on protein loss ...... 155

8.2.4 Protease effect on protein loss ...... 155

8.2.5 Heat treatment effects on protein loss and carbohydrate Hydrolysis ...... 156

8.2.6 Analytical methods ...... 156

8.3 Results and Discussion ...... 159

8.3.1 Ionic strength (IS) effect ...... 159

8.3.2 Protease effect on soy flour protein during enzymatic processing 162

8.3.3 Proteinaceous release and carbohydrate hydrolysis of commercial soy flours of different toasting extents ...... 167

8.3.4 Effects of heat treatment on enzymatic processing of 7B-grade soy flour ...... 169

8.3.4.1 Proteinaceous release and distribution among SPC, SPI and hydrolysate ...... 169

8.3.4.2 Sugar release ...... 172

8.3.4.3 Material balances ...... 173

8.4 Conclusion ...... 178

IX. IMPROVED CARBOHYDRASE PRODUCTION BY ASPERGILLUS NIGER FERMENTATION FOR SOYBEAN MEAL CARBOHYDRATE HYDROLYSIS FOR USE AS FERMENTATION FEEDSTOCK ...... 179

9.1 Introduction ...... 179

9.2 Materials and methods ...... 180

9.2.1 Materials ...... 180

9.2.2 Preculture and fermentation ...... 181

9.2.3 Batch fermentation: pH and solid loading study ...... 182

9.2.4 Fed-batch fermentation ...... 183

9.2.5 pH gradient study ...... 183

9.2.6 Enzyme analysis ...... 183

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9.3 Results and discussion...... 187

9.3.1 Comparison of enzyme production at different fermentation Condition ...... 187

9.3.2 Effect of solid loading ...... 189

9.3.3 Effect of fed-batch addition ...... 191

9.3.4 Effect of pH gradient ...... 193

9.3.5 Sucrase production ...... 195

9.4 Conclusion ...... 195

X. CONCLUSIONS AND RECOMMENDATIONS ...... 197

10.1 Conclusions ...... 197

10.2 Recommendations ...... 201

REFERENCES ...... 205

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LIST OF TABLES

Table Page

2.1 Guayule Bagasse Composition ...... 15

2.2 Soybean hull composition ...... 18

2.3 Soybean meal composition ...... 20

4.1 Fermentation inhibitor concentrations generated at different CO2-H2O pretreatment conditions, measured in suspensions of 50 g/L pretreated guayule bagasse before enzyme hydrolysis ...... 54

4.2 Critical inhibitor concentrations for ethanol production ...... 65

4.3 Comparison of sugar conversion and inhibitor generation by different pretreatment/hydrolysis methods ...... 70

5.1 Acetic acid, 5-hydroxymethyl-2-furaldehyde (HMF) and furfural concentrations found in aqueous suspension of 50 g/L soybean hulls unpretreated or pretreated by the CO2-H2O based method at different conditions ...... 97

5.2 Comparison of pretreatment vessel bare module costs for the CO2- H2O based pretreatment at different pressures and for the dilute acid pretreatment (vessel volume = 30 m3) ...... 100

6.1 Matrix for the factorial design for studying the effects of heat pretreatment, citric acid pretreatment, and FPU supplementation, and the code values of variables ...... 111

6.2 Sugar composition in soybean meal ...... 115

7.1 Best-fit kinetic model parameters ...... 147

8.1 Enzyme activities measured for different enzyme broths (EBs) used in this study and their ionic strengths ...... 154

8.2 Effect of ionic strength of enzyme broth on proteinaceous release during enzymatic processing of soy flour ...... 160

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8.3 Protease effect on proteinaceous releases in hydrolysates before and after SPI collection ...... 163

8.4 Effects of heat treatment (160 °C, 2h) on proteinaceous release, solid recovery in SPC, protein distribution among SPC, SPI and hydrolysate, and monomeric sugar concentrations in hydrolysate for systems with 250 g/L soy flour loading ...... 171

LIST OF FIGURES

Figure Page

1.1 Process overview for (A) Guayule and Soybean hull study and (B) Soybean meal study ...... 6

2.1 Different components of soybean to be studied in this research, Here RFOs = Raffinose Family Oligosaccharides = Stachyose, Raffinose, and Sucrose ...... 17

2.2 Schematic representation of the effectiveness of pretreatment for increasing the sugar yield in enzymatic hydrolysis ...... 26

2.3 Experimental setup of CO2-H2O based pretreatment ...... 28

2.4 Formation of acetic acid, HMF, and furfural during pretreatment (Note: Furfural and HMF are produced by dehydration reaction of sugars at high temperature and acidic condition) ...... 30

2.5 Overall process overview for the proposed research ...... 32

4.1 Effects of (a) pretreatment temperature and (b) time on HMF, furfural and acetic acid formation; data for (a) averaged from systems of all pressures with 30-min pretreatment time and data for (b) from all pressures at 195 C ...... 55

4.2 (a) Effects of pretreatment time on the reducing sugar conversion at two pretreatment pressures: 1250 and 1800 psi, but a fixed temperature (195°C) and water content (66.7%); (b) – (d) effects of pretreatment temperature and pressure on (b) hemicellulose, (c) cellulose and (d) total reducing sugar conversions for 30 min pretreatment at 66.7% water content. The conversions reported were for 48-h enzymatic hydrolysis at 50°C and pH 4.8...... 59

4.3 a) Effect of pretreatment pressure on the physical explosion at 180°C and 30 min pretreatment time on reducing sugar subsequent enzymatic hydrolysis; b) Contribution of physical explosion on reducing sugar conversion at different pretreatment temperature and pressure, pretreatment time was 30 min...... 61

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4.4 Profiles of (a) cell growth, (b) pH change, (c) glucose consumption, (d) ethanol production, (e) furfural removal and (f) HMF removal observed in K. marxianus fermentations made with no (control) and 3 levels of inhibitors corresponded to those from suspensions of 50, 100 and 200 g/L guayule bagasse pretreated by the CO2-H2O method at 195°C, 1800 psi, 66.7% water content and 30 min...... 64

4.5 Correlation of sugar conversions and concentrations with combined inhibitor concentrations generated at different pretreatment conditions. Reducing sugar, cellulose and hemicellulose conversions obtained after enzyme hydrolysis of pretreated guayule biomass are shown. Furfural, HMF and acetic acid are the inhibitors considered. Pretreatment conditions for individual (vertical) sets of data are labeled above the top figure, in terms of pressure (psi), temperature (C) and time (min) used...... 67

4.6 Correlation between cellulose conversion and HMF concentration (Left) and correlation between hemicellulose conversion and furfural concentration (Right) shown for different pretreatment temperature (C)-time (min) groups at varying pressures...... 69

5.1 Profiles of reducing sugar release from ground and unground soybean hulls hydrolyzed by the A. niger enzyme broth; in both systems: solid loading = 50 g/L, enzyme loading = 5 mL/g, pH = 4.8 and T = 50C. 82

5.2 Profiles of reducing sugar release during enzymatic hydrolysis of unpretreated, ground soybean hulls showing (a) the enzyme loading effect, by different loadings of the A. niger enzyme broth, i.e., 0 (control), 2, 4, 8 and 12 mL per g hulls, and (b) comparison of A. niger versus T. reesei broth, at the same enzyme loading of 4 mL per g hulls. The other conditions were kept constant: soybean hull loading = 50 g/L, pH = 4.8, T = 50°C...... 83

5.3 Monomeric sugar release profiles observed in some of the enzymatic hydrolysis experiments described in Figure 5.2...... 85

5.4 Figure 5.4 Correlation (a) between glucose concentration and cellulase loading and (b) between xylose concentration and xylanase loading after 95 h enzymatic hydrolysis of unpretreated ground soybean hull by A. niger broth...... 86

5.5 Mannose release profile during enzymatic hydrolysis of unpretreated ground soybean hull; enzymatic hydrolysis condition were described in Figure 5.2...... 87

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5.6 (a) Pretreatment effect on the reducing sugar release profiles during enzymatic hydrolysis, shown for different pretreatment methods and conditions. (b) Comparison of total reducing sugar concentrations released after 72 h enzymatic hydrolysis. A. niger enzyme broth and ground soybean hulls were used; the enzyme loading was 5 mL/g. The enzymatic hydrolysis conditions were soybean hull loading = 50 g/L, pH = 4.8, T = 50°C...... 90

5.7 Effects of CO2-H2O based pretreatments on monomeric sugar release: (a) glucose, (b) xylose, (c) galactose, (d) arabinose, and (e) mannose. Enzymatic hydrolysis conditions were the same as those given in Figure 5.6...... 93

5.8 (a) Effect of pretreatment temperature (at fixed pressure 1250 psi) and (b) pressure (at fixed temperature 180°C) on monomeric sugar release after 72 h enzymatic hydrolysis (at same conditions as those in Figure 5.6)...... 95

5.9 Comparison of monomeric sugar release from 50 g/L pretreated and unpretreated soybean hulls, after 72 h hydrolysis with A. niger broth (loadings specified) at pH 4.8 and 50°C...... 98

5.10 Physical explosion effect evaluated at two pretreatment conditions: (A) 750 psi and 180°C, and (B) 1250 psi and 180°C, shown by comparison of the reducing sugar release profiles during enzymatic hydrolysis of pretreated soybean hulls; enzyme loading – 5 mL/g A. niger broth, and solid loading – 50 g/L...... 101

6.1 Effect of particle size on total reducing sugar yield released by enzymatic hydrolysis of soybean meal...... 116

6.2 Effects of heated pretreatments (90 °C, 2 h) with different concentrations of EDTA, HMP and citric acid, respectively, on total reducing sugar yield from enzymatic hydrolysis of the pretreated soybean meal...... 118

6.3 Effects of heat (H) and citric acid (CA) pretreatments and cellulase supplementation (FPU) on reducing sugar yield after 24 h enzymatic hydrolysis of soybean meal...... 121

6.4 Effects of heat (H) and citric acid (CA) pretreatments and SPEZYME (primarily cellulase) supplementation (FPU) on monosaccharide yields after 24 h enzymatic hydrolysis of soybean meal: (a) glucose, (b) xylose, (c) galactose, (d) arabinose, and (e) fructose + mannose. 125

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6.5 Monosaccharide releases from soybean meal observed after the pretreatments (prior to enzymatic hydrolysis, 0 h), by separate and combined heat and citric acid pretreatments, and after (24 h) enzymatic hydrolysis by A. niger enzymes (without SPEZYME supplementation)...... 127

7.1 Effect of different enzyme loading (U/g) on monomeric sugar yield in enzymatic hydrolysis of soybean flour...... 137

7.2 Comparison of model-predicted and experimentally measured yields of glucose, xylose, galactose, arabinose and fructose+mannose in hydrolysate after 48 h hydrolysis...... 140

7.3 Carbohydrate composition of soybean flour from model parameters. 142

7.4 Monomeric sugar release profiles by (a) SPEZYME; with, per g SF, 10 FPU cellulase, 23.7 U xylanase, 0.5 U pectinase, 0.1 U α- galactosidase, and 0.01 U sucrase; (b) A. niger enzyme; with, per g SF, 0.5 FPU cellulase, 156.1 U xylanase, 10.3 U pectinase, 11.4 U α- galactosidase, and 2.9 U sucrase; (c) 0.7 FPU cellulase, 412.1 U xylanase, 16.8 U pectinase, 14.3 U α-galactosidase, and 5.3 U sucrase; and (d) 11.1 FPU cellulase, 538.5 U xylanase, 19.5 U pectinase, 12.7 U α-galactosidase, and 18.2 U sucrase...... 144

7.5 Comparison of model predicted and experimental yield of monomeric sugar release from kinetic model...... 148

8.1 Contributions of ionic strength to increased proteinaceous release in supernatants before and after SPI separation (same experimental procedures as for Table 8.2)...... 162

8.2 SDS-PAGE results for SPC, SPI and final hydrolysates collected from the enzyme-free control (Ct) and 3 systems with A. niger enzyme broths (labeled with their protease loadings: 73, 557 and 490 BAEE U/g SF, as given in Table 3): (A) Lanes 1-4, SPC (Ct, 73, 557 and 490 BAEE U/g SF); Lanes 5-8, SPI (Ct, 73, 557 and 490 BAEE U/g SF); Lane 9, marker; Lanes 10-11, hydrolysate H (Ct and 73 BAEE U/g SF), (B) Lane 2, 73 BAEE U/g SF (DF 40); Lane 3, 557 BAEE U/g SF (DF 4); Lane 4, 490 BAEE U/g SF (DF 4); and Lane 5, marker Sample dilution factors (DF) used for the analysis are also given in the lane labels...... 166

8.3 (a) Unrecovered proteinaceous concentrations (g/L) in final hydrolysates after SPI collection and (b) reducing sugar conversions 168 observed with commercial soy flours of different extents of toasting

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(TN > BS > 7B-grade SF); processing was done without (enzyme-free controls) and with 1 mL EB2 per g soy flour......

8.4 Material balances compared for controls (without A. niger enzyme broth) with and without heat treatment (160 °C, 2h) of 7B-grade soy flour...... 175

8.5 Material balances compared for enzyme processing (1 mL EB4/g-SF) of 7B-grade soy flour with and without heat treatment (160 °C, 2h). 176

9.1 Comparison of pectinase, α-galactosidase and cellulase production in different fermentations; F1 = 2x C,2x N, pH 6; F2 = 2x C, 2x N, pH 7; F3 = 5x C, 2x N, pH 6; F4 = 5x C, 5x N, pH 7; F5 = 3x C, 2x N, pH 6, Fed batch; F6 = 5x C, 2x N, pH 6, Fed batch; F7 = 5x C, 2x N, pH gradient = 0.0156 pH drop per h; F8 = 5x C, 2x N, pH gradient = 0.0292 pH drop per h; and F9 = 5x C, 2x N, pH gradient = 0.0357 pH drop per h...... 188

9.2 Effect of solid loading in enzyme production; F1 = 2x C, 2x N, pH 6; F2 = 2x C, 2x N, pH 7; F3 = 5x C, 2x N, pH 6; and F4 = 5x C, 5x N, pH 7...... 191

9.3 Effect of fed-batch soybean hull addition in enzyme production; F5 = 3x C, 2x N, pH 6, Fed batch (2x C initially + 1x C at 72 h); and F6 = 5x C, 2x N, pH 6, Fed batch (2x C initially + 1x C at 48 h + 1x C at 72 h + 1x C at 96 h)...... 193

9.4 Effect of pH gradient in enzyme production; F7 = 5x C, 2x N, pH gradient = 0.0156 pH drop per h; F8 = 5x C, 2x N, pH gradient = 0.0292 pH drop per h; and F9 = 5x C, 2x N, pH gradient = 0.0357 pH drop per h...... 194

9.5 Comparison of sucrase production in different fermentation...... 195

10.1 Summary of the research...... 201

10.2 Future recommendations of the developed process...... 204

CHAPTER I

INTRODUCTION

1.1 Background and Significance of the Study

Biorefinery using sustainable and renewable resources can reduce fossil fuel dependency and its associated climate change effect. With the growth of human population, finding food sources is another important challenge. Improving economy and rising per capita income results in changes in dietary habits in developing countries which has led to ever more demand for animal protein. IMPACT global food model developed at IFPRI (International Food Policy Research Institute) predicts that the demand for meat could increase by 58 percent by the year 2020, demand for poultry is expected to rise by 85 percent, and that of pig meat by 55 percent of the current level [1].

The agricultural sector will have to produce an adequate number of cereal crops to meet the world food energy requirements. However, cereals are low in protein content and lack many essential amino acids, so it has to be supplemented with a protein source [2].

Different biomass provides different scopes for use as biorefinery feedstock and food source for human and animal. For use as biorefinery feedstock, lignocellulosic biomass is cheaper and does not compete with food uses when compared with the food crops that have been used in the production of the first-generation biofuels [3–5]. Bagasse of guayule (Parthenium argentatum Gray), soybean hull and soybean meal are an

1 excellent source of lignocellulosic biomass to support biorefinery. Guayule has been planted for industrial production of hypoallergenic latex and, potentially, resins. The rubber and resin components, however, constitute less than 20% of total dry biomass weight [6,7]. That leaves at least 80% as waste bagasse. Soy is one of the most cultivated crops in the world, primarily for soybean oil production. Soybean hull makes up about 8-

10% of the soybean weight [8]. It is, therefore, a major by-product of soybean processing.

The global soybean production was 313 million metric tons in 2015 [9]; accordingly, about 30 million tonnes of soybean hulls were generated. The bagasse composition has been reported as around 50% carbohydrate which contains mainly cellulose and hemicellulose and also has high (~35%) lignin content [10–12]. Soybean hull has very high carbohydrate content (65-70%) which contain pectin besides cellulose and hemicellulose and the lignin content is lower than guayule (1-4%) [13–15]. Pretreatment is often needed to make lignocellulosic biomass more accessible for enzyme attack [16].

Pretreatment can reduce the crystallinity of cellulose microfibrils and separate or remove the protection barriers due to hemicellulose and lignin [17]. Lignocellulose pretreatment methods such as dilute acid, alkali, wet oxidation, and steam explosion have been reported. They are effective to different extents in improving the sugar yield in enzymatic hydrolysis but also release inhibitory levels of compounds like acetic acid, hydroxymethylfurfural (HMF) and furfural to complicate the use of concentrated hydrolysate in fermentation processes [18–21].

Soybean has a reasonable amino acid profile and can be one of the best sources of protein in animal feed and fish meal [22]. It also contains a large proportion of carbohydrate. Soybean meal contains approximately 50% crude proteins and 30-35%

2 carbohydrates. Soymeal carbohydrates are mainly composed of approximately 10-15% soluble sugars and 20-25% non-starch polysaccharides which are cellulose, hemicellulose and some pectin polysaccharides [23]. Soymeal products available commercially are typical of three categories: Soymeal, Soy Protein Concentrate (SPC) and Soy Protein

Isolate (SPI). SPC is currently produced by washing soy flour with water, possibly containing pH buffer and organic solvent. The water-soluble carbohydrates (and other soluble and colloidal materials) are removed from the remaining protein-rich solids. SPC, thus produced, has a protein content of 65-67% and retains the insoluble soy carbohydrates and other materials such as non-starch polysaccharides (hemi/cellulose and pectin) and lignin [22]. The protein yield (i.e., the portion of initial proteins retained in the product) for SPC production is high (75%-95% depending on the method), but it is not ideal for fish because of the presence of hard to digest polymers.

SPI, on the other hand, is prepared by first dissolving proteins in aqueous solution, together with water-soluble carbohydrates and others. This causes disintegration of soy meal particles and allows removal of insoluble constituents by centrifugation.

Proteins in the supernatant are then made insoluble by adjusting the pH of the protein- containing a solution and collected by centrifugation. The SPI product, thus prepared, has higher protein content (about 90%) but is costly to produce and the dry weight yield is only 30% (protein yield about 60%) [22]. Despite the protein content of SPC being comparable to that in the fish meal, the presence of considerable amount of indigestible fibers and cellulose during the SPC preparation by conventional method prevents it from being viable. An economic process of producing SPC having higher digestibility to fish is necessary in order to effectively substitute fish meal by soy meal products. Digestibility

3 of the SPC can be increased if cellulose and other indigestible carbohydrates can be hydrolyzed to soluble sugars.

In young animals with the still undeveloped digestive tract, feeding unprocessed soy protein results in physiological and intestinal morphological changes. Although many herbivorous and omnivorous aquatic species seem to tolerate the higher dietary level of soy products without any significant detrimental effect on the growth performance, carnivorous fish, like Atlantic salmon, cannot digest high dietary content due to the lack of appropriate digestive enzymes. Accumulation of intestinal gas and flatulence in carnivorous animals result from the presence of alpha-linked oligosaccharides such as raffinose and stachyose, which are non-reducing sugars composed of one or two galactose units linked to sucrose. Due to the lack of alpha 1,6-galactosidase enzymes in the intestinal mucosa of the monogastric animals, raffinose and stachyose, if present in the diet, remained unabsorbed in the small intestine and metabolized by the intestinal microflora in the large intestine resulting gas production which causes flatulence. During the processing of soy protein, removal of these indigestible oligosaccharides would contribute to a significant increase in the nutritional value of the product. Enzymatic extraction of soybean meal can be a very attractive approach which will be able to hydrolyze the oligomers and polymeric carbohydrate and hence increase the protein content without having the digestion issue.

1.2 Objectives and Overview of the Study

The main objective of this project is to improve the process for increasing the value of each biomass by the enzymatic process and pretreatment. In guayule and

4 soybean hull, having carbohydrate is the major content, the process will be improved to hydrolyze carbohydrate for use as a fermentation feedstock. Combined pretreatment and enzyme hydrolysis will be studied for getting high sugar conversion. CO2-H2O based pretreatment at different operating condition will be studied for both guayule and soybean hull. We hypothesize that pretreatment condition or severity depends on the complexity of the biomass structure i.e. extent of lignin content and cellulose/hemicellulose content, and on the balance of sugar conversion with minimal fermentation inhibitors formation.

Since soybean meal has a complex mixture of carbohydrate and has value in both protein and sugar viewpoint, the goal is to demonstrate the applicability of using an enzymatic process for removal of the indigestible carbohydrates from the soybean meal and increase the protein content of the resulting soy protein product. The hypothesis for this section is that a complex mixture of enzyme, containing at least cellulase, xylanase, pectinase, α- galactosidase and sucrase produced by fungal fermentation will efficiently hydrolyze all the components of polymeric carbohydrate into monomeric sugar with enriched protein content. The process will also be improved by using different pretreatment strategy to increase the enzyme hydrolysis efficiency and to decrease protein loss in the final sugar- rich hydrolysate.

Guayule/ Fungal Fermentation Enzyme Soy hull

Enzyme CO2-H2O based Pretreated Hydrolysis Pretreatment Biomass

Inhibition Study Monomerized Sugar

(A)

Fungal Soy hull Enzyme Fermentation

Soybean Meal Enzyme Soy Protein Hydrolysis Concentrate

Chemical additives Pretreated and/or heat Biomass Monomerized treatment Sugar

(B)

Figure 1.1 Process overview for (A) Guayule and Soybean hull study and (B) Soybean

meal study.

The overview of the process is shown in Figure 1.1. The whole study is subcategorized in three biomasses-guayule, soybean hull and soybean meal. Figure 1.1

(A) shows the process overview to improve the process to increase the value of guayule and soy hull biomass. Guayule has mainly cellulose and hemicellulose which requires cellulase and xylanase enzyme during enzyme hydrolysis, and it can be obtained from

6 commercial cellulase. CO2-H2O based pretreatment will be studied to increase the degree of enzyme hydrolysis. Soybean hull has cellulose, hemicellulose, and pectin and for soybean hull hydrolysis, the enzyme will be produced from Aspergillus niger and

Trichoderma reesei fermentation using soybean hull as a substrate which will contain at least cellulase, xylanase, pectinase, α-galactosidase and sucrose enzyme. Effect of CO2-

H2O based pretreatment will be studied to increase the hydrolysis efficiency. The study of enzymatic processing of soybean meal is divided into three stages, first is the study to improve enzymatic hydrolysis by pretreatment, second is to study the effect of different enzyme and substrate interaction in enzyme hydrolysis by modeling approach and third is to improve the enzyme production by Aspergillus niger fermentation. Different factors

(heat treatment, citric acid, additional cellulase) will be studied to improve the sugar yield without increasing the enzyme loading. Once efficient hydrolysis is accomplished, insoluble protein product (SPC) will be centrifuged, and the hydrolysate containing soluble sugar can be used for fermentation feedstock. Even though most of the proteins are expected to be precipitated at the isoelectric pH, some of the proteins might still be present in the hydrolysate due to the presence of different types of proteins with different molecular size. These soluble proteins at the isoelectric point will be recovered by heating the hydrolysate. Different factors i.e. protease enzyme, ionic strength, the extent of heat treated soy flour will be evaluated reduce the protein loss in the hydrolysate.

1.3 Hypothesis and Experimental Design

For achieving these goals effectively, several technical aspects of the process are addressed and are subdivided into six main chapters. First two chapters talk about the effect of CO2-H2O based pretreatment of guayule and soybean hull on enzyme hydrolysis

7 along with inhibitors production during pretreatment and their effect on fermentation.

Last four sections talk about the process improvements for increasing the value of soybean meal by pretreatment, enzyme production, enzyme hydrolysis and by reducing protein loss in enzyme hydrolysis.

The first section is to evaluate the CO2-H2O based pretreatment condition, i.e. pressure, temperature for achieving high sugar-low toxic hydrolysate production in combined pretreatment and enzyme hydrolysis. Guayule has mainly cellulose and hemicellulose which requires cellulase and xylanase enzyme during enzyme hydrolysis, and it can be obtained from commercial cellulase. Having higher lignin content will require relative harsher pretreatment condition hence higher possibility of acetic acid,

HMF and furfural formation. Process condition will be optimized considering higher sugar conversion in enzymatic hydrolysis and minimal inhibitors formation during pretreatment. Inhibitory effects of acetic acid, HMF and furfural are studied in -

Kluyveromyces marxianus fermentation. This section sets out to answer some important questions, what are ranges of pressure and temperature for high sugar conversion, what is the combination of pressure-temperature combination which gives high sugar-low toxic hydrolysate and what is the optimum condition at which the ethanol production by K. marxianus fermentation will not get inhibited by higher substrate loading?

The second section is to understand the different source of enzyme (T. reesei and A. niger) in effectively hydrolyzing soybean hull carbohydrates and effect of

CO2-H2O based pretreatment for improving sugar conversion. Since lignin content in soybean hull is low, pretreatment condition will be milder than guayule pretreatment.

This section will answer the following questions, will we need different combination of

8 enzyme than for guayule hydrolysis since soybean hull has pectin and galacto- oligosaccharides along with cellulose and hemicellulose? Is having lower lignin content will allow us to use gentler pretreatment condition (lower T, P) in CO2-H2O based pretreatment and what are the corresponding inhibitors production which will show the effectiveness of using this pretreatment?

Soybean meal has a more complex structure having very high pectin and galacto- oligosaccharides content; it also has cellulose and hemicellulose content. For potential enhancements in enzymatic hydrolysis, three chelating agents, i.e., ethylenediaminetetraacetic acid (EDTA), sodium hexametaphosphate (HMP), and citric acid are examined at a heated condition. Multivalent cations such as Ca2+ are known to be able to form ionic bonding/crosslinking with the polygalacturonate backbones of pectin

[24,25]. Use of chelators (EDTA and HMP) has been shown to improve extraction of soybean okara pectin into aqueous media [26,27]. In addition to chelating, citric acid may act as a dilute acid pretreatment agent. This section will answer the questions, can we use this pretreatment to improve all the monomeric sugar conversion without increasing enzyme loading, and can they improve the pectin hydrolysis along with cellulose hydrolysis?

Hydrolyzing soybean meal carbohydrates enzymatically to fermentable sugars requires complex enzyme system with at least pectinase, xylanase, cellulase, α- galactosidase and sucrase activities. A better understanding of how enzyme compositions affect the hydrolysis of different soybean flour carbohydrates (cellulose, hemicellulose, pectin, and galacto-oligosaccharides) is important for further improvement. This has been investigated in this study by modeling the effect of enzyme composition on the kinetic

9 release of individual monomeric sugars from different carbohydrate components. The models are valuable for understanding the hydrolysis mechanisms and be helpful for further reactor design, batch and continuous process design and optimization of overall economics.

The next section is to understand and minimize protein loss associated with the new enzyme-based soy flour processing. Possible factors were considered thoroughly and investigated in this study. A. niger enzymes with different protease activity levels were used to evaluate the extent of protein loss due to proteolytic effects, in addition to the effects of other non-enzymatic factors. Different commercially heat-processed soy flour/meal products were compared for the performance of enzyme processing regarding both carbohydrate hydrolysis and protein loss. Finally, the effects of controlled heat treatment applied in this laboratory on soy flour without previous commercial toasting were studied at different enzyme-processing stages, i.e., before enzyme hydrolysis, solid

(SPC) and a liquid fraction (SPI and sugar-containing hydrolysate), final SPI and hydrolysate by comparing the overall material balance.

From modeling of enzyme hydrolysis, enzyme composition needed to achieve higher sugar conversion of enzyme hydrolysis was evaluated, and from that, the limiting enzymes were found as pectinase and α-galactosidase. Enzyme production by Aspergillus species is sensitive to the nutrient and environmental conditions. In this study, different soybean hull loading and different starting pH in fermentation was evaluated. In the previous study, 20 g/L soybean hull was used, while in this current study higher solid loading (up to 100 g/L) was studied at two different pH level. Effect of fed-batch soybean addition was also evaluated to continual production of the enzyme. Since, in Aspergillus

10 niger fermentation, the different enzyme can be produced at different pH range, to evaluate that dependency, difference pH gradient (with different pH drop rate per h) was studied to maximize all the all production.

The entire project can be simplified into following series of hypothesis and tested statements:

1. Guayule has higher lignin content. For achieving better pretreatment higher

severe condition is required which also has a negative effect on toxic

compound production. Will study of different pretreatment condition will

allow us to select T-P combination to have high sugar-low toxic hydrolysate?

2. Whether the produced fermentation inhibitors will inhibit the fermentation

and which compound is mainly responsible for our process and can we use

higher substrate loading of guayule without having inhibition problem?

3. Can the enzyme mixture produced by fungal fermentation effectively

hydrolyze soybean hull carbohydrate?

4. Soybean hull has lower lignin content than guayule, will this allow us to use

gentler pretreatment condition in CO2-H2O based pretreatment?

5. Soybean meal has pectin as the major structural component which will require

different pretreatment strategy. Will a chelating agent or very mild acid

pretreatment condition will improve the overall enzyme hydrolysis efficiency?

6. Cellulase enzyme in the broth produced by A. niger is not high. Whether that

contributes to the lower glucan conversion in enzyme hydrolysis and increase

additional cellulase loading will improve the glucan conversion.

7. In soybean meal hydrolysis, complex enzyme mixture is required for the

complex carbohydrate in soybean meal. Hydrolysis of particular fraction

might depend on the interactor of other enzyme and substrate.

8. What are the limiting enzyme we have in our produced enzymes and what are

the total enzyme requirements of the different enzyme to achieve complete

monomerization of all the carbohydrates?

9. Whether ionic strength, protease enzyme in the broth or the extent of heat

treatment affects the protein loss in the process and how that will affect in the

final sugar conversion?

10. Higher carbohydrate enzyme production is required for the process

improvement. What are the factors can contribute to better enzyme production

in A. niger fermentation, i.e. higher substrate loading, fed-batch substrate

addition?

11. Enzyme production depends on the inducing effect of substrate and different

enzyme different preferred pH for their enzyme production. Can maintaining

different pH gradient/profile will improve all the enzyme production?

In this study, we investigated different aspects of biorefinery i.e. pretreatment, enzyme hydrolysis and enzyme production for different types of biomass. This work will allow us to differentiate the requirement of the severity of pretreatment types and condition and enzyme composition regarding biomass composition. The work to model the enzyme hydrolysis of soybean meal and very high enzyme production along with the reduction of protein loss could be major steps forward for soybean bioprocessing. The enzyme production and enzyme hydrolysis in this work has the huge potential to use in

12 nanocellulose and soy protein nanoparticles production for rubber filler application, adhesive preparation and to use in hydrolyzing soy molasses and soy okara. The produced enzyme can also be very potential additives in poultry feed industry.

CHAPTER II

BACKGROUND

2.1 Guayule Biomass

Guayule (Parthenium argentatum Gray) has been used for industrial production of hypoallergenic latex and, potentially, resins. The environmentally friendly aqueous- based extraction process is one of the methods used to extract rubber that grinds the harvested shrub to produce a rubber particles suspension in buffer (guayule latex) [6].

10–20 wt% of the plant dry weight are comprised of natural rubber and resins which more than 80% of the biomass as a waste crop residue. The waste residual lignocellulosic co-product, referred to as bagasse, could be further developed into bioenergy feedstock, composite boards, soil amendments, and construction-related materials [7]. Because the feedstock is currently a “waste” generated for the latex production, it is essentially “free” to use as a fermentation feedstock. Bagasse of guayule is thus an excellent source of lignocellulosic biomass to support biorefinery. The bagasse composition has been reported as 19-34% cellulose, 13-22% hemicellulose, and 29-37% lignin, where the variations may be associated with different biomass sources and the extraction processes used for latex and resin collection [10–12]. Table 2.1 shows the composition of guayule bagasse (acetone extracted, without resin extraction) reported by Chundawat et al. [28] which has been used in this research.

Table 2.1 Guayule Bagasse Composition [28]

Component Composition (%, Dry basis)

Cellulose 27.1 Hemicellulose 20.7 Klason Lignin 36.1 Acid Soluble Lignin 1.4 Ash 1.8 Acetic Acid 4.3 Other (Including resin) 8.6

2.2 Soybean

Soybean is one of the most cultivated crops in the world, primarily for soybean oil production. The global soybean production was 313 million metric tons in 2015 [9]. The protein is particularly valuable, with all essential amino acids [29]. Soybean contains about 20% oil (dry basis) and is the largest source of edible oils in the world. In typical soy processing, the protein ends up in the powdered soy flour or meal after oil extraction and dehulling (hull contains approximately 8-10% of soybean weight). Containing about

80% of the original dry bean weight, soybean flour comprises approximately 50% protein, 30-35% carbohydrate and other minor components [30]. The high protein content and good amino acid profile and digestibility of soybean meal [31] make it an ideal protein source for feed and food [29,32]. Soy flour can be further processed to make products with enriched protein content, i.e., soy protein concentrate (SPC) and soy protein isolate (SPI). SPC has 60-68% protein content, made by removing the soluble carbohydrate in soy flour by processes like dilute acid wash or alcohol (20-80%) wash at pH around the isoelectric point of soy protein [33,34]. SPI has about 90% protein content

15 and is produced by serial steps of dissolving protein at high pH, separating the solution from remaining solids, and then re-precipitating protein from the solution at the isoelectric point [33]. The series of SPI production steps typically result in relatively low protein recovery (40%-70%) [33]. On the other hand, the high contents of non-starch polysaccharides (NSPs) and galacto-oligosaccharides in soy flour/meal and SPC can cause indigestibility concerns to monogastric animals such as fish, pig and poultry [35–

37]. Three main components of soybean; soy hull, soy meal and soy protein will be focused in this research for process improvement to increase their values to support biorefinery. Figure 2.1 shows the components of soybean to be studied in this research.

Figure 2.1 Different components of soybean to be studied in this research, Here RFOs =

Raffinose Family Oligosaccharides = Stachyose, Raffinose, and Sucrose.

2.2.1 Soybean hull carbohydrate

Soybean hull makes up about 8-10% of the soybean weight [8]. It is, therefore, a major by-product of soybean processing. About 30 million tonnes of soybean hulls were generated. The hull is composed mainly of cell wall carbohydrates that can potentially be

17 used as low-cost renewable feedstock for fuel and chemical production by bioprocesses.

Compositions reported for soybean hulls varied: cellulose 30-50%, hemicellulose 12-

25%, pectin 6-15% and Klason lignin 1-4% [13–15] (Table 2.2). The variation can be attributed, at least partly, to the different locations and cultivars planted. Their main usage has been the use as pellets to feed the cattle and pigs. Soybean hulls have not received much attention so far for use as biorefinery feedstock except some use as animal feed. The low lignin content and high total carbohydrate content makes soybean hull as very potent and easier feedstock for the production biomass-derived chemicals.

Table 2.2 Soybean hull composition [15,38–41]

Components Composition (%, Dry Basis) Cellulose 30-50 Hemicellulose 12-25 Pectin 6-15 Oligosaccharide 9-14 Lignin 1-4 Protein 9-15 Ash 1-6

2.2.2 Soybean meal carbohydrate

Soymeal contains approximately 30-35% of carbohydrates which are usually divided into two main categories based on their physiochemical properties. Table 2.3 shows the soybean meal composition. Firstly, nonstructural carbohydrates, which can often be divided into three groups such as low molecular weight sugars

(monosaccharides), oligosaccharides and storage polysaccharides, make up approximately half of the total carbohydrates in the soybean meal. Secondly, structural

18 polysaccharides, which includes dietary fiber components such as cellulosic, noncellulosic and pectic polysaccharides which also known as non-starch polysaccharides (NSPs) [42]. The primary sugar found in the soybean meal is sucrose which can be as high as 25-35% of the total carbohydrates. In addition to sucrose, other oligosaccharides are also present albeit in lower concentrations, with stachyose as the main oligosaccharides followed by raffinose and verbascose [43]. Even though these oligosaccharides are low molecular weight sugars, they deserve special attention due to their structure and contribution to the nutritional attributes of the soybean meal.

The soybean meal in this research had 30.2 ± 3.9% total carbohydrate (on a wet basis, with 9.38 ± 0.18% moisture), including 12.8 ± 0.6% soluble carbohydrate and 17.4

± 3.5% structural carbohydrate. Individual monomeric sugar contents are, glucose, 9.4 ±

1.2%; xylose, 1.9 ± 0.4%; galactose, 6.1 ± 1.3%; arabinose, 2.9 ± 0.2%; 4.5 ± 0.3%; mannose, 2.1 ± 0.6% and galacturonic acid, 3.3 ± 0.5% [44]. Xylose is essentially all from hemicellulose (xylan); fructose is from soluble carbohydrates (stachyose, raffinose, and sucrose). Mannose and arabinose can come from both pectin and hemicellulose (for arabinose, approximately 0.9% from hemicellulose and 1.7% from pectin [45]; for mannose, no values reported separately from hemicellulose and pectin); and glucose and galactose are from both soluble carbohydrates (stachyose, raffinose, and sucrose) and structural carbohydrates. Structural glucose (5.1 ± 0.9%) are predominantly from cellulose; although hemicellulose and pectin contain approximately 1.2% [30]. For galactose, pectin is the main structural carbohydrate source with some possible contribution from hemicellulose [30].

Table 2.3 Soybean meal composition [30,45–47].

Components Composition (%, Dry Basis)

Soluble Oligosaccharides 12 - 15

(i) Stachyose (i) 4 - 6 (ii) Raffinose (ii) 1 - 1.3 (iii) Sucrose (iii) 6 - 8 (iv) Verbascose (iv) 0.2 - 0.3

Cellulose 1 – 6.2

Hemicellulose 4 – 5.2

Pectin 10 – 12.2

(i) Galactose (i) 4 – 5 (ii) Arabinose (ii) 0.3 – 2.4 (iii) Mannose (iii) 0.9 – 1.3 (iv) Rhamnose (iv) 0.3 – 0.8 (v) Galacturonic acid (v) 3 – 4.8

Starch 0.5 – 2.7

Lignin 0 – 1.6

Protein 49 - 54

Fat 2.8 - 4

Ash 4.5 - 6

2.2.3 Soy protein

Soy proteins mainly contain 10% albumins and 90% globulins. Albumins are extracted by water, and globulins can be extracted by dilute salt solutions [48]. Soy globulins can be isolated based on their sedimentation coefficients and consist of four major water-extractable fractions, 2S, 7S, 11S and 15S, where the S is in Svedberg units as determined by sedimentation chromatography. The smaller the Svedberg number, the smaller the molecular weight of protein. 15-22% of soy protein contains 2S with a

20 molecular weight ranging from 8 to 21.5 KDa which mainly contains trypsin inhibitors and cytochromes enzymes [49]. The 11S (glycinin) and 7S (β-conglycinin) fractions are storage proteins and constitute ~35% and ~30% respectively of the total protein content

[50]. The glycinin is a hexamer with the molecular mass varies between 340-375 kDa and has six acidic and six basic subunits [51,52]. Glycinin is usually insoluble because of its extensive disulfide bonds. High and low pH disrupts its quaternary structure of the 11S molecule is disrupted by high and low pH, and at temperatures above 80°C (denaturation temperature is about 80°C, at neutral pH) [53]. β-Conglycinin (7S) is a trimeric protein with molecular weight of 140 – 210 KDa and composed of three subunits, α (~67 kDa), α'

(~71 kDa), and β (~ 50KDa) [49]. Soy protein contains only about 11% 15S, with a molecular weight of 506- 600 kDa. 15S either can exist as a native protein in the seed or can be formed during isolation of the proteins. During the frozen and thawed process or freeze-drying process, 11S can convert to 15S fractions [54].

2.3 Enzyme hydrolysis of different carbohydrate

2.3.1 Cellulose hydrolysis

Cellulose is composed of insoluble, β- 1-4 linked glucose units with an average degree of polymerization. Cellulose can be hydrolyzed by cellulase which is an enzyme complex composed of three major classes of enzymes: endo-gluconases, exo-gluconases, and β-glucosidases [55]. Enzymatic degradation of cellulose to reducing sugar is accomplished by the synergistic action of these three enzymes, which involves cellulase adsorption onto the surface of the cellulose, the biodegradation of cellulose to reducing sugars and desorption of the cellulase. The process of enzyme hydrolysis continues by

21 shortening the chain length leading to the loss of the residual substrate. The endoglucanases adsorb on the surface of the long chain polymer and attack the interior of the chains of cellulose and create several units cellobiose dimer. The cellobiose dimer units are removed by exoglucanase from the non-reducing end of the cellulose chain. The

β-glucosidase component now acts on the cellobiose by breaking β- 1-4 bond to release individual molecules of glucose [56]. The cellulose fibrils have both crystalline and amorphous region in which about 70 % of the cellulose is crystalline. At first amorphous part of cellulose is attacked by the enzymes and the crystalline region gets hydrolyzed gradually after losing their peripheral parts [57].

2.3.2 Hemicellulose hydrolysis

Hemicelluloses are branched heteropolysaccharides representing 15–35% of plant biomass and which may contain both pentoses (β-D-xylose, α-L-arabinose), hexoses (β-

D-mannose, β-D-glucose, α-D-galactose) and uronic acids (α-D-glucuronic, α -D-4-O- methylgalacturonic and α-D-galacturonic acids). Hemicellulose also contains acetyl groups. Other sugars, i.e. α-L-rhamnose and α-L-fucose may also be present in small amounts, and the hydroxyl groups of sugars can be partially substituted with acetyl groups [58]. The most relevant hemicelluloses are xylans, galactomannan, and glucomannans, with xylans (the β-1,4-linked backbone of xylan polymers) being the largest structural fragments. Xylanases are a group of enzymes responsible for the hydrolysis of xylan. The main enzymes involved are endo-1,4-β-xylanase (1,4-β-D-xylan xylanohydrolase; EC 3.2.1.8) and β-xylosidase (1,4-β-D-xylan xylohydrolase; EC

3.2.1.37), which cleave β-1,4 glycosidic bonds in the xylan backbone [59].

2.3.3 Pectin hydrolysis

In addition to cellulose and hemicellulose, the cell wall of plant cells also consists polymeric substances called pectins. These pectins are complex and heterogeneous large molecule bearing carboxylic groups esterified with methanol [60]. Main constituents of pectins are homogalacturonan chain, which consists of α (1, 4)-linked D-galacturonic acids and side chains rhamnogalacturonan I and rhamnogalacturonan II. Pectin molecules are highly branched at the rhamnogalacturonan part by a large number of side chains such as arabinans, galactans, and arabinogalactans, which are linked by β-(1, 4) linkages to rhamnoses. In the main chain, the arabinoses are α-(1-5) linked while the galactoses are joined by β-(1-4) linkages. Beside these neutral sugars, the side chains can also contain d- glucopyranose, l-fucopyranose and xylopyranose; and in rhamnogalacturonan II, d- adipose, 2-O-methyl-D-xylose, and 2-O-methyl-L-fucose are present [61].

Pectins can be degraded using a group of pectinolytic enzymes consisting of esterases, hydrolases, and lyases. Esterases are mainly responsible for degradation of the main backbone consisting homogalacturonan and hydrolases are involved in the degradation of side chains [62]. Aspergillus niger fermentation has been widely used to produce pectinase enzymes which are used in many industrial processes to degrade the pectic substances. This fungus synthesizes different types of required pectinases such as polygalacturonase, pectin lyase, pectin esterases, etc. [63].

2.3.4 Galacto-Oligosaccharides hydrolysis

The primary sugar found in the soybean meal is sucrose which can be as high as

25-35% of the total carbohydrates. In addition to sucrose, other oligosaccharides are also

23 present in lower concentrations, with stachyose as the main oligosaccharides followed by raffinose and verbascose [43]. Even though these oligosaccharides are low molecular weight sugars, they deserve special attention due to their structure and contribution to the nutritional attributes of the soybean meal. Stachyose, raffinose, and verbascose are galacto-oligosaccharides, which consists of terminal sucrose linked with 1 (raffinose), 2

(stachyose) or 3 (Verbacose) galactose monomers by α-1,6 linkage and the bond between terminal sucrose and the galactose is α-1,3 [23]. These can be hydrolyzed to D-galactose and sucrose by enzyme α-galactosidase (α-GAL) [64]. The intestinal tract of nonruminants does not produce this enzyme and are dependent on microbes in the lower intestine for digestion. However, the high concentration of these oligosaccharides are reported to depress digestion efficiency and additionally, the microbial breakdown of oligosaccharides produces gas which causes flatus in rats, humans, and swine and may increase the possibility of diarrhea, abdominal discomfort and nausea [65]. The other component, nonstructural polysaccharides are primarily storage polysaccharides.

Soybean contains a lower amount of starch compared to the other beans and peas. Starch concentration varies in the range of 0.2-2% depending on the conditions they are cultivated [42].

2.4 Pretreatment

The task of hydrolyzing lignocellulose to fermentable monosaccharides is technically problematic because the digestibility of cellulose is hindered by many physico-chemical structural and compositional factors. Owing to these structural characteristics of biomass, pretreatment is a very important step for producing fermentable sugars in the hydrolysis step. The main reason of the pretreatment is to break

24 down the lignin structure, hydrolyzing hemicellulose to some extent and disrupt the crystalline structure of cellulose for enhancing enzymes accessibility to the cellulose during hydrolysis step (Figure 2.2) [66]. The choice of certain pretreatment has a large impact on all subsequent steps in the overall conversion scheme regarding cellulose digestibility, generation of toxic compounds potentially inhibitory for yeast/bacteria, stirring power requirements, energy demand in the downstream process and wastewater treatment demands [67].

To hydrolyze the cellulosic and hemicellulosic content in guayule bagasse enzymatically to fermentable sugar, a mixture of cellulase and xylanase is needed, but only 15% sugar yield was obtained after enzyme hydrolysis without any pretreatment

[12,68]. Combining effective pretreatment and enzyme hydrolysis is a desirable approach. Pretreatment is often needed to make lignocellulosic biomass more accessible for enzyme attack [16]. Pretreatment can reduce the crystallinity of cellulose microfibrils and separate or remove the protection barriers due to hemicellulose and lignin [17]. There has been a number of pretreatment methods used previously- biological, physical, chemical, and thermochemical pretreatments. In biological pretreatment processes, microorganisms such as brown-, white-, and soft-rot fungi are used to degrade lignin and hemicellulose in waste materials [67]. Such processes offer advantages such as low- capital cost, low energy, no chemicals requirement, and mild environmental conditions.

However, the main drawback to develop biological methods is the low hydrolysis rate obtained in most biological materials compared to other technologies [69]. The increase of the specific surface area and the reduction of the degree of polymerization can be achieved through physical pretreatment by reducing particle size and crystallinity of

25 lignocellulosic [70]. The power requirement of this pretreatment is proportional to final particle size and also depends on the biomass characteristics, so power requirement is relatively high [71]. There are different types of chemical pretreatment such as acid pretreatment, alkali pretreatment, organosolv, and ionic liquid (IL) pretreatment have been used. In acid pretreatment, the hemicellulosic fraction of the biomass is solubilized and make the cellulose more accessible to enzymes [70]. Alkaline pretreatment is favorable for removal of lignin and hemicellulose which increase the accessible surface area and for low inhibitor formation and can be operated at room temperature [71].

Figure 2.2 Schematic representation of the effectiveness of pretreatment for increasing

the sugar yield in enzymatic hydrolysis

2.4.1 CO2-H2O based pretreatment

CO2-H2O based pretreatment is a promising pretreatment method that has been used successfully for several types of biomass [11,72–74]. This pretreatment does not use

26 any chemical other than CO2, which is inexpensive, non-flammable, non-toxic and readily available. Figure 2.3 shows the experimental setup of CO2-H2O based pretreatment. In this pretreatment method, a preheated reactor containing wet biomass is pressurized by CO2 and held at a set pressure and temperature condition for some time; the pressure is then rapidly released to create a physical explosion effect. The pressurized

CO2-H2O can form weak carbonic acid to slightly hydrolyze the biomass and improve subsequent enzyme hydrolysis [73]. Explosive release of pressure can disrupt the biomass structure and decrease the cellulose crystallinity to increase the enzyme accessibility to the solid substrate [75]. Almost all of the hemicellulose content can be retained in the

CO2-H2O pretreated biomass. Regarding this, Srinivasan et al. [11] have compared the

CO2-H2O and dilute acid methods for pretreatment of guayule bagasse. CO2-H2O pretreatment was carried out in six conditions covering wide ranges of temperature (100-

200°C), pressure (2500-4000 psi) and cooking/holding time (30-60 min); dilute acid pretreatment was performed at 180°C for 5 min using 0.75% H2SO4. They found that the bagasse hemicellulose content remained essentially the same before and after the CO2-

H2O pretreatment under all tested conditions whereas about 77% hemicellulose was removed into the acid stream by the dilute acid pretreatment. Recovery of dilute carbohydrate from the acid solution can be difficult and economically unfavorable.

Pretreatment is particularly important for guayule bagasse because of its high lignin content [11]. For the value-added use, all types of carbohydrate in soybean hulls should be monomerized into readily fermentable sugars. The optimal/effective pretreatment condition depends on biomass composition, particularly the lignin content.

The biomass with higher lignin content may require a comparatively harsher condition.

Soybean hull has very low lignin content (1-4%). Presumably, its pretreatment can be effective at mild conditions. This is supported by the finding of a previous study on thermo-mechanical extrusion pretreatment of soybean hull, where the barrel temperature did not need to be higher than 80C to achieve high sugar yield in the following enzyme hydrolysis [76]. There are significant benefits if soybean hull can indeed be pretreated at mild conditions. Pretreatment at lower pressures can substantially decrease the reactor cost and the use of milder conditions can minimize generation of degradation products, compared to the higher-temperature acid pretreatment [18].

Figure 2.3 Experimental setup of CO2-H2O based pretreatment

2.4.2 Fermentation Inhibitor generation

For lignocellulose biomass pretreatment, other methods, i.e. dilute acid pretreatment, alkali, wet oxidation, and steam explosion have also been reported. They

28 are effective to different extents in improving the sugar yield in enzymatic hydrolysis but also release inhibitory levels of compounds like acetic acid, hydroxymethylfurfural

(HMF) and furfural to complicate the use of concentrated hydrolysate in fermentation processes [18–21]. Luo et al. identified more than 35 fermentation inhibitors from pretreatment of hybrid poplar by using dilute nitric acid [20]. The inhibitors have been categorized as sugar degradation products (most importantly, furfural and HMF), lignin degradation products, carboxylic acids (mainly acetic acid and formic acid), and inorganic salts. [19]. Among them, HMF, furfural and acetic acid are the most studied inhibitors for different biomass pretreatments [77–80]. Figure 2.4 shows the source of the generation of these inhibitors during pretreatment. Inhibitor production depends on the pretreatment condition. For example, the acetate groups in hemicellulose can be liberated in acidic conditions to form acetic acid; HMF and furfural can be produced by the dehydration reaction of six carbon sugars and five carbon sugars, respectively, in acidic conditions at elevated temperature [81]. A high sugar concentration in feed medium is highly desirable for fermentation because it can give high product concentration for easier collection and minimize water requirement and downstream cost of wastewater treatment [82]. Therefore, these fermentation inhibitors cannot be managed by using dilute hydrolysate. Detoxification has been studied and found effective, but the associated cost is generally too high [83–85].

Cellulose Glucose

Lignocellulose Galactose HMF

Hemicellulose Mannose

Hydrolysis of Acetyl content Xylose Furfural Acctic Acid

Figure 2.4 Formation of acetic acid, HMF, and furfural during pretreatment (Note:

Furfural and HMF are produced by dehydration reaction of sugars at high temperature

and acidic condition)

2.4.3 Mechanism of Inhibition in fermentation

Acetic acid is derived from the acetyl groups in hemicellulose. At low pH, in the fermentation medium, the acetic acid (pKa=4.75) is in the undissociated form, is liposoluble and diffuses into the cells. In the cell (pH=7.4) the acid dissociates causing a lowering of cell pH that inhibits cell activity. The furans and phenols are aromatic compounds that have different functional groups (e.g., acid, ketone, or aldehyde) and hence different potential inhibitory activity Furfural affect cell growth and respiration

[86]. HMF is considered less toxic than furfural in fermentation, and its concentration in

(hemi)cellulose hydrolysates is usually low. HMF has been reported to be converted at a lower rate than furfural, which might be due to lower membrane permeability, and cause a longer lag-phase in growth [87].

2.5 Enzyme production

Juhasz et al. [88] have characterized the cellulases and hemicellulases produced by Trichoderma reesei RUT C30 on various carbon sources and found that it can produce an enzyme complex consisting of cellulase and hemicellulases such as xylanase, mannanase, α-galactosidase, α-arabinosidase, β-xylosidase and acetyl xylan esterase.

Aspergillus niger fermentation has been widely used to produce pectinase enzymes which are used in many industrial processes to degrade the pectic substances. This fungus synthesizes different types of required pectinases such as polygalacturonase, pectin lyase, and pectin esterases. [63]. This also produces cellulase and hemicellulases for degradation of cellulose and hemicelluloses in the soybean meal. A. niger is certified as

GRAS (Generally Regarded As Safe), making it a suitable metabolite for use in the food processing industry.

One of the major issues of industrial usage of the enzyme is the production cost and indirectly influences the finished product cost. The global enzyme industry was worth almost $4.8 billion in 2013 and was estimated to be increased to $7.1 billion by

2018 [89]. Therefore, the cheap substrate (lignocellulosic biomass source, i.e. soybean hull) for enzyme production can be very attractive and will be beneficial for overall biorefinery processing. Soybean hull has been shown to induce all the enzyme production

(cellulase, xylanase, pectinase, polygalacturonase, α-galactosidase, and sucrase) by different Aspergillus niger strains, Aspergillus foetidus, T. reesei, which can hydrolyze all the carbohydrate content of soybean meal [90–92].

Production of soy flour degrading pectinases can be induced by the inclusion of soy flour in the medium as a part of the substrate. The enzyme mixtures produced by the

Aspergillus niger fermentation can be used to separate the indigestible carbohydrates into the solution by degrading them to monosaccharides.

The overall process overview is presented in Figure 2.5. The process inside the dotted box will be used for guayule, and soybean hull processing.

Figure 2.5 Overall process overview for the proposed research

CHAPTER III

MATERIALS AND METHODS

3.1 Materials

Guayule biomass used in this study was provided by Yulex Corporation

(Carlsbad, CA). The company chopped the whole guayule shrub and removed latex by wet milling. The remaining bagasse (without resin extraction) was used in this study. The bagasse sample was previously analyzed to contain 27.1% glucan, 16.4% xylan, 1.7% galactan, 2.6% arabinan, 36.1% Klason lignin, 1.4% acid-soluble lignin, 1.8% ash, 4.3% acetic acid and 8.6% other (including resin) materials [28]. Soybean hulls and different soy flour grades (7B grade soy flour, Toasted Nutri Soy, Bakers Soy and soybean meal) were provided by the Archer Daniels Midland Company. The hull pieces were approximately 100 μm thick and most had a diameter of 2–4 mm, with more than 90% being larger than 850 μm. Some hulls were ground and sieved through a 20 mesh (<850

μm) screen in this laboratory. Both of the ground and original soybean hulls were used in this study. All of the soy flour was in powder form majority of which were below 75 µm.

Kluyveromyces marxianus NRRL Y1195 was the yeast used for the study of inhibitor effects. Trichoderma reesei Rut C30 (NRRL 11460), Aspergillus niger NRRL

341, and Aspergillus niger NRRL 322 were used for enzyme hydrolysis of soy materials.

The yeast and fungal strains were obtained from the Agricultural Research Service (ARS)

Culture Collection (NRRL) of the United States Department of Agriculture (USDA). The

CO2 used for pretreatment was purchased from Praxair Inc. (Akron, OH). Proteose peptone (from meat, Type I, for microbiology), MgSO47H2O (99%), MnSO44H2O

(99%), ZnSO47H2O (ACS reagent grade), FeSO47H2O (reagent grade), CaCl22H2O

(reagent grade), urea (98%), NaN3 (> 99%) and dinitrosalicylic acid (DNS, 98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The water used was Milli-Q water

(18.2 MΩ-cm at 25 °C; Milli-Q Direct 8, Millipore S.A.S., Molsheim, France). All the other chemicals used were purchased from Fisher Scientific, Inc. (Waltham, MA, USA).

3.2 CO2-H2O based pretreatment

The pretreatment was done in a high-pressure stainless-steel vessel (250 mL, with

1.5’’ internal diameter, 8’’ height, and ½’’ wall thickness). The reactor was preheated to the studied temperature by a high-temperature heating tape. 15 g ground biomass were evenly added, under mixing, with 30 mL deionized water. The wet biomass was transferred to a cellulose extraction thimble (Whatman®, Piscataway, NJ) and then loaded into the preheated reactor. A 260D syringe pump from ISCO (Lincoln, Nebraska) was used to pump liquid CO2 into the reactor. The reactor was held at the pressure and temperature for a specific period of time. Then the outlet valve (SS-83KS4 ¼” 2-way ball valve; Swagelok, Solon, OH) was opened to quickly release the pressure. The pretreated biomass was collected for further study. The pressure studied in this work was in the range of 550-4900 psi, temperature 80°C-195°C and hold time 10-60 min while the water content was fixed at 66.7%. To study the explosion effect, additional experiments were made using the same pretreatment procedure except that the pressure was released very

34 slowly, at an approximate rate of 4 psi/s, so as not to cause the explosion. All pretreatments at different conditions were done with at least two repeated batches.

3.3 Enzyme Hydrolysis

Enzymatic hydrolysis was also done with pretreated material to evaluate the pretreatment effectiveness, and with unpretreated material to find out the effectiveness of the enzyme mixture in releasing monomeric sugars. The hydrolysis experiments were made in 250 mL Erlenmeyer flasks with different solid loading and enzyme loading which were described in the respective studies. Sodium azide (0.05%) was added to prevent microbial contamination during the hydrolysis. pH was adjusted to 4.8.

Enzymatic hydrolysis was conducted for a studied period of time in a shaker (Thermo

Scientific MaxQ 5000 Incubating/Refrigerating floor shaker, Ashville, NC) operating at

50°C and 250 rpm shaking speed. Duplicate samples were taken periodically and centrifuged at 10,000 rpm (9,300×g, Eppendorf 5415D) for 10 min to separately collect the solids and supernatant. The supernatant (hydrolysate) was analyzed for total reducing sugar and individual monomeric sugar concentrations.

3.4 Cultivation

3.4.1 Cultivation of Kluyveromyces marxianus for guayule inhibition study

The K. marxianus culture was maintained with regular subcultures on agar containing 3 g/L yeast extract, 3 g/L malt extract, 5 g/L peptone, and 10 g/L glucose. The preculture of K. marxianus was grown at 42°C in a 250 mL Erlenmeyer flask containing

50 mL medium with the following composition: 5 g/L yeast extract, 5 g/L peptone, 2 g/L

3.4.2 Cultivation of Aspergillus niger for enzyme production

Fungal strains were stored on potato dextrose agar (PDA) at 4 °C. The inoculum was prepared by aseptically adding three loops of cells from a mature slant to 150 mL preculture medium in 500 mL shake flask, and incubating for 48 h at 25 °C in a shaker

(Thermo Scientific MaxQ 5000 Incubating/Refrigerating floor shaker, Ashville, NC) at

200 rpm. The medium composition was: 1.4 g/L (NH4)2SO4, 2.0 g/L KH2PO4, 0.3 g/L

MgSO4·7H2O, 0.4 g/L CaCl2·2H2O, 0.3 g/L urea, 1.0 g/L proteose peptone, 0.2 g/L

Tween 80, 20 g/L soybean hull, 0.005 g/L FeSO4·7H2O, 0.0016 g/L MnSO4·H2O, 0.0014 g/L ZnSO4·7H2O, and 0.002 g/L CoCl2·2H2O. The preculture was used to inoculate the stirred tank fermentors at 10% (v/v). The fermentors were operated with 1-1.5 L working volume under controls of DO, pH, agitation, temperature and foaming, the last by automatic addition of Trans-278 (Trans-Chemco, Inc., Bristol, WI). The production medium was differed by carbon and nitrogen content than the seed culture medium. The

DO was maintained at above 20% air saturation by automatic oxygen addition in all of the fermentation experiments. The agitation rate was maintained at 350-450 rpm. At a later stage of all the fermentation, DO started to increase, and all the fermentation were harvested when the DO was between 70-80%. Daily samples were taken for enzyme analysis. All the samples were centrifuged at 10,000 g for 10 min to remove the solid biomass, and the supernatants collected were stored at -20 C before analysis.

3.5 Analytical Methods

3.5.1 Carbohydrate composition analysis of soybean hull and soybean meal

Soluble and structural carbohydrates in soybean hull and meal were determined using the standard NREL procedure [93]. Soybean hull and meal were first extracted with water and the water extract collected was later analyzed for the soluble carbohydrate composition. After water extraction, the remaining solids were subjected to two steps of acid hydrolysis to determine the structural carbohydrate content. Loss of monomeric sugars due to acid degradation was considered. The procedures were done with triplicate soybean meal samples. Total reducing sugar concentrations were measured by the DNS method, and individual sugar concentrations were measured by the high-performance liquid chromatography (HPLC), as described in the following section.

3.5.2 Total reducing sugar measurement

Total reducing sugar concentration was determined by the standard dinitrosalicylic (DNS) acid method based on color change of reducing sugar in the sample when heated with the DNS solution [94]. This method is based on the principle that 3,5-dinitrosalicylic acid is reduced to 3-amino-5-nitrosalicylic acid in the presence of reducing sugar. reagent was prepared by dissolving 10 g 3,5-dinitrosalicylic acid, 16 g

NaOH and 300 g sodium potassium tartrate (Rochelle salt) in 1 L deionized water. DNS reagent (3 mL) was mixed with 1 mL sample in a test tube. The mixture was heated in a boiling water bath for 5 min and then added with water to a total volume of 25 mL. After being cooled to ambient temperature, the mixture was measured for absorbance at 550 nm by using a UV/Vis spectrophotometer (UV-1601, Shimadzu Corporation, Columbia,

MD). Glucose solutions were used as calibration standards for conversion of absorbance to total reducing sugar concentration.

3.5.3 High performance liquid chromatography (HPLC)

The concentrations of individual sugars present in the hydrolysate were measured by HPLC. This analysis was done using a Shimadzu machine equipped with pump (LC-

10AT), column oven (CTO-20A), refractive index detector (RID-10A) and a system controller (SCL-10A). Samples were prepared with proper dilution and filtered through

0.22 µm nylon filters. Stachyose, raffinose, cellobiose, glucose, xylose, galactose, arabinose, mannose and fructose were separated using a SUPELCOGEL Pb column (30 cm × 7.8 mm, sulfonated polystyrene divinylbenzene packing material) and its guard column, at 80C with HPLC grade water as the mobile phase at a flow rate of 0.5 mL/min. Total run time was 40 min. Peak area of each sugar was converted to concentration using calibration established with respective sugar standards.

HPLC was also used for measuring acetic acid, HMF and furfural concentrations.

BIORAD Aminex HPX-87H column was used with 0.005N H2SO4 as the mobile phase.

The column temperature was 35C; flow rate of the mobile phase was 0.6 mL/min. A UV detector was used to detect HMF and furfural at 254 nm wavelength and acetic acid at

210 nm. Total run time was 60 min.

3.5.4 Proteinaceous content determination

The Kjeldahl method [95] was used to measure the nitrogen (N) contents of samples. A 50 mL sample containing 10 to 200 mg/L proteinaceous substances was

38 added to a flask and digested with 10 mL reagent containing 134 mL/L concentrated sulfuric acid, 134 g/L potassium sulfate, and 7.3 g/L cupric sulfate. The digestion was carried out to completion until the reaction mixture became a clear solution. Then 30 mL water and 10 mL of a distillation reagent containing 500 g/L NaOH and 25 g/L

Na2S2O35H2O were added to the digested sample. This mixture was then distilled using a distillation unit (RapidStill 1, Labconco, Kansas City, MO) to produce ammonia gas, which was absorbed in a 0.1 N boric acid solution. The boric acid solution was back- titrated using a 0.1 N H2SO4 to determine the concentration of ammonia absorbed, which was used to calculate the N content of the starting solid sample. The N content was multiplied by 6.25 [96] to estimate the proteinaceous content.

3.5.5 Intracellular protein content measurement for cell growth

As an indicator of cell concentration, the intracellular protein concentration was measured by using the Bradford protein assay (Bio-Rad Protein Assay Kit #500-002,

Bio-Rad Laboratories, Hercules, CA) based on the binding of Coomassie Brilliant Blue

G-250 dye with the proteins containing basic and aromatic amino acid residues. The culture sample was centrifuged, and cell pellet washed with deionized water. The washed cell suspension was added with an equal volume of 0.2 M NaOH and heated at 100 °C for 20 min. The supernatant (containing released intracellular protein) was collected by centrifugation and used for intracellular protein concentration measurement by the

Bradford protein assay, with bovine serum albumin as the standard.

3.5.6 Enzyme Assay

Five groups of extracellular enzymes were analyzed: cellulase, xylanase, pectinase, -galactosidase, and sucrase. Tests were all done in triplicate and results reported as the average values with standard deviations. By definition, one unit of enzyme activity corresponds to the activity that gives the target product at a rate of 1

3.5.6.1 Cellulase

The cellulase assay used was modified from that reported by Ghose [97]. It was found to be the most suitable for samples with cellulase activities in the range of 0.05

FPU/mL to 3 FPU/mL. The analysis procedure was as follows: (1) Cut Whatman No. 1 filter paper into pieces of 6  1 cm, ~ 50 mg/piece. Roll and insert a piece (1 cm in height) into a 25 mL tube. Add 1.4 mL 0.05 M sodium citrate buffer (pH 4.8) and 100 µL sample under analysis to the tube. The filter paper should be completely immersed in the solution. (2) Prepare the blank in the same way but without the filter paper. (3) Incubate the samples and blanks in a water bath at 50C for 1 h. (4) Add 3 mL regular DNS solution that consisted of 10 g/L 3,5-dinitrosalicylic acid, 16 g/L sodium hydroxide

(NaOH) and 300 g/L sodium-potassium tartrate to each sample and blank to stop the enzyme reaction. (5) Incubate the DNS-added tubes in boiling water (100C) for 10 min.

(6) Add deionized water to make the total volume 25 mL, mix, and then measure the absorbance of reaction supernatant at 540 nm with a spectrophotometer. Cellulase

40 activity was calculated using the following equation by determining the amount (mg) of reducing sugar released, using the pre-establish calibration with pure glucose solutions of different concentrations as standards.

glucose released (푚𝑔) 1 푚푚표푙 1000 휇푚표푙 Cellulase (퐹푃푈/푚퐿) = × × (60 푚𝑖푛 )(0.1 푚퐿 enzyme sample) 180 푚𝑔 1 푚푚표푙

= 0.925 × glucose released (푚𝑔).

3.5.6.2 Xylanase

The method reported by Bailey et al. [98] was adopted for the xylanase assay.

Samples were diluted to have xylanase activities in the range of 0.5-2 U/mL. The procedure was as follows: (1) Prepare 1 wt% substrate solution/suspension: mix 2 g beechwood xylan (Sigma Aldrich, St. Louis, MO) in 180 mL 0.05 M sodium citrate buffer (pH 5.3); heat the stirred mixture till the water vapor became apparent but not boiling; turn off heating and stir the mixture overnight; add 20 mL 0.05 M sodium citrate buffer (pH 5.3); and then store the substrate mixture at -20C for future use. (2) Add 100

µL test sample and 900 µL xylan substrate mixture to a 25 mL test tube. (3) Prepare the

(enzyme-free) blank with only 900 µL xylan substrate. (4) Incubate the samples and blanks in a water bath at 50C for 5 min. (5) Add 3 mL regular DNS solution to each sample and blank to stop the enzyme reaction. Moreover, add 100 µL test sample to the corresponding blank (to account for the potential turbidity introduced by the sample).

DNS analysis was then done to determine the amount (mg) of reducing sugar released, using D-xylose solutions as standards. The xylanase activity was calculated using the following equation:

푈 xylose released (푚𝑔) 1 푚푚표푙 1000 휇푚표푙 Xylanase ( ) = × × 푚퐿 (5 푚𝑖푛 )(0.1 푚퐿 enzyme sample) 150.13 푚𝑔 1 푚푚표푙

= 13.32 × xylose released (푚𝑔).

3.5.6.3 Pectinase

The pectinase methods were established in this laboratory [99]. The procedure for pectinase was similar to that described above for the xylanase assay, with four differences. First, the substrate solution/suspension was prepared by mixing 0.5 g citrus pectin (Sigma Aldrich, St. Louis, MO) in 100 mL 0.1 M sodium citrate buffer and then adjusting the pH to 4.8. Second, the samples and blanks were incubated at 50C for 30 min (instead of 5 min as in the xylanase assay). Third, the DNS solution used did not contain sodium-potassium tartrate to prevent precipitation of residual substrate. Fourth, the calibration for determining the released amount of reaction product(s) is made with standard solutions of D-galacturonic acid (monohydrate). The activity was calculated according to the following equation:

푈 Pectinase ( ) = 1.57 × galacturonic acid released (푚𝑔) 푚퐿

3.5.6.4 Sucrase

The sucrase assay used in this study was modified from a method reported by

Uma et al. [100]. The method was best for samples with invertase activities in the range of 0.2 - 2.0 U/mL. The procedure was very similar to that for the pectinase assay, with the following differences: (1) sucrose was used for preparing the substrate solution; (2) the enzyme reaction at 50C was allowed for 20 min, and (3) the regular (tartrate-

42 containing) DNS solution was used. Glucose standards were used for DNS analysis calibration. The sucrase activity was calculated as:

푈 Sucrase ( ) = 2.78 × glucose released (푚𝑔). 푚퐿

3.5.6.5 -Galactosidase

The -galactosidase activity measurement was by a method modified from

Kumar et al.[101]. The samples were analyzed after dilution to have -galactosidase activities of 0.05 - 0.2 U/mL. The procedure was as follows: (1) prepare the substrate solution by dissolving 0.033 g p-nitrophenyl-α-D-galactopyranoside (Sigma Aldrich, St.

Louis, MO) in 100 mL 0.1 M sodium citrate buffer (pH 4.8); (2) mix 100 µL test sample with 900 µL substrate solution; (3) prepare the (enzyme-free) blank with only 900 µL substrate solution; (4) incubate samples and blanks at 50C for 10 min; (5) add 2 mL 0.5

M sodium carbonate (pH 9.8) to each sample and blank to stop the reaction and develop the color from released p-nitrophenol; (6) add 100 µL test sample to the blank; and (7) measure the absorbance at 405 nm. Calibration established with pure p-nitrophenol standards was used for quantitation of the enzyme-released p-nitrophenol. The - galactosidase activity was calculated by the following equation:

푈 α − Galactosidase ( ) = 7.19 × 푝 − nitrophenol released (푚𝑔) 푚퐿

CHAPTER IV

CO2-H2O BASED PRETREATMENT AND ENZYME HYDROLYSIS OF GUAYULE

BIOMASS FOR HIGH SUGAR-LOW TOXIC HYDROLYSATE GENERATION

4.1 Introduction

Bagasse of guayule (Parthenium argentatum Gray) is an excellent source of lignocellulosic biomass to support a biorefinery. The rubber and resin components, however, constitute less than 20% of total dry biomass weight [6]. That leaves at least

80% as waste bagasse. For energy-related uses, there have been developments to convert guayule bagasse to pelletized fuel, gaseous and liquid fuels, bio-oil, and ethanol [102].

The bagasse composition has been reported as 19-34% cellulose, 13-22% hemicellulose, and 29-37% lignin, where the variations may be associated with different biomass sources and the extraction processes used for latex and resin collection [11,28].

Converting the relatively high (~50%) carbohydrate content to monomeric sugars for use as a fermentation feedstock for making ethanol and other value-added bioproducts can improve the overall guayule economics. While the most expensive aspect of many cellulosic ethanol processes is growth, transport, and grinding of the biomass, this cost can be less than zerofor the case of guayule biomass because biorefining transforms a waste disposal problem into a valuable raw material.

Enzyme hydrolysis is considered a very promising route for converting polymeric carbohydrates to monomeric sugar for biorefinery use [5,103–105]. However, pretreatment is often needed to make lignocellulosic biomass more accessible for enzyme attack [70]. Pretreatment can reduce the crystallinity of cellulose microfibrils and separate or remove the protection barriers due to hemicellulose and lignin [70,106].

Pretreatment is particularly important for guayule bagasse because of its high lignin content [11,12,75]. Guayule bagasse has been subjected to the following pretreatments: ammonia fiber expansion (AFEX) [28], dilute acid, delignification, and CO2-H2O based pretreatment [11] for improved sugar conversion in enzyme hydrolysis. Pretreatments by alkali, wet oxidation, and steam explosion have also been reported for other biomass.

While they all can be effective to varying extents in improving the sugar yield in enzymatic hydrolysis, they may also release inhibitory levels of compounds like acetic acid, hydroxymethylfurfural (HMF) and furfural to complicate the use of concentrated hydrolysate in downstream fermentation processes [107,108]. Luo et al. [20] identified 37 fermentation inhibitors from pretreatment of hybrid poplar by using dilute nitric acid.

Among them, HMF, furfural, and acetic acid are the most studied inhibitors for various biomass pretreatments [80,109]. Inhibitor production depends on the pretreatment conditions; for example, the acetate groups in hemicellulose can be liberated in acidic conditions to form acetic acid and HMF and furfural can be produced by the dehydration reaction of six carbon sugars and five carbon sugars, respectively, in acidic conditions at elevated temperature [19]. For use in fermentation media, high sugar concentrations are highly desirable for achieving higher product concentrations to allow easier collection and lower downstream cost of wastewater treatment [82]. Therefore, problems associated

45 with these fermentation inhibitors cannot be simply managed by using dilute hydrolysate.

Detoxification has been studied and found effective but the associated cost is generally too high [84].

Compared to other pretreatment methods, the pressurized CO2-H2O based pretreatment is potentially gentler in the formation of inhibitory compounds. The process involves: raising reactor temperature, loading wet bagasse into the reactor, pressurizing the system with CO2, holding the condition for a period of time, and then quickly releasing the pressure to create an explosion effect. In this pretreatment, biomass reacts in hot liquid water with CO2 as an acid catalyst [110]. Explosive release of pressure may cause physical disruption of biomass structure to improve the enzyme digestibility of pretreated biomass [12,75]. At atmospheric conditions, CO2 can be easily separated from the hydrolysate and recycled. Also, unlike other pretreatment methods, the CO2-H2O based process retains nearly all hemicellulose in the pretreated biomass [11] and, thus, can significantly increase the total sugar yield in the enzymatic hydrolysate.

Previously, guayule pretreatment by this method has been studied over the following ranges of operation conditions: 500-5000 psi pressure, 95-235°C temperature,

37-85% water content (out of the total solid and fluid mass), and 10-840 min reaction time [11,12,75]. The conditions of 175-180 °C, 3800 psi, 60-66.7% water, and 30-35 min reaction were reported to enable highest sugar conversion in the subsequent enzymatic hydrolysis. However, the generation of inhibitory compounds by the CO2-H2O based pretreatment at different conditions and their effect on bioethanol production have not been examined at all. The main objective of this study was to determine the effect of

CO2-H2O pretreatment conditions on the formation of acetic acid, HMF, and furfural.

How different concentrations of these inhibitors affect the ethanol fermentation by

Kluyveromyces marxianus was also investigated. The results of sugar conversion, inhibitor generation, and tolerable inhibitor threshold were considered together to identify the optimum pretreatment condition for preparing guayule bagasse for biorefinery use.

4.2 Materials and Methods

4.2.1 Materials

Guayule biomass used in this study was provided by Yulex Corporation

SPEZYME® CP (Genencor, Finland) with 32.3 Filter Paper Unit (FPU) per mL cellulase activity, 26.7 IU/mL β-glucosidase activity and 12.4 U/mL xylanase activity was used for enzymatic hydrolysis [11]. Kluyveromyces marxianus NRRL Y1195 was the yeast used for the study of inhibitor effects. The yeast was obtained from the Agricultural Research

Service (ARS) Culture Collection (NRRL) of the United States Department of

Agriculture (USDA). The CO2 used for pretreatment was purchased from Praxair Inc.

(Akron, OH). All other laboratory grade chemicals used in this study were purchased from Fisher Scientific (Pittsburgh, PA).

4.2.2 CO2-H2O based pretreatment

The pretreatment was done in a high-pressure stainless steel vessel (250 mL, with

1.5’’ internal diameter, 8’’ height, and ½’’ wall thickness). The reactor was preheated to the studied temperature by a high-temperature heating tape. Then 15 g (dry weight) guayule biomass that had been previously soaked with 30 mL water was placed into the reactor. A 260D syringe pump from ISCO (Lincoln, Nebraska) was used to pump liquid

CO2 into the reactor. The reactor was held at the pressure and temperature for a specific period of time. Then the outlet valve (SS-83KS4 ¼” 2-way ball valve; Swagelok, Solon,

OH) was opened to quickly release the pressure. The pretreated biomass was collected for further study. The pressure studied in this work was in the range of 550-4900 psi, temperature 160°C-195°C and hold time 10-60 min while the water content was fixed at

66.7%. To study the explosion effect, additional experiments were made using the same pretreatment procedure except that the pressure was released very slowly, at an approximate rate of 4 psi/s, so as not to cause the explosion.

4.2.3 Enzymatic hydrolysis

Enzymatic hydrolysis of pretreated guayule biomass was conducted in 250 mL

Erlenmeyer flasks. The pretreated biomass was measured for water content and then added to a SPEZYME CP solution in 50 mM citrate buffer at pH 4.8, to prepare systems with 50 g/L biomass (dry weight concentration) in 100 mL total liquid volume. The amount of commercial enzyme added was to give 1.82 FPU/mL cellulase, 1.52 IU/mL β- glucosidase and 0.7 U/mL xylanase activities [12]. Hydrolysis was done at 50°C and pH

4.8 for 48 h with 250 rpm mixing in a shaker (MaxQ 5000 Incubating/Refrigerating floor

48 shaker; Thermo Scientific, Ashville, NC). Sodium azide was added at 0.05% (w/v) to prevent microbial contamination during hydrolysis. After hydrolysis, the samples were centrifuged at 10,000 rpm (9,300×g, Eppendorf 5415D) for 10 min. The supernatant

(hydrolysate) collected was measured for total reducing sugar, glucose, xylose and arabinose.

4.2.4 Determination of acetic acid, HMF and furfural in pretreated biomass

To determine the formation of acetic acid, HMF and furfural during the pretreatment, suspensions of 50 g/L pretreated biomass were prepared following the same procedure for preparing enzyme hydrolysis samples but without enzyme addition. The biomass suspension was mixed thoroughly and then centrifuged at 9,300×g for 10 min to collect supernatant for analysis of these compounds by High Pressure Liquid

Chromatography (HPLC). The HPLC analysis is described in the Analyses section.

4.2.5 K. marxianus cultivation for inhibition study

Experiments were done to evaluate potential inhibition of the yeast fermentation by the degradation compounds produced during the CO2-H2O based pretreatment and, if inhibition occurred, to determine the critical inhibitor concentrations. The pretreatment was done at 195°C and 1800 psi for 30 min. The pretreated biomass was mixed with water at a concentration of 250 g/L. The suspension was then filtered to collect the wash water containing all soluble substances, including the inhibitory compounds acetic acid,

HMF and furfural and some sugars released during the pretreatment. This wash water was diluted to have three levels of inhibitors which corresponded to inhibitors found in suspensions of 50, 100 and 200 g/L pretreated bagasse. These dilutions and an inhibitor-

49 free “control” (deionized water) were used to prepare media for K. marxianus cultivation as described in the next paragraph. Acetic acid, HMF and furfural concentrations in the cultivation media prepared were measured prior to the fermentation.

50 mL medium with the following composition: 5 g/L yeast extract, 5 g/L peptone, 2 g/L

NH4Cl, 1 g/L KH2PO4, 0.3 g/L MgSO4.7H2O and 30 g/L glucose [80]. The culture was mixed at 250 rpm in the MaxQ 5000 shaker. After 16 h, the culture was harvested and inoculated at 4% (v/v) to 250 mL flasks with different media (50 mL) for the inhibition study. Besides the soluble compounds from the pretreated biomass, the media had the same composition as the preculture medium except that 30 g/L glucose was replaced by a mixture of 20 g/L glucose and 10 g/L xylose to simulate the main sugar ratio (2:1 glucose: xylose) in actual guayule hydrolysate. The yeast culture was grown at 42°C for

72 h, under 150 rpm shaking [80]. Samples were taken daily to follow and compare the pH change, cell growth, sugar consumption, ethanol production, and HMF and furfural concentration changes observed in systems of different levels of inhibitors.

4.2.6 Analyses

Total reducing sugar concentration was determined by the standard 3,5- dinitrosalicylic (DNS) acid method [94] using glucose as the calibration standard.

Glucose, xylose, arabinose, acetic acid, HMF, furfural, and ethanol were measured by

HPLC (Shimadzu LC 10A) with refractive index (RI) and UV detectors. For glucose,

50 xylose, and arabinose, a SUPELCOGEL Pb column (30 cm × 7.8 mm) was used at 80C with the RI detector and a mobile phase of 0.5 mL/min HPLC grade water. The sample injection volume was 10 µL and total run time was 40 min. For acetic acid and ethanol,

20 µL samples were injected and analyzed using an ion exchange column Aminex HPX-

87H (Bio-Rad, Hercules, CA) at 35C, with 0.6 mL/min 0.005N H2SO4 as the mobile phase. Acetic acid was detected by the UV detector at 210 nm and ethanol by the RI detector. The total run time was 40 min. For HMF and furfural measurements, a

SUPELCOSIL LC-18 column was used at 30°C and detected by UV at 280 nm. The mobile phase was 0.5 mL/min methanol: water (35:65), injection volume 5 µL and total run time 25 min. As an indicator of cell concentration, the intracellular protein concentration was measured by using the Bradford protein assay (Bio-Rad Protein Assay

Kit #500-002, Bio-Rad Laboratories, Hercules, CA) based on the binding of Coomassie

Brilliant Blue G-250 dye with the proteins containing basic and aromatic amino acid residues. The culture sample was centrifuged and cell pellet washed with deionized water. The washed cell suspension was added with an equal volume of 0.2 M NaOH and heated at 100 °C for 20 min. The supernatant (containing released intracellular protein) was collected by centrifugation and used for intracellular protein concentration measurement by the Bradford protein assay, with bovine serum albumin as the standard.

The guayule bagasse had 27.1% (w/w) glucan, 16.4% xylan, and 2.6% arabinan; glucan was predominantly in cellulose while xylan and arabinan were in hemicellulose.

Cellulose, hemicellulose and total reducing sugar conversions were therefore estimated using the following equations:

퐶푔 퐶푒푙푙푢푙표푠푒 푐표푛푣푒푟푠𝑖표푛 (%) = × 100 50 × 퐺푙푢푐푎푛%(= 27.1%)

퐶(푥+푎) 퐻푒푚𝑖푐푒푙푙푢푙표푠푒 푐표푛푣푒푟푠𝑖표푛 (%) = × 100 50 × (푥푦푙푎푛 + 푎푟푎푏𝑖푛푎푛)%(= 19.0%)

퐶 푅푒푑푢푐𝑖푛𝑔 푠푢𝑔푎푟 푐표푛푣푒푟푠𝑖표푛 (%) = 푅푆 × 100 50 × 푡표푡푎푙 푐푎푟푏표ℎ푦푑푟푎푡푒%(= 47.8%)

Ci is the concentration (g/L) of sugar i in the hydrolysate and subscripts g, x, a and RS denote glucose, xylose, arabinose and reducing sugar, respectively.

4.3 Results and Discussion

4.3.1 HMF, furfural and acetic acid formation at different CO2-H2O pretreatment conditions

Pretreatment temperature evaluated was limited to the range of 160°C-195°C.

Below 160°C the pretreatment was significantly less effective for guayule bagasse; for example, Moharreri et al. [12] reported that at 3800 psi and 66.7% water, decreasing temperature from 160°C to 145°C caused the reducing sugar yield in final enzyme hydrolysate to drop from 42% to 28%. Above 195°C the pretreatment could cause much more sugar degradation [111]. Under the CO2-H2O operation conditions studied here, the biomass was pretreated in a biphasic environment with a water-rich liquid phase and a

CO2-rich vapor phase [12,112].

HMF, furfural, and acetic acid concentrations measured in suspensions of 50 g/L guayule biomass pretreated at the various pretreatment conditions are summarized in

Table 4.1. The inhibitor concentrations observed were in the following ranges: 0.007-

0.126 g/L HMF, 0.006-0.397 g/L furfural, and 0.085-4.01 g/L acetic acid. HMF is a degradation product of hexose (glucose) and furfural of pentose (xylose, arabinose) [19].

According to Table 4.1, the pressure effects on HMF, furfural and acetic acid formation were rather complicated. With the shortest pretreatment time (10 min), even at the highest temperature (195 C), concentrations of all inhibitors increased with increasing pressure (from 1250 psi to 1800 psi). With the longest pretreatment time (60 min) and the highest temperature (195 C), the inhibitor concentrations did not change significantly with the pressure increase (from 1250 psi to 1800 psi). With the mid-level pretreatment time (30 min), the inhibitor concentrations appeared to either decrease with increasing pressure or showed increase-then-decrease trends. Many of the trends were, however, statistically insignificant (p > 0.05, as given in Table 4.1) according to one-way ANOVA.

The more significant trends of decreasing furfural concentrations in systems of higher temperature, 180°C and 195°C for 30 min, (p = 0.01-0.039) could be due to its further degradation to formic acid [18].

Table 4.1 Fermentation inhibitor concentrations generated at different CO2-H2O pretreatment conditions, measured in suspensions of 50 g/L pretreated guayule bagasse before enzyme hydrolysis

Time T P HMF Furfural Acetic acid (min) (C) (psi) (g/L) p (g/L) p (g/L) p

10 195 1250 0.010±0.002 0.009 0.074±0.016 0.328 0.85±0.07 0.398

1800 0.027±0.001 0.108±0.020 1.35±0.46

30 160 1800 0.007±0.001 0.213 0.007±0.001 0.264 0.79±0.01 0.001

3400 0.021±0.008 0.013±0.005 1.11±0.01

4900 0.010±0.001 0.006±0.001 0.86±0.01

180 1800 0.035±0.002 0.123 0.125±0.009 0.01 1.50±0.06 0.001

3400 0.061±0.003 0.108±0.004 3.34±0.03

4900 0.048±0.010 0.057±0.006 2.52±0.04

195 1250 0.095±0.018 0.517 0.379±0.058 0.039 2.88±0.93 0.423

1800 0.075±0.010 0.174±0.013 4.01±0.01

3400 0.073±0.011 0.171±0.010 3.67±0.01

60 195 1250 0.128±0.014 0.889 0.397±0.008 0.122 3.16±0.44 0.918

1800 0.126±0.001 0.376±0.001 3.23±0.47

Unlike pressure which had rather weak and often insignificant effects, temperature and pretreatment duration had significant (p < 0.05) and stronger effects on

HMF, furfural, and acetic acid formation. For easier presentation, these effects of temperature (at 30-min reaction time) and time (at 195 C) are shown in Figures 4.1 (a) and (b) with averages and standard deviations of the data from all pressures investigated.

Since the generation of these inhibitors is reaction dependent, their concentrations were all very low at the lowest temperature (160 C) and shortest reaction time (10 min). The inhibitor concentrations all increased with increasing temperature and time, although acetic acid generation appeared to plateau after 30 min. Even with the data averaged from several pressures, the temperature and time effects were statistically significant (p < 0.05) for HMF and acetic acid concentrations. Higher p values were found for furfural concentrations (0.14 and 0.08 for temperature and time effects, respectively), presumably coming form higher standard deviations at a higher temperature because of further degradation of furfural [18].

HMF Furfural Acetic acid HMF Furfural Acetic acid 0.40 5.0 0.50 5.0

4.0 0.40 4.0 0.30

3.0 0.30 3.0 0.20

2.0 0.20 2.0

Acetic acid (g/L) acid Acetic

Acetic acid (g/L) acid Acetic HMF, Furfural (g/L) Furfural HMF, 0.10 (g/L) Furfural HMF, 1.0 0.10 1.0

0.00 0.0 0.00 0.0 150 170 190 0 10 20 30 40 50 60 Temperature (C) Pretreatment time (min) (a) (b)

Figure 4.1 Effects of (a) pretreatment temperature and (b) time on HMF, furfural and

acetic acid formation; data for (a) averaged from systems of all pressures with 30-min

pretreatment time and data for (b) from all pressures at 195 C.

4.3.2 Effects of pretreatment conditions on sugar conversion from enzyme hydrolysis

The conversions of total reducing sugar, cellulose, and hemicellulose achieved after 48 h enzyme hydrolysis of the bagasse pretreated at different conditions are shown in Figure 4.2. Conversions usually maxed out by 24 h [11]; 48 h was used here to ensure maximum hydrolysis. Pretreatment time is important for reactor sizing as shorter pretreatment time can significantly decrease the reactor size and cost. The effects of pretreatment time (10, 30 and 60 min) on reducing sugar conversion in enzyme hydrolysis are shown in Figure 4.2 (a) for 195°C and 2 pressures (1250 psi and 1800 psi).

The effects were clearly pressure dependent; for example, at 1250 psi the conversion increased significantly from 58.8% (± 2.1%) with 10 min to 84.3% (± 3.4%) with 30 min but it did not further increase with the prolonged pretreatment, while at 1800 psi the conversion did not change much at all 3 times (76.5%-83.8%) and the 10-min pretreatment already enabled a high conversion. 30-35 min was reported to be optimal in previous studies of CO2-H2O pretreatment of guayule bagasse but the effect was not studied at lower pressures like 1250 psi [12,75].

Figures 4.2 (b), (c), and (d) are 3-D plots showing simultaneously the effects of pretreatment temperature and pressure on hemicellulose, cellulose and reducing sugar conversions, respectively. The plots are generated by MATLAB from experimentally measured conversions at various pretreatment temperatures (160, 180 and 195 C) and pressures (550, 1250, 1800, 3400 and 4900 psi) and fixed time (30 min) and water content (66.7%). All three conversions could reach very high levels: 92.2 ± 1.5% hemicellulose conversion was obtained experimentally at 180°C and 4900 psi while 99.2

± 1.3% cellulose conversion and 86.9 ± 1.1% total reducing sugar conversion were both 56 achieved at 195°C and 3400 psi. The lower maximum reducing sugar conversion than the cellulose conversion was due to degradation of hemicellulosic sugars at the high temperature. The mildly acidic conditions facilitated pretreatment and afforded very high conversions obtained in the enzymatic hydrolysis of pretreated bagasse [58].

All three conversions are also shown in Figure 4.2 to be much more sensitive to pretreatment temperature than pressure. Temperature dependencies of the three conversions have clear differences. With increasing temperature in the range studied

(160°C-195°C), the cellulose conversion increased monotonically while the hemicellulose conversion increased up to 180°C but then decreased clearly at a higher temperature (180 °C-195 °C). Kabel et al. [106] studied the effect of pretreatment severity (regarding temperature and acidic condition) on xylan solubility. They found that the degradation of xylose to furfural increased with increasing severity and the effect was more prominent at temperatures over 180°C. This report supported the finding of lower hemicellulose conversions in this study due to degradation of the hemicellulosic sugars released during the pretreatment at >180°C. Note that at the same pressures, hemicellulose conversions at 180°C were consistently larger than the corresponding cellulose conversions but became consistently lower at 195°C. Cellulose conversion, however, continued to increase with increasing temperature in the range studied here. The pretreatment at harsher conditions helped remove the protection barriers offered by hemicellulose and lignin to make the cellulose more accessible to enzyme attack [113].

Moharreri et al. [12] examined the textural properties of guayule biomass samples. They found that with the CO2-H2O pretreatment (180°C, 3800 psi, 66.7% water content and 35 min), the biomass surface area increased to 3.03 m2/g, which was much higher than the

57 surface areas of 0.27 m2/g in nonpretreated biomass and 0.60 m2/g in the sample pretreated hydrothermally at corresponding conditions, i.e., 180°C, 66.7% water and 35 min. The much higher surface area accessible for enzyme attack helped to obtain almost complete cellulose conversion from the CO2-H2O pretreated bagasse. The dependency of conversions on pressure is not as high as that on temperature but is still significant. As an example for the temperature effect, at 1800 psi, the cellulose conversion increased from

19.0±1.3% at 160°C to 74.7±4.8% at 180°C and further to 95.2±0.9% at 195°C. As for the pressure effect, at 180°C, the cellulose conversion increased from 43.8 ± 1.7% at 550 psi to 74.7 ± 4.8% at 1800 psi and then to 81.1 ± 1.1% at 3400 psi. The reducing sugar conversion generally plateaued at temperatures above about 180°C, reflecting the combined effects of increasing cellulose conversion and decreasing hemicellulose conversion at higher temperatures (180-195 °C) as described above.

100%(a) (b) 80%

60%

40%

20%

Totalreducing sugar (%) 0% 0 20 40 60 Pretreatment time (min) 1250 psi 1800 psi (c) (d)

Figure 4.2 (a) Effects of pretreatment time on the reducing sugar conversion at two

pretreatment pressures: 1250 and 1800 psi, but a fixed temperature (195°C) and water

content (66.7%); (b) – (d) effects of pretreatment temperature and pressure on (b)

hemicellulose, (c) cellulose and (d) total reducing sugar conversions for 30 min pretreatment at 66.7% water content. The conversions reported were for 48-h enzymatic

hydrolysis at 50°C and pH 4.8.

4.3.3 Explosion effect

In this pretreatment method, the sudden large pressure drop is used to create an explosion which is thought to rupture biomass structure and increase the sugar conversion in enzyme hydrolysis. To evaluate this effect, the comparison was made for pretreatments with rapid versus slow pressure release at otherwise the same pretreatment conditions.

Figure 4.3 (a) shows the difference between rapid and slow pressure release at different pressure and 180°C and 30 min pretreatment time. Figure 4.3 (b) shows the contribution of the physical explosion on total reducing sugar conversion in enzyme hydrolysis at different temperature and pressure. Both figures show that the difference between the rapid and slow pressure release (explosion effect) is significant when the pretreatment was done in the lower pressure-temperature range. When the pretreatment condition was milder, the physical explosion contributed more and at relatively harsher condition

(above 1800 psi and 180 °C), there is no or little effect of the physical explosion. Highest physical explosion effect was 50.9%, obtained at 160°C and 3400 psi. However, the actual reducing sugar conversion is very low. Therefore, in the higher-pressure level, this pretreatment is mainly attributed by reaction effect, large pressure drop does not have significant explosion effect. However, in lower pressure level hydrolysis efficiency is achieved by the combination of reaction and explosion effect in pretreatment. At 550 psi and 1250 psi, explosion effect contributed approximately 8% and 15% respectively in total reducing sugar yield. Here, lower contribution from the lower pressure is because it might need enough pressure drop to cause more explosion effect. Less reaction effect can yield lower fermentation inhibitors production during pretreatment.

100% (a) 90%

80%

70%

60%

50% conversion conversion (%) Reducing Reducing sugar 40%

30% 0 1000 2000 3000 4000 5000 Pressure (psi) Rapid pressure release Slow pressure release

(b)

Figure 4.3 a) Effect of pretreatment pressure on the physical explosion at 180°C and 30

min pretreatment time on reducing sugar subsequent enzymatic hydrolysis; b)

Contribution of the physical explosion on reducing sugar conversion at different

pretreatment temperature and pressure, pretreatment time was 30 min.

4.3.4 Inhibition study

Figure 4.4 shows the profiles of cell growth, pH change, glucose consumption and ethanol production in four K. marxianus fermentations. (Xylose was found not consumed

61 by this yeast under the anaerobic fermentation conditions.) These fermentations were made with media containing zero (control) and 3 other levels of inhibitors generated from guayule bagasse pretreated by the CO2-H2O method at 195°C, 1800 psi, 66.7% water content for 30 min. The 3 inhibitor levels corresponded to those present in the wash water from 50, 100 and 200 g/L pretreated bagasse. 20 g/L glucose was added to all fresh media; however, some glucose was released during the pretreatment, giving slightly increasing initial glucose concentrations, i.e., 20, 20.9, 21.8 and 23.6 g/L, in media prepared with wash water from increasing pretreated bagasse concentrations, i.e., 0 (for control), 50, 100 and 200 g/L bagasse, respectively. The changes of furfural and HMF concentrations during the fermentations are also shown in Figure 4.4. After cell inoculation, the initial acetic acid concentration was measured at 3.61 ± 0.21 g/L in the medium prepared with wash water from 50 g/L pretreated bagasse, 6.83 ± 0.34 g/L in that from 100 g/L pretreated bagasse, and 13.31 ±0.21 g/L in that from 200 g/L pretreated bagasse. Changes in acetic acid concentrations along the fermentations were not tracked.

The cell growth profiles in Figure 4.4 (a) indicated some levels of inhibition in all the systems containing soluble components generated by the pretreatment. The system with wash water from 100 g/L pretreated bagasse showed a much-prolonged lag phase

(about 14 h) and the system with wash water from 200 g/L pretreated bagasse did not show clear cell growth. Compared to the control, the system with wash water from 50 g/L pretreated bagasse had slower cell growth (with a maximum specific growth rate of 0.035 h-1, compared to 0.047 h-1 in the control) and a lower maximum cell concentration (0.39 g/L intracellular protein, compared to 0.51 g/L in the control). The glucose consumption was however comparable (Figure 4.4 (c)). The cell yield (in terms of intracellular protein)

62 from glucose was 0.020 g/g for the control, and 0.015 g/g and 0.005 g/g for the systems with wash water from 50 and 100 g/L pretreated bagasse, respectively. The pH decrease was significantly smaller in the system with wash water from 50 g/L pretreated bagasse

(to about 4.8) than in the control (to about 4.0), while the ethanol production reached about 20% higher in the former system (10.1 g/L ethanol, compared to 8.1 g/L in the control). These observations suggested that in the system with wash water from 50 g/L pretreated bagasse, the sugar consumed was channeled less to cell mass and metabolic acid(s) but more favorably to ethanol. The same shift occurred in the system with wash water from 100 g/L pretreated bagasse where even less cell mass was formed and the maximum ethanol concentration reached, though delayed, was again higher than in the control. The maximum volumetric productivity of ethanol was estimated as 0.62 g/L-h in the control, and 0.73 and 0.71 g/L-h in the systems with wash water from 50 and 100 g/L pretreated bagasse, respectively. Ethanol yields from glucose in these 3 systems were

0.41 (control), 0.48 and 0.41 g/g, respectively. Nonetheless, the inhibition in the system with wash water from 200 g/L pretreated bagasse was very strong; no cell growth, glucose consumption or ethanol production was apparent.

The inhibited cell growth in the 3 systems with pretreatment-generated inhibitors correlated also with the furfural and HMF removal profiles shown in Figures 4.4 (e) and

(f). In the system with wash water from 50 g/L pretreated bagasse, both furfural and

HMF were already removed by 14 h, presumably consumed by the yeast. In the system with wash water from 100 g/L pretreated bagasse, HMF persisted till 21 h, corresponding to the prolonged >14-h lag phase before cell growth occurred (Figure 4.4 (a)). In the system with wash water from 200 g/L pretreated bagasse, furfural and HMF only showed

63 slight removal after 45 h, consistent with the no or minimal bioactivities indicated by the other parameters measured.

5.5 0.7 (a) (b) 0.6 5 0.5 Control Control 4.5 0.4 50 g/L 50 g/L

100 g/L pH 100 g/L 0.3 200 g/L 4 200 g/L 0.2 3.5

Intracellular protein (g/L) protein Intracellular 0.1 0 3 0 20 40 60 80 0 20 40 60 80 Time (h) Time (h)

25 (c) 12 (d)

20 10

8 15 Control 6 Control 10 50 g/L 50 g/L 100 g/L Glucose (g/L) Glucose 100 g/L

Ethanol (g/L) Ethanol 4 200 g/L 200 g/L 5 2 0 0 0 20 40 60 80 0 20 40 60 80 Time (h) Time (h)

(e) (f) 1.0 0.30 0.25 0.8 0.20 0.6 50 g/L 50 g/L 0.15 100 g/L 0.4 100

HMF (g/L) HMF 200 g/L

Furfural (g/L) Furfural g/L 0.10 0.2 0.05 0.0 0.00 0 20 40 60 80 0 20 40 60 80 Time (h) Time (h)

Figure 4.4 Profiles of (a) cell growth, (b) pH change, (c) glucose consumption, (d) ethanol production, (e) furfural removal and (f) HMF removal observed in K. marxianus fermentations made with no (control) and 3 levels of inhibitors corresponded to those from suspensions of 50, 100 and 200 g/L guayule bagasse pretreated by the CO2-H2O method at 195°C, 1800 psi, 66.7% water content and 30 min.

For comparison, the critical inhibitor concentrations reported in the literature for ethanol production by some commonly used microorganisms including K. marxianus are summarized in Table 4.2. Note the previous study for K. marxianus was made with individual inhibitors, not the combined inhibitors in this current work. Nonetheless, the

HMF and furfural concentrations generated by the CO2-H2O based pretreatment were far lower than those critical concentrations reported. The inhibition observed here was likely associated with acetic acid: maximum ethanol production, though delayed, was not inhibited in the system with about 6.8 g/L acetic acid (prepared with wash water from

100 g/L pretreated bagasse) but was completely inhibited in the system with 13.3 g/L acetic acid (prepared with wash water from 200 g/L pretreated bagasse). This dependency of inhibition on acetic acid concentration was consistent with the reported 50% inhibition by 10 g/L acetic acid.

Table 4.2 Critical inhibitor concentrations for ethanol production

Inhibit Escherichia Saccharomyce Zymomonas Pichia stipitis K. marxianus K. marxianus or coli LY01 s cerevisiae mobilis CP4 (NRRL Y1195) (NRRL Y1195) (pZB5) (This study) g/L Inhibit % g/L Inhibit % g/L Inhibit % g/L Inhibit % g/L Inhibit % g/L Inhibit %

HMF 8 50a 4 40c 0.05 16d 0.5 10e 4.01 50g 0.23 *

Furfur 3.5 38a 1.6 27c 0.95 40d 2.0 17e 2.5 50g 0.72 * al Acetic 15 21b 6 74d NA NA 7.1 100f 10 50g 13.3 100 acid

Note: NA – not available;

* Effect could not be evaluated, present together with acetic acid at a much higher concentration (13.3 g/L), but concentration was very low compared to the inhibitory levels reported in other studies; a. [114]; b. [115]; c. [116]; d. [117]; e. [109]; f. [118]; g. [80]

4.3.5 Compromise between inhibitor formation and sugar conversion

The choice of pretreatment conditions should consider both high sugar conversion and low inhibitor generation. Figure 4.5 shows how the reducing sugar, cellulose and hemicellulose conversions and the reducing sugar concentration achieved would change with the combined inhibitor (HMF + furfural + acetic acid) concentration generated at various pretreatment conditions, using suspensions of 50 g/L guayule bagasse as examples. The total reducing sugar conversion and concentration results showed clear increasing-then-plateauing profiles with increasing combined inhibitor concentration.

Among all of the conditions that gave the plateau level of reducing sugar conversion (and concentration), the pretreatment at 1800 psi, 180 C and 30 min is clearly the condition that produced the lowest concentration of inhibitors. At the lower 160 C temperature tested, the inhibitor formation was much lower (0.8-1.2 g/L combined inhibitors) but the reducing sugar conversion was also below 40%, less than half of the plateau level. At the higher 195 C tested, the combined inhibitor concentrations more than doubled that at the

1800 psi, 180 C and 30 min condition, unless the pretreatment was kept very short at 10 min. Nonetheless, at 1800 psi, 195 C, and 10 min, both the reducing sugar conversion and inhibitor formation were somewhat comparable to those at 1800 psi, 180 C and 30 min (with about 8% lower conversion and 11% lower combined inhibitor concentration).

30 60 30

30 30 30 10 30 60 30

- - -

------

min

180

160 195 195

195 160 160 195 180 195 195

195

- - -

------



psi

4900

1800

1800 1250 4900 3400 1800 3400 1250 1800 3400 1800 1250

100% 20

80% 15 60% 10 40% Reducing sugar

Conversion Reducing sugar

5 concentration(g/L)

Sugar conversion (%) 20% Concentration 0% 0 0.00 1.00 2.00 3.00 4.00 5.00 100%

80%

60%

40% Cellulose

Sugar conversion (%) 20% Hemicellulose 0% 0.0 1.0 2.0 3.0 4.0 5.0 Combined inhibitors (g/L)

Figure 4.5 Correlation of sugar conversions and concentrations with combined inhibitor concentrations generated at different pretreatment conditions. Reducing sugar, cellulose

and hemicellulose conversions obtained after enzyme hydrolysis of pretreated guayule

biomass are shown. Furfural, HMF, and acetic acid are the inhibitors considered.

Pretreatment conditions for individual (vertical) sets of data are labeled above the top

figure, in terms of pressure (psi), temperature (C) and time (min) used.

Cellulose and hemicellulose conversion profiles, versus the combined inhibitor concentration, were generally similar to those described above for the reducing sugar.

However, the hemicellulose conversion appeared to plateau at lower inhibitor

67 concentrations (0.81-0.94 g/L), particularly for the systems pretreated with 195 C. Also, while the hemicellulose conversion tended to decline gradually after plateauing, cellulose conversion would increase slightly after the initial rapid increasing trend. The differences can be seen clearly in Figure 4.6 where cellulose conversion is plotted against the HMF concentration (Left) and hemicellulose conversion against the furfural concentration

(Right) because HMF is converted from glucose while furfural from pentoses. Cellulose conversion and HMF formation increased almost correspondingly until the harsher pretreatment condition at 195 C and 30 min when the conversion reached about 99% and then started to decrease with further increase in the HMF concentration presumably due to more glucose degradation to HMF. On the other hand, hemicellulose conversion peaked at milder pretreatment conditions of 180 C-30 min and 195 C-10 min, and then decreased with further increase in the furfural concentration indicating more pentose degradation to furfural. Overall, the sugar loss due to the production of these inhibitors was low and the pentose loss is higher than the glucose loss (but still very low compared to those by other pretreatment processes as described in the next section). The glucose loss due to HMF production was only in the range of 0.24% - 1.11%, estimated stoichiometrically according to the dehydration reaction. The pentose loss due to furfural production was estimated at 0.27% - 7.60%.

100% 100%

80% 80%

60% 60% 160 °C-30 min 160 °C-30 min 180 °C-30 min 40% 180 °C-30 min 195 °C-10 min 40% 195 °C-10 min

Cellulose Cellulose conversion (%) 195 °C-30 min 195 °C-30 min 20% Hemicellulose conversion (%) 20%

0% 0% 0.00 0.05 0.10 0.15 0.00 0.20 0.40 HMF (g/L) Furfural (g/L)

Figure 4.6 Correlation between cellulose conversion and HMF concentration (Left) and correlation between hemicellulose conversion and furfural concentration (Right) shown

for different pretreatment temperature (C)-time (min) groups at varying pressures.

4.3.6 Comparison with other pretreatment/hydrolysis methods

According to the previous section, the pretreatment at 1800 psi, 180 °C, and 30 min is optimal in producing highest reducing sugar conversion (82.8%) with lower inhibitor amounts: 1.8 mg HMF, 6.3 mg furfural and 76 mg acetic acid, per g reducing sugar released. Sugar conversion and inhibitor generation results in this optimized CO2-

H2O based pretreatment are compared in Table 4.3 with the literature values reported for other pretreatment methods. The comparisons are made with other biomass because no previous literature has reported the inhibitor generation from guayule. Per g biomass pretreated/hydrolyzed, the optimized CO2-H2O pretreatment generated much lower amounts of HMF (0.7 mg) and furfural (2.5 mg) than the other methods (1.7 and 12.3 mg

HMF and 8.7 and 24.9 mg furfural by steam explosion and hot liquid water pretreatment of poplar, respectively; and 44.8 mg HMF and 7.4 mg furfural by dilute acid pretreatment of spruce). A similar conclusion, though with different numerical values, can be drawn when the comparison is made on the basis of per g sugar released by the pretreatment/hydrolysis. The acetic acid formation appeared comparable but this value would depend on the extent of acetylation of the biomass. Acetic acid content in guayule biomass is 4.3% whereas in poplar biomass it is 3.4% and in spruce, it is 1.4% by mass

[28,119,120]. The CO2-H2O based method also afforded a significantly higher sugar conversion from guayule than other pretreatment methods did from other biomass. The minimized inhibitor generation by the optimized CO2-H2O pretreatment was therefore even more obvious in some cases.

Table 4.3: Comparison of sugar conversion and inhibitor generation by different pretreatment/hydrolysis methods

Sugar HMF Furfural Acetate HMF Furfural Acetate Method Biomassb conversion mg/(g biomass) mg/(g sugar released)

CO -H O 2 2 Guayule 83% 0.7 2.5 30.2 1.8 6.3 76.1 (this work) Steam explosion 59% 1.7 8.7 27.0 4.3 21.6 67.2 [121] Poplar Liquid hot water 65% 12.3 24.9 43.7 27.8 56.4 98.9 [121]

Dilute acida [87] Spruce 38% 44.8 7.4 37.6 186.7 31.1 156.4 Notes: a. No enzyme used after dilute acid hydrolysis b. Biomass composition: Guayule – 27.1% cellulose, 19% hemicellulose and 35% lignin; Poplar – 43.5% cellulose, 24.5% hemicellulose and 19% lignin; Spruce – 43.4% cellulose, 19.8% hemicellulose and 12% lignin.

4.4 Conclusion

Pressure effect on inhibitor formation was less significant compared to the effects of temperature and pretreatment duration. With the pretreatment conditions investigated, the inhibitor concentrations were found in the following ranges, per 50 g/L pretreated guayule: 0.007-0.126 g/L HMF, 0.006-0.397 g/L furfural, and 0.085-4.01 g/L acetic acid.

Correspondingly, the HMF formation was estimated to cause 0.24% - 1.11% glucose loss while furfural formation caused 0.27% - 7.60% pentose loss. For sugar conversions, pretreatment temperature also had a stronger effect than pressure. The 30 min pretreatment time gave higher reducing sugar conversion (more than 80%) for a given T

(195 °C) and P (1250 and 1800 psi). Increasing the time to 60 min did not further improve sugar conversion. The inhibition study with K. marxianus on media prepared with different levels of soluble materials from guayule pretreatment showed that both cell growth and ethanol production were stopped when the total inhibitor concentration was about 14 g/L, corresponding to that in hydrolysate from 200 g/L pretreated guayule. The furfural and HMF concentrations present were far lower than the critical inhibition concentrations reported in the literature. Acetic acid was likely mainly responsible for the observed inhibition Among the conditions examined, the pretreatment at 180°C, 1800 psi and 30 min was considered optimal in giving the highest total reducing sugar conversion of 82.8% ± 2.8% and low inhibitor formation: 1.8 mg HMF, 6.3 mg furfural and 76 mg acetic acid, per g reducing sugar released. This optimized CO2-H2O pretreatment compared very favorably with other pretreatment methods with reported inhibitor generation. Affording low inhibitor formation and high sugar conversion, this pretreatment provides a great opportunity for biorefinery use of guayule bagasse.

CHAPTER V

CO2-H2O BASED PRETREATMENT AND ENZYMATIC HYDROLYSIS OF

SOYBEAN HULLS

5.1 Introduction

Soybean hull makes up about 8-10% of the soybean weight [8]. It is, therefore, a major by-product of soybean processing. The global soybean production was 313 million metric tons in 2015 [9]; accordingly, about 30 million tonnes of soybean hulls were generated. Compositions reported for soybean hulls varied: cellulose 30-50%, hemicellulose 12-25%, pectin 6-15% and Klason lignin 1-4% [13–15]. The variation can be attributed, at least partly, to the different locations and cultivars planted. However, soybean hull has not received much attention so far for use as biorefinery feedstock.

For this value-added use, all types of carbohydrate in soybean hulls should be monomerized into readily fermentable sugars. Combining effective pretreatment and enzyme hydrolysis is a desirable approach. CO2-H2O based pretreatment does not use any chemical other than CO2, which is inexpensive, non-flammable, non-toxic and readily available. While binding of cellulose and hemicellulose with lignin get softened at a higher temperature in the presence of moisture, the pressurized CO2-H2O can form weak carbonic acid to slightly hydrolyze the biomass and improve subsequent enzyme hydrolysis [73,76]. Explosive release of pressure can disrupt the biomass structure and

72 decrease the cellulose crystallinity to increase the enzyme accessibility to the solid substrate [75]. Almost all of the hemicellulose content can be retained in the CO2-H2O pretreated biomass. Regarding this, Srinivasan et al. [11] have compared the CO2-H2O and dilute acid methods for pretreatment of guayule bagasse. CO2-H2O pretreatment was carried out in six conditions covering wide ranges of temperature (100-200°C), pressure

(2500-4000 psi) and cooking/holding time (30-60 min); dilute acid pretreatment was performed at 180°C for 5 min using 0.75% H2SO4. They found that the bagasse hemicellulose content remained essentially the same before and after the CO2-H2O pretreatment under all tested conditions whereas about 77% hemicellulose was removed into the acid stream by the dilute acid pretreatment. Recovery of dilute carbohydrate from the acid solution can be difficult and economically unfavorable. For soybean hulls, acid and alkali pretreatments have also been reported to significantly decrease the hemicellulose content in pretreated solids [14]. Furthermore, conventional pretreatment methods like the dilute acid pretreatment are prone to produce fermentation inhibitors such as acetic acid, furfural, and hydroxymethylfurfural (HMF), which may need costly processes to remove before the concentrated hydrolysate is used as fermentation substrate

[122]. The mild hydrolysis effect associated with the CO2-H2O pretreatment is not expected to produce high amounts of these inhibitors. Nonetheless, this potential advantage of using the CO2-H2O pretreatment method has not been demonstrated in previous studies. This is examined in this work including determination of the effects of pretreatment conditions.

In previous studies with the CO2-H2O pretreatment, high pressure (1450-5800 psi) and temperature (up to 235°C) were used [74,75,110,123,124]. These studies suggested

73 that the optimal/effective pretreatment condition depends on biomass composition, particularly the lignin content. The biomass with higher lignin content may require a comparatively harsher condition. For example, for guayule biomass with about 35% lignin, the optimal CO2-H2O pretreatment condition to get higher glucose and pentose yields in subsequent enzyme hydrolysis were found to be 175C and 3800 psi [75].

However, for rice straw with 5.7% lignin, the pretreatment at 110C and 4350 psi already gave much improvement in the following enzymatic cellulose hydrolysis [74]. Further,

Zheng et al. obtained a good glucose yield of 72.6% from enzyme hydrolysis of lignin- free Avicel pretreated by the CO2-H2O method at a far lower temperature 35C and 3000 psi [72]. Soybean hull has very low lignin content (1-4%). Presumably, its pretreatment can be effective at mild conditions. This is supported by the finding of a previous study on thermo-mechanical extrusion pretreatment of soybean hull, where the barrel temperature did not need to be higher than 80C to achieve high sugar yield in the following enzyme hydrolysis [76]. There are significant benefits if soybean hull can indeed be pretreated at mild conditions. Pretreatment at lower pressures can substantially decrease the reactor cost and the use of milder conditions can minimize generation of degradation products, compared to the higher-temperature acid pretreatment [18].

However, CO2-H2O pretreatment of complex biomass at both lower pressure and temperature has not been investigated. This work is the first to evaluate the feasibility of using the CO2-H2O method as an environmentally friendly and more economical pretreatment process particularly for low-lignin biomass such as soybean hull.

Previous studies on soybean hull pretreatment and enzyme hydrolysis have mainly focused on its cellulose portion and the released glucose [39,76,125]. However,

74 more than 40% of the carbohydrate in soybean hulls is in the forms of hemicellulose and pectin, which require different enzymes (at least xylanase and pectinase) to hydrolyze

[91,92,103,126]. Recognizing this property, we studied here also the effects of enzyme mixtures containing varying levels of cellulase, xylanase, pectinase and α-galactosidase activities. The enzyme broths were produced in this laboratory by fungal fermentation of

Aspergillus niger and Trichoderma reesei, respectively. Soybean hull was used as the fermentation substrate to ensure optimal induction of necessary enzyme activities. This study of enzyme broth effect was first made, with analysis of the released monomeric sugars. The CO2-H2O pretreatment study was then made at different pressure and temperature conditions to determine their effect on outcomes of subsequent enzyme hydrolysis. The objective of the pretreatment study was to identify the optimum condition that affords high sugar release while generating low amounts of fermentation inhibitors.

5.2 Materials and Methods

5.2.1 Materials

Soybean hulls were provided by the Archer Daniels Midland Company.

The hull pieces were approximately 100 µm thick and most had a diameter of 2-4 mm, with more than 90% being larger than 850 µm. Some hulls were ground and sieved through a 20 mesh (< 850 µm) screen in this laboratory. Both of the ground and original soybean hulls were used in this study. Enzyme broths used in the enzymatic hydrolysis were produced by submerged fermentation of Trichoderma reesei Rut C30 (NRRL

11460) and Aspergillus niger NRRL 341, respectively, using ground soybean hulls as substrate. A 3 L fermentor with 1 L working volume was used for the fermentation.

Besides 20 g/L ground soybean hulls, the medium for T. reesei fermentation had proteose peptone, 1 g/L; urea, 0.3 g/L; (NH4)2SO4, 1.4 g/L; K2HPO4, 2 g/L; CaCl2.2H2O, 0.4 g/L;

MgSO4·7H2O, 0.3 g/L; Tween 80, 0.2 g/L; CoCl2.6H2O, 0.02 g/L; FeSO4.7H2O, 5 mg/L;

MnSO4.4H2O, 1.6 mg/L; and ZnSO4.7H2O, 1.4 mg/L [90]. The medium for A. niger fermentation was the same except for the doubled concentrations of nitrogen sources, i.e., proteose peptone, urea and (NH4)2SO4. Fermentation was inoculated by 10% seeds grown in the same medium. For the T. reesei fermentation, the initial pH was 6. It was allowed to decrease naturally to 4 and then kept constant at 4 by automatic acid/base addition. The

A. niger fermentation was made at a constant pH of 5 throughout. The temperature was

25 ± 1 ºC; agitation was by a 6-blade turbine at 350 rpm. Dissolved oxygen concentration

(DO) was maintained at above 20% by automatic supplementation of pure oxygen. The cell free enzyme broth was collected by centrifugation at 12,000×g for 15 min. Enzyme activities in the harvested T. reesei broth were: cellulase, 2.2 ± 0.2 FPU/mL; xylanase, 91

± 4 U/mL; pectinase, 3.6 ± 0.8 U/mL; α-galactosidase, 0.19 ± 0.02 U/mL; and sucrase,

0.05 ± 0.02 U/mL. The A. niger broth contained cellulase, 0.61 ± 0.03 FPU/mL; xylanase, 60 ± 5 U/mL; pectinase, 4.0 ± 0.3 U/mL; α-galactosidase, 4.87 ± 0.07 U/mL; and sucrase, 5.09 ± 0.07 U/mL. The CO2 used for CO2-H2O pretreatment was purchased from Praxair Inc. (Akron, OH, USA). All of the other chemicals used were purchased from Fisher Scientific Inc.

5.2.2 Pretreatment of soybean hull

Soybean hulls were pretreated in this study by either autoclaving or the CO2-H2O based method. For the autoclave pretreatment, 2.5 g ground soybean hulls were added to

40 mL deionized water in a 250 mL Erlenmeyer flask and autoclaved at 121 C for 15

76 min. The pretreated mixture was cooled to room temperature and then stored under refrigeration for later use in enzymatic hydrolysis. For the CO2-H2O pretreatment, 15 g ground soybean hulls were evenly added, under mixing, with 30 mL deionized water. The wet hulls were transferred to a cellulose extraction thimble (Whatman®, Piscataway, NJ) and then loaded into the preheated reactor. CO2 was next pumped by a 260D syringe pump (ISCO, Lincoln, NE) to pressurize the reactor to the studied level. The reactor was held at the temperature and pressure for 30 min. The pressure was then released rapidly to create the physical explosion effect. The studied temperature range was 80-180C and the pressure range was 650-1800 psi. The pretreated soybean hulls were collected and used in the subsequent enzymatic hydrolysis. To study the explosion effect, additional experiments were made using the same pretreatment procedure except that the pressure was released very slowly, at an approximate rate of 4 psi/s, so as not to cause the explosion. All pretreatments at different conditions were done with at least two repeated batches.

5.2.3 Enzymatic hydrolysis

Enzymatic hydrolysis was studied with soybean hulls without and with different pretreatments. The hydrolysis experiments were made in 250 mL Erlenmeyer flasks.

Soybean hulls (2.5 g dry weight) were added to 50 mL enzyme solution, i.e., at a solid loading of 50 g/L. The enzyme solution was prepared from deionized water and clarified broth of A. niger or T. reesei fermentation, with different volume ratios to adjust the enzyme loading studied. Sodium azide (0.05%) was added to prevent microbial contamination during the hydrolysis. pH was adjusted to 4.8. Enzymatic hydrolysis was conducted for a studied period of time in a shaker (Thermo Scientific MaxQ 5000 77

Incubating/Refrigerating floor shaker, Ashville, NC) operating at 50°C and 250 rpm shaking speed. Duplicate samples were taken periodically and centrifuged at 10,000 rpm

(9,300×g, Eppendorf 5415D) for 10 min to collect the solids and supernatant separately.

The supernatant (hydrolysate) was analyzed for total reducing sugar and individual monomeric sugar concentrations.

For studying the effect of particle size, enzymatic hydrolysis was compared with ground (passed through a 20 mesh screen) and unground soybean hulls using the A. niger enzyme broth at an enzyme loading of 5 mL broth per g hulls. To study the enzyme loading effect, hydrolysis of ground soybean hulls was compared at different loadings of the A. niger broth, i.e., 0 (enzyme-free control), 2, 4, 8 and 12 mL per g hulls. The comparison was also made for the hydrolysis effectiveness between the T. reesei and A. niger broths in systems with 4 mL of each broth per g soybean hulls.

Enzymatic hydrolysis was also done with pretreated soybean hulls to evaluate the pretreatment effectiveness. The moisture content of each pretreated biomass was determined. This moisture content was taken into account when preparing the 50 g/L solid loading for the corresponding enzymatic hydrolysis experiment. The A. niger broth was used for these experiments with pretreated hulls. For effectiveness comparison of different pretreatment methods/conditions, the A. niger broth was adjusted to provide, per g hulls, 3 FPU cellulase (and, proportionally, other activities in the broth). This corresponded to a constant enzyme loading of 5 mL A. niger broth per g hulls. In a separate experiment to get higher sugar yield, a commercial enzyme Spezyme CP

(Dupont, Cedar Rapids, IA) was used at 18 FPU/g enzyme loading to hydrolyze 200 g/L pretreated hulls prepared by the CO2-H2O method (1800 psi and 180C).

5.2.4 Evaluation of fermentation inhibitor production

Production of the fermentation inhibitors acetic acid, HMF, and furfural by the

CO2-H2O pretreatment was evaluated for all of the pretreatment conditions used in this study. To do so, samples were taken from the pretreated biomass that had been mixed to improve sample representativeness. The samples taken were centrifuged at 10,000 rpm

(9,300×g) for 10 min. The supernatants collected were then analyzed for concentrations of these inhibitors by High-Pressure Liquid Chromatography (HPLC) as described in the next section.

5.2.5 Analytical methods

Monomer sugar composition of soybean hull (ground soybean hulls collected between 80 and 120 mesh sieves) was determined using the standard procedure prescribed by National Renewable Energy Laboratory (NREL, Golden, CO) [93]. Briefly, acid hydrolyzed samples were used to determine total reducing sugar content and monomeric sugar content. Monomeric sugar loss due to acid degradation was considered.

All procedures were done with triplicate soybean hull samples.

Total reducing sugar concentration was determined by the standard dinitrosalicylic (DNS) acid method based on the color change of reducing sugar in the sample when heated with the DNS solution [94]. Glucose was used as the calibration standard for the DNS analysis. The concentrations of individual sugars present in the hydrolysate were also measured by HPLC. This analysis was done using a Shimadzu machine equipped with a pump (LC-10AT), column oven (CTO-20A), refractive index detector (RID-10A) and a system controller (SCL-10A). Samples were prepared with

79 proper dilution and filtered through 0.22 µm nylon filters. Stachyose, raffinose, cellobiose, glucose, xylose, galactose, arabinose, mannose and fructose were separated using a SUPELCOGEL Pb column (30 cm × 7.8 mm, sulfonated polystyrene divinylbenzene packing material) and its guard column, at 80C with HPLC grade water as the mobile phase at a flow rate of 0.5 mL/min. Total run time was 40 min. Peak area of each sugar was converted to concentration using calibration established with respective sugar standards.

HPLC was also used for measuring acetic acid, HMF and furfural concentrations.

BIORAD Aminex HPX-87H column was used with 0.005N H2SO4 as the mobile phase.

The column temperature was 35C; flow rate of the mobile phase was 0.6 mL/min. A UV detector was used to detect HMF and furfural at 254 nm wavelength and acetic acid at

210 nm. Total run time was 60 min.

5.3 Results and Discussion

5.3.1 Sugar composition in soybean hull

Total reducing sugar content in soybean hull obtained by the NREL procedure with two-step acid hydrolysis was 64 ± 2% by weight, measured by the DNS analysis with glucose as standard. The HPLC analysis showed that the glucose content, 35.7 ±

1.3%, was the highest among all monosaccharide components. Other major sugars included: xylose, 13.2 ± 0.8%; galactose, 5.9 ± 0.5%; arabinose, 6.5 ± 0.2%; and mannose, 5.7 ± 1.4%. These results are very similar to those reported by Miron et al.

[13], who hydrolyzed soybean hulls with 24 N H2SO4 at 21C for 1 h and then with 1 N

H2SO4 at 100C for 5 h. Their analysis gave the following monosaccharide composition:

80 glucose, 39.5 ± 0.1%; xylose, 8.8 ± 0.1%; galactose, 4.80 ± 0.04%; arabinose, 5.55 ±

0.03%; and mannose, 7.55 ± 0.04% [13]. Glucose comes mainly from cellulose while hemicellulose contains xylose, arabinose, and mannose. Soybean hulls also contain pectin whose main components are uronic acid, galactose, arabinose, rhamnose, and fucose

[38,127,128].

5.3.2 Effect of grinding soybean hull on enzymatic hydrolysis

The release profiles of total reducing sugar during enzymatic hydrolysis of unground and ground soybean hulls are shown in Figure 5.1. Corresponding to the size reduction from 2-4 mm to < 850 µm, the grinding improved the reducing sugar release rate and the released concentration by 20% during the first 24 h. After 72 h, the released total reducing sugar concentration for the ground soybean hulls reached 8.0 ± 0.4 g/L

(27% conversion) whereas it was 6.8 ± 0.4 g/L (23% conversion) for unground hulls.

Hulls used here were not pretreated and the conversions achieved were low. Nonetheless, grinding was shown to improve the hydrolysis effectiveness. Ground hulls were used in all of the subsequent experiments.

9 30% 8 25% 7 6 20% 5 15% 4 3 10%

2 Conversion(%)

ReducingSugar (g/L) 5% 1 0 0% 0 24 48 72 Time (h) Unground Ground

Figure 5.1 Profiles of reducing sugar release from ground and unground soybean hulls

hydrolyzed by the A. niger enzyme broth; in both systems: solid loading = 50 g/L,

enzyme loading = 5 mL/g, pH = 4.8 and T = 50C

5.3.3 Soybean hull hydrolysis by different loadings of A. niger and T. reesei enzyme

Summarized in Figure 5.2(a) are the profiles of reducing sugar release from unpretreated, ground soybean hulls when hydrolyzed by different loadings of the A. niger enzyme broth, i.e., 0 (control), 2, 4, 8 and 12 mL per g hulls (all made up with deionized water to have the same total liquid volume of 50 mL for hydrolysis of 2.5 g hulls). The profiles are compared in Figure 5.2(b) for hydrolysis with 4 mL/g A. niger and T. reesei enzyme broths, respectively. In Figure 5.2(a), the addition of A. niger broth is shown to significantly improve the release of reducing sugar as compared to the enzyme-free control system. Further, among the enzyme-containing systems, the released reducing sugar concentration increased linearly with increasing enzyme loading: at 95 h, the

82 reducing sugar concentration (g/L) = 0.203 × (enzyme loading, mL/g) + 4.69, R2 = 0.995

(correlation figure not shown). This equation does not hold for the enzyme-free control and the intercept 4.69 g/L is much larger than the concentration (up to about 1.3 g/L) seen in the control. The finding suggests that certain carbohydrate component(s) in soybean hull was readily hydrolyzable by the A. niger enzyme, almost completely hydrolyzed even at the lowest enzyme loading of 2 mL/g, while other components required higher enzyme activity and were hydrolyzed in proportion to the enzyme loading. In Figure 5.2

(b), the T. reesei broth is shown to be more effective than the A. niger broth in hydrolyzing soybean hulls, giving an almost 3 times higher reducing sugar concentration after 95 h. This finding can be attributed to the higher cellulase and xylanase activities in the T. reesei broth.

8 (a) Control 18 (b) 60% 25% 2 mL/g A. niger 7 4 mL/g 16 T. reesei 8 mL/g 50% 6 20% 12 mL/g 14 5 12 40% 15% 10 4 30% 8 3 10%

6 20% Conversion(%)

2 Conversion(%) ReducingSugar (g/L)

ReducingSugar (g/L) 4 5% 10% 1 2 0 0% 0 0% 0 50 100 0 50 100 Time (h) Time (h)

Figure 5.2 Profiles of reducing sugar release during enzymatic hydrolysis of unpretreated, ground soybean hulls showing (a) the enzyme loading effect, by different loadings of the

A. niger enzyme broth, i.e., 0 (control), 2, 4, 8 and 12 mL per g hulls, and (b) comparison

of A. niger versus T. reesei broth, at the same enzyme loading of 4 mL per g hulls. The other conditions were kept constant: soybean hull loading = 50 g/L, pH = 4.8, T = 50°C.

The release profiles of main individual sugars are shown in Figure 5.3 for some selected systems from those described above. Stachyose (approximately 0.8%) and raffinose (approximately 0.4%) were the oligomers present in soybean hulls initially.

They were degraded to monomers (galactose, glucose and fructose) within the first day of hydrolysis (stachyose and raffinose concentrations not shown). The glucose and xylose profiles are shown in Figure 5.3(a) and Figure 5.3(b). Between the two A. niger systems shown, the one with higher enzyme loading (12 mL/g) achieved the higher release of glucose and xylose. The T. reesei system had the highest released concentrations, particularly for glucose, as expected from its significantly higher cellulase activity (2.2

FPU/mL, as compared to 0.6 FPU/mL in the A. niger broth). The trends of glucose and xylose release are consistent with those of total reducing sugar release results described above (Figure 5.2). This consistency is not surprising because glucose and xylose are the major constituents in soybean hull (35.7 ± 1.3% glucose and 13.2 ± 0.8% xylose).

5 40% 3 50% 4 3 2 20% 2

Xylose Xylose (g/L)

Glucose(g/L) Conversion(%) 1 Conversion(%) 0 0% 0 0% 0 50 100 0 50 100 Time (h) Time (h) Control A. niger: 4 mL/g Control A. niger: 4 mL/g A. niger: 12 mL/g T. reesei: 4 mL/g A. niger: 12 mL/g T. reesei: 4 mL/g 2 3 100% 1.5 50% 80% 2 1 60% 40%

0.5 1

Galactose (g/L) Galactose

Conversion(%) Mannose(g/L) Conversion(%) 20% 0 0% 0 0% 0 50 100 0 50 100 Time (h) Time (h) Control A. niger: 4 mL/g Control A. niger: 12 mL/g T. reesei: 4 mL/g A. niger: 4 mL/g

Figure 5.3 Monomeric sugar release profiles observed in some of the enzymatic

hydrolysis experiments described in Figure 5.2

Also, as expected, among all the systems hydrolyzed by different loadings of A. niger enzyme, the glucose and xylose release increased roughly linearly with increasing cellulase and xylanase loadings, respectively (Figure 5.4).

3.0 3 2.5 3 2.0 2 1.5 2

Xylose Xylose (g/L) 1.0

Glucose Glucose (g/L) 1 0.5 1 0.0 0 0 200 400 600 800 0 2 4 6 8 Xylanage loading (U/g) Cellulase loading (FPU/g)

Figure 5.4 Correlation (a) between glucose concentration and cellulase loading and (b)

between xylose concentration and xylanase loading after 95 h enzymatic hydrolysis of

unpretreated ground soybean hull by A. niger broth

The galactose and mannose profiles are shown in Figure 5.3(c) and Figure 5.3(d).

Among the four monosaccharides, mannose was clearly the easiest to hydrolyze; about

90% conversion was achieved in all of the three enzyme-containing systems shown in

Figure 5.3(d). Mannose came predominantly from hemicellulose in soybean hulls

[76,113]. Although the specific enzyme activity for mannan hydrolysis was not analyzed here, the A. niger broth appeared to be more effective in mannose release; even at a 2 mL/g loading (Figure 5.5), the A. niger broth released more mannose at 24 h (2.2 g/L) than the T. reesei broth at a 4 mL/g loading (1.8 g/L). On the other hand, galactose was significantly harder to release, reaching only about 30% conversion after 96 h in all of the enzyme-containing systems. For soybean hulls, galactose came mainly from the pectin but also from oligosaccharides like stachyose and raffinose. With the A. niger broth, the release profiles were essentially the same for a wide range of enzyme loading from 2 mL/g to 12 mL/g, and the release plateaued after 24 h. The A. niger broth again appeared

86 to release galactose faster initially than the T. reesei broth, possibly because of the much higher α-galactosidase activity in the former (4.87 U/mL, versus 0.19 U/mL in the latter).

The results however suggested that pectinase could only attack very limited portion(s) of the pectin in unpretreated soybean hulls. It is important to note that this situation was improved significantly after proper pretreatment of the soybean hulls, as described in the following section.

3 100% Control 2.5 A. niger: 2 mL/g A. niger: 4 mL/g 80% A. niger: 8 mL/g 2 A. niger: 12 mL/g T. reesei: 4mL/g 60% 1.5

40% Mannose(g/L) 1 Conversion(%)

0.5 20%

0 0% 0 50 100 Time (h)

Figure 5.5 Mannose release profile during enzymatic hydrolysis of unpretreated ground

soybean hull; enzymatic hydrolysis condition were described in Figure 5.2.

5.3.4 Pretreatment effect

5.3.4.1 Reducing sugar release

The pretreatment effect was studied with seven CO2-H2O pretreatment systems, one autoclave pretreatment system, and a control system without pretreatment. Autoclave

pretreatment was done at 121C for 15 min. For the CO2-H2O pretreatment, the moisture content and hold time (at the pretreatment pressure and temperature) were kept constant

87 at 66.7% and 30 min, respectively. These were found optimum for the CO2-H2O pretreatment of guayule bagasse in a previous study [75]. The highest temperature tested was set at 180C which was close to the optimum temperature (175°C) for guayule pretreatment. Higher temperatures were not considered because they tend to generate more fermentation inhibitors from sugar degradation [18].

Enzymatic hydrolysis in the pretreatment study was done with 5 mL of the A. niger enzyme broth per g soybean hull, which corresponded to 3 FPU/g cellulase, 300

U/g xylanase, 19.8 U/g pectinase, 24.4 U/g α-galactosidase and 25.5 U/g sucrase. The 3

FPU/g cellulase activity used in this study was very low compared to the 15-20 FPU/g usually used for other lignocellulosic biomass [129]. The lower enzyme dosage was chosen so that the enhancements under different pretreatment methods and conditions could be clearly differentiated. Enzymatic hydrolysis was done for 72 h and samples were taken at 0 (before enzyme addition), 24, 48 and 72 h.

Reducing sugar concentration profiles over the 72 h enzymatic hydrolysis and the final conversion attained after 72 h are compared in Figure 5.6. The autoclave pretreatment improved the reducing sugar release after 72 h by almost 37% (10% more conversion), compared to the unpretreated control. However, at 0 h (before enzyme addition) the reducing sugar concentration was already higher, which showed the occurrence of some hydrolysis during the autoclave pretreatment. The subsequent sugar release was slightly higher than the control. The improved overall sugar release came mostly from the thermal hydrolysis during the autoclaving.

Hydrolysis during pretreatment (prior to enzyme addition) did not occur for the

CO2-H2O pretreatments done at 80°C and 130°C (Figure 5.6(a), 0 h data) but at 180°C, it was even higher than that by the autoclave pretreatment. The CO2-H2O pretreatments at milder conditions, i.e., 750 psi-80°C and 1250 psi-80°C, improved the reducing sugar release only slightly; the reducing sugar concentrations after 72 h enzymatic hydrolysis were 6-11% higher and the release rates during the first 24 h were 0.23-0.25 g/L-h, slightly higher than that of the unpretreated system (0.21 g/L-h). The pretreatments at

130°C and 180°C, however, gave very high sugar release rates during the first 24 h (0.31-

0.43 g/L-h) compared to the other systems. After 72 h enzymatic hydrolysis, 77-84% improvements in the released reducing sugar concentration (20%-23% more conversion) were obtained, over that of the unpretreated control. Also, the approximately 50% reducing sugar conversion obtained here with an enzyme loading of only 3 FPU/g (plus other activities proportionally present in the A. niger broth) was comparable to the conversion obtained from unpretreated hulls using an enzyme loading of 8.8 FPU/g (plus other activities in the T. reesei broth) (Figure 5.2(b)). Therefore, the CO2-H2O based pretreatment at proper conditions could significantly reduce the required enzyme loading, here by almost two thirds.

For the CO2-H2O pretreatments done at 130°C, the temperature was close to the autoclaving temperature (121°C). The CO2-H2O pretreatments were however much more effective in improving enzymatic hydrolysis, reaching 47-50% conversion after 72 h, higher than the 37% conversion attained in the autoclave pretreatment system.

Presumably, the presence of CO2 offered a higher reaction effect (mild acid hydrolysis) in the CO2-H2O pretreatment. Also, the explosion effect created by the sudden pressure drop

89 at the end of the CO2-H2O pretreatment might have contributed a mechanical effect on the soybean hull structure, which in turn helped the enzyme hydrolysis.

18 (a) Unpretreated 16 Control Autoclave 14 pretreatment 750 psi-80°C 12 10 1250 psi-80°C

8 750 psi-130°C 6 1250 psi-130°C

Reducing sugar (g/L) 4 750 psi-180°C 2 0 0 20 40 60 80 Time (h)

18 (b) 60% 16 50% 14 12 40% 10 30% 8

6 20% Conversion Conversion (%)

Reducing sugar (g/L) 4 10% 2

0 0%

Control

Autoclave

750 psi-80°C 750

Pretreatment

Unpretreated

1250 psi-80°C 1250 psi-130°C 750 psi-180°C 750

1250 psi-180°C 1250 psi-180°C 1800 1250 psi-130°C 1250

Figure 5.6 (a) Pretreatment effect on the reducing sugar release profiles during enzymatic hydrolysis, shown for different pretreatment methods and conditions. (b) Comparison of

total reducing sugar concentrations released after 72 h enzymatic hydrolysis. A. niger enzyme broth and ground soybean hulls were used; the enzyme loading was 5 mL/g. The enzymatic hydrolysis conditions were soybean hull loading = 50 g/L, pH = 4.8, T = 50°C.

5.3.4.2 Monomeric sugar release

Effect of the CO2-H2O based pretreatments on the individual monomeric sugar release in enzyme hydrolysis is presented in Figure 5.7. In the unpretreated control, not much hydrolysis occurred after 24 h; only the readily available carbohydrate was hydrolyzed. All pretreatment systems showed the improved release of monomeric sugars, except for mannose where the pretreatments at high temperature (180C) gave lower mannose yields (presumably due to degradation during pretreatment). Also, the sugar release continued beyond 24 h in at least some of the pretreatment systems; this longer- term release was especially clear for glucose and xylose. These findings indicated that the pretreatments helped open up the hull structure and make the substrate more accessible and hydrolyzable by the enzyme.

As mentioned earlier, some hydrolysis occurred during the pretreatment step for some pretreatment conditions. By comparison of the 0 h concentrations of these sugars in the pretreatment systems and those in the control, it is shown that this hydrolysis during pretreatment mainly released more xylose and arabinose. On the other hand, the 0 h mannose concentration was higher in the control than in all pretreatment systems. This suggested that the released mannose was very prone to degradation by the pretreatments.

It was previously reported that mannose is more susceptible to degradation than glucose but no direct comparison was found between mannose, xylose, and arabinose [130].

Xylose, arabinose, and mannose are all main components of hemicellulose. It is clear that the hydrolysis occurring during the CO2-H2O pretreatment was mainly on the hemicellulose content in hulls.

Regarding the release profiles over time, the release of minor components galactose, arabinose, and mannose occurred predominantly during the first 24 h, except for the systems pretreated at the lowest temperature 80°C; the release of glucose and xylose, however, continued more appreciably beyond 24 h. Overall, the highest releases of glucose (4.70 ± 0.02 g/L, 28% conversion), xylose (3.66 ± 0.11 g/L, 59% conversion) and galactose (1.93 ± 0.04 g/L, 70% conversion) were obtained in the system pretreated with the highest pressure-temperature (1800 psi-180°C) condition. The high temperature systems, however, did not give the highest arabinose release and gave the lowest mannose release.

5 (a) Glucose (b) Xylose Unpretreated 4 Unpretreated 750 psi-80°C 750 psi-80°C 4 1250 psi-80°C 3 1250 psi-80°C 3 750 psi-130°C 750 psi-130°C 1250 psi-130°C 2 1250 psi-130°C 2 750 psi-180°C 750 psi-180°C

1250 psi-180°C 1 1250 psi-180°C Concentration(g/L)

1 Concentration(g/L) 1800 psi-180°C 1800 psi-180°C 0 0 0 12 24 36 48 60 72 0 12 24 36 48 60 72 Time (h) Time (h) (c) Galactose (d) Arabinose 2.5 Unpretreated 2.5 Unpretreated 750 psi-80°C 2.0 750 psi-80°C 1250 psi-80°C 2.0 1250 psi-80°C 1.5 750 psi-130°C 1.5 750 psi-130°C 1250 psi-130°C 1250 psi-130°C 1.0 750 psi-180°C 1.0 750 psi-180°C 1250 psi-180°C 1250 psi-180°C Concentration(g/L) 0.5 0.5 1800 psi-180°C Concentration(g/L) 1800 psi-180°C

0.0 0.0 0 12 24 36 48 60 72 0 12 24 36 48 60 72 Time (h) Time (h) (e) Mannose 3.5 Unpretreated 3.0 750 psi-80°C 1250 psi-80°C 2.5 750 psi-130°C 2.0 1250 psi-130°C 1.5 750 psi-180°C

1.0 1250 psi-180°C Concentration(g/L) 0.5 1800 psi-180°C 0.0 0 12 24 36 48 60 72 Time (h)

Figure 5.7 Effects of CO2-H2O based pretreatments on monomeric sugar release: (a) glucose, (b) xylose, (c) galactose, (d) arabinose, and (e) mannose. Enzymatic hydrolysis

conditions were the same as those given in Figure 5.6.

The effects of temperature and pressure on the outcomes of CO2-H2O based pretreatments are illustrated in Figure 5.8 with the sugar concentrations obtained after 72 h enzymatic hydrolysis. The temperature effect is shown with systems pretreated at 1250 psi (Figure 5.8(a)) while the pressure effect with systems at 180°C (Figure 5.8(b)); nonetheless, the general trends were similar if compared at other pressures and temperatures. Regarding the potential pressure effect, the ANOVA analysis showed that these pressures had a significant effect (p = 0.043) only on the glucose release by pretreatments at 180°C, as seen here in Figure 5.8(b) by the increased-then-plateaued glucose release with increasing pressure. The pressure effect was insignificant for the release of all other sugars at this temperature (180°C) and all sugars (including glucose) at other 2 pretreatment temperatures (80°C and 130°C), with p values ranging from 0.117 to 0.986. The pretreatment temperature, on the other hand, had more significant effects.

For pretreatments done at 750 psi, the p values for temperature effect on sugar release were all smaller than 0.05, in the range of 0.006-0.042. For pretreatments at 1250 psi, the temperature effect was also significant on release of glucose, xylose and arabinose (p =

0.005-0.02) but not significant on release of galactose (p = 0.367) and mannose (p =

0.078). As shown in Figure 5.8(a), raising the pretreatment temperature from 80°C to

130°C increased the released concentrations of all monomeric sugars (glucose, xylose, galactose, arabinose and mannose) but further raising it to 180°C had different effects on different sugars: it increased glucose release slightly but decreased the releases of others particularly mannose and arabinose. The decrease was presumably due to more sugar degradation during pretreatment at higher temperature. It has been reported that at high temperature and acidic conditions, sugars could be converted to furfural and HMF and

94 then furfural to formic acid and HMF to levulinic acid and formic acid [81,130]. In this study, mannose was found to be the most sensitive to such degradation in the CO2-H2O based pretreatment, followed by arabinose.

(a) Temperature effect (1250 psi) (b) Pressure effect (180°C) 5 5 Glucose Glucose 4 4 Xylose Xylose 3 3 Galactos e Galactose 2 2 Arabinos e

Concentration(g/L) 1 Concentration(g/L) 1

0 0 60 80 100 120 140 160 180 700 950 1200 1450 1700 Temperature (°C) Pressure (psi)

Figure 5.8 (a) Effect of pretreatment temperature (at fixed pressure 1250 psi) and (b) pressure (at fixed temperature 180°C) on monomeric sugar release after 72 h enzymatic

hydrolysis (at same conditions as those in Figure 5.6)

5.3.4.3 Acetic acid, HMF and furfural formation during pretreatment

In biorefinery fermentations using biomass hydrolysate as a substrate, the presence of acetic acid, HMF and furfural in the hydrolysate has been reported to have negative effects on some microorganisms [18,20]. During pretreatment and chemical hydrolysis, acetic acid can be produced from hydrolysis of the acetyl group in hemicellulose and HMF, and furfural can be formed from the dehydration reaction of hexose and pentose sugars, respectively, at higher temperatures and acidic conditions

[18–20]. The acetic acid, HMF and furfural concentrations observed in the current study at different pretreatment conditions are summarized in Table 5.1. These concentrations were measured in suspensions of 50 g/L soybean hulls in the water. Increasing

95 temperature generally increased the formation of these inhibitory compounds but the increases were more apparent only at the high temperature of 180°C. At this high temperature, increasing pressure also generally increased the inhibitor formation, particularly apparent for HMF and furfural. Except for the pretreatment at the harshest condition (1800 psi and 180°C), HMF and furfural concentrations were very low (< 5 mg/L).

For comparison, Cassales et al. [39] studied acid hydrolysis of soybean hulls. For hydrolysis at a solid loading of 20%, they observed that the use of 1.7% H2SO4 at 153 °C for 60 min generated about 6 g/L combined inhibitory compounds (i.e., acetic acid, HMF, and furfural), which was higher than the 4 g/L tolerable threshold generally reported for several microorganisms. They then used a less harsh condition: 118 °C, 2.7% H2SO4, and

40 min. Under this condition, the combined inhibitor concentration decreased to 3 g/L.

By the CO2-H2O based pretreatment used in this study, the total combined inhibitor concentration, after adjustment to the equivalent 20% solid loading, would be 2.5 g/L at the harshest pretreatment condition of 1800 psi and 180°C. This is lower than those reported by Cassales et al. [39] for acid hydrolysis of soybean hulls. More importantly, the inhibitor concentration results suggested 1250 psi and 130°C as the optimal pretreatment condition. At this condition, the combined inhibitor concentration was below 0.3 g/L (at 20% solids loading), which is one order of magnitude lower than the concentrations reported by Cassales et al. [39]. No inhibitory effects are expected when the enzymatic hydrolysate produced from hulls pretreated under this condition is used as a fermentation substrate.

Table 5.1 Acetic acid, 5-hydroxymethyl-2-furaldehyde (HMF) and furfural

concentrations found in an aqueous suspension of 50 g/L soybean hulls unpretreated or

pretreated by the CO2-H2O based method at different conditions

Pretreatment condition Acetic acid (g/L) HMF (mg/L) Furfural (mg/L)

Unpretreated 0.036±0.003 0.14±0.06 ND 750 psi-80°C 0.027±0.002 0.05±0.05 ND 1250 psi-80°C 0.040±0.003 0.26±0.12 0.21±0.05 750 psi-130°C 0.085±0.005 0.51±0.09 0.20±0.05 1250 psi-130°C 0.071±0.006 0.21±0.21 0.18±0.10 750 psi-180°C 0.334±0.178 2.63±1.15 2.16±0.19 1250 psi-180°C 0.357±0.006 4.30±0.71 1.74±0.00 1800 psi-180°C 0.570±0.012 16.65±0.33 33.82±0.67 Note: ND – not detectable

5.3.4.4 Optimal pretreatment condition and vessel cost analysis

According to all the results described above, the optimal condition for the CO2-

H2O based pretreatment of soybean hulls is at/near 1250 psi and 130°C. The condition was mild regarding generation of minimal levels of inhibitors and the hulls pretreated at this condition gave the highest total reducing sugar concentration in the subsequent enzymatic hydrolysis, among all of the pretreatment conditions studied as shown in

Figure 5.6(b). Monomeric sugar release after 72 h enzymatic hydrolysis of hulls pretreated at this optimal condition is compared in Figure 5.9 with the release from the unpretreated control. All of the monomeric sugar concentrations obtained were significantly higher in the pretreated system. Even when compared with the unpretreated system hydrolyzed with 2.5-fold more enzyme (12 mL/g, compared to 5 mL/g in the pretreated system), glucose, xylose and mannose releases in the pretreated system were

97 improved by 55%, 35%, and 52%, respectively. Improvements in galactose and arabinose releases were even higher, by 105% and 683%, respectively.

1 Concentration(g/L) 0 Glucose Xylose Galactose Arabinose Mannose

Unpretreated (4 mL/g) Unpretreated (12 mL/g) Pretreated (1250 psi, 130 deg.C, 5 mL/g)

Figure 5.9 Comparison of monomeric sugar release from 50 g/L pretreated and unpretreated soybean hulls, after 72 h hydrolysis with A. niger broth (loadings specified)

at pH 4.8 and 50°C

Because the CO2-H2O based pretreatment is done at relatively high pressures, further considerations are given for the vessel costs at different pressures. The cost is also compared with the conventional dilute acid pretreatment vessel. The bare module cost of process vessel can be estimated using the following equations [131]:

퐵푎푟푒 푀표푑푢푙푒 퐶표푠푡 = 푃푢푟푐ℎ푎푠푒 푐표푠푡 (퐶푃) × (2.25 + 1.82 × 퐹푀 × 퐹푃)

2 퐶푃 = 3.4974 + 0.4485 × 퐿표𝑔(푉) + 0.1074 × (퐿표𝑔(푉))

Where FM is a material factor depending on the material of construction required, FP is a pressure factor depending on the vessel pressure P (and diameter D), and V is the vessel

3 volume (m ). For the CO2-H2O based pretreatment, a stainless steel clad material (FM =

1.7) is sufficient to handle the slightly acidic condition associated; for the dilute acid

98 pretreatment, nickel alloy (FM = 7.1) is needed for the highly corrosive environment

[131]. FP can be calculated according to the following equation [131]:

퐷 {(푃 + 1) × + 0.00315} (2 × [850 − 0.6 × (푃 + 1)]) 퐹 = (1) 푃 0.0063

Where P is the pressure in bar (1 bar = 14.5 psi) and D is the vessel diameter (m). The processing volume also depends on the length (L) of the vessel. The optimal L/D ratio varies for different pressure ranges, to give the minimal overall cost that accounts for other factors such as the mixer cost, weight, and floor area requirement. For pressures lower than 250 psi, the optimal L/D is 3; for pressures higher than 500 psi, the optimal

L/D increases to 5 [132].

Considering all of the above, the bare module costs can be estimated, e.g., for processing 100,000 metric ton soybean hulls per year using a 30 m3 vessel. The cost comparison is summarized in Table 5.2 for the CO2-H2O based pretreatment under the 3 pressures (750, 1250 and 1800 psi) tested in this study and for the dilute acid pretreatment. As expected, the vessel bare module cost for the CO2-H2O based pretreatment increases with pressure, i.e., about $1.20M, $1.95M and $2.82M at 750,

1250 and 1800 psi, respectively, as of 2016 (Chemical Engineering Plant Cost Index

551.7). For the dilute acid pretreatment vessel (P = 141 psi [133]), the cost is estimated at

$1.38M. The mild CO2-H2O based pretreatment at 750 psi would, therefore, require a significantly cheaper vessel than the more corrosive dilute acid pretreatment. Also, for the dilute acid pretreatment, an additional cost, approximately 9% of the vessel cost, is required for conditioning the vessel, which is not needed for the CO2-H2O based pretreatment vessels [133]. Furthermore, about 10% xylose and 1% glucose loss would

99 occur associated with the dilute acid pretreatment [133]. Among the CO2-H2O based pretreatments, the optimum condition for obtaining higher sugar conversion by the combined pretreatment and enzyme hydrolysis is at 1250 psi and 130°C (e.g., reducing sugar conversion = 50% with the A. niger enzyme of 3 FPU/g loading). The pretreatment at a lower pressure of 750 psi and the same temperature (130°C) gives slightly lower sugar conversion (e.g., 47% with the same enzyme) but requires much lower vessel costs.

This lower pressure pretreatment may be a more preferred process in practice.

Table 5.2 Comparison of pretreatment vessel bare module costs for the CO2-H2O based pretreatment at different pressures and for the dilute acid pretreatment (vessel volume =

30 m3)

Pressure Material Pressure Factor Bare Module Pretreatment L/D (psi) Factor (FM) (FP) Cost ($) CO2-H2O 750 5 1.7 10.6 $1,203,532 CO2-H2O 1250 5 1.7 17.6 $1,951,864 CO2-H2O 1800 5 1.7 25.7 $2,820,026 Dilute acid 141 3 7.1 2.9 $1,375,922

5.3.5 Effect of physical explosion

The CO2-H2O based pretreatment can provide a combination of reaction and physical explosion effects. CO2 can form weak carbonic acid which, particularly at high temperatures, can hydrolyze the hemicellulose content and open up the solid structure.

On the other hand, physical explosion offers a mechanical mechanism to rupture the more rigid pectin and cellulose structures, whereby rendering the substrate more accessible and hydrolyzable to the enzyme [70]. Results described for systems pretreated by the CO2-

H2O based method in the previous sections are all from pretreatments with fast pressure

100 release at the end to create the physical explosion effect. A study was done to evaluate the contribution of the explosion effect. The results are shown in Figure 5.10, where the reducing sugar release profiles during the enzymatic hydrolysis are compared for systems with and without the explosion step. The comparison is made at two pressure-temperature conditions: 750 psi-180°C (Figure 5.10(A)) and 1250 psi-180°C (Figure 5.10(B)). For both conditions, the explosion was found to improve the pretreatment effect. More specifically, the explosion increased the final reducing sugar concentration (after 72 h enzymatic hydrolysis) by almost 14% and 20% at 1250 psi and 750 psi, respectively. The

24-h enzymatic hydrolysis rate was also faster in the exploded systems.

20 16 14 15 12 10 10 8 6 5 4

2 Total reducing sugar (g/L) sugar reducing Total 0 Total(g/L) sugar reducing 0 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Time (h) Time (h) Rapid pressure release Slow pressure release Rapid pressure release Slow pressure release

Figure 5.10 Physical explosion effect evaluated at two pretreatment conditions: (A) 750 psi and 180°C, and (B) 1250 psi and 180°C, shown by comparison of the reducing sugar release profiles during enzymatic hydrolysis of pretreated soybean hulls; enzyme loading

– 5 mL/g A. niger broth, and solid loading – 50 g/L

5.3.6 Enzymatic hydrolysis of pretreated hulls at higher enzyme loading

In the previous sections, the enzymatic hydrolysis was all done with low enzyme loadings so that the pretreatment effects at different conditions could be more clearly

101 differentiated. To verify that high conversions could indeed be obtained by the CO2-H2O pretreated soybean hulls, an experiment was done with a higher enzyme loading. 200 g/L pretreated hulls (1800 psi, 180°C) was hydrolyzed using Spezyme CP at 18 FPU/g

(versus 3 FPU/g in the experiments reported in previous sections). Monomeric sugar concentrations after 72 h hydrolysis were (g/L): glucose – 69.0 ± 1.4; xylose – 26.1 ± 0.3; galactose – 4.8 ± 1.0; arabinose – 7.7 ± 0.7, and mannose – 9.9 ± 0.3. These corresponded to approximate conversions of 97% for glucose, 98% for xylose, 41% for galactose, 59% for arabinose and 87% for mannose (based on the monomeric sugar composition measured for soybean hulls). The overall monomeric sugar conversion was about 89%.

Pectinase activity might be low in the commercial enzyme; thus, galactose conversion was less complete in this hydrolysis. With the A. niger broth at much lower (1/6th) FPU loading, galactose, arabinose and mannose conversions reached 70%, 31%, and 60%, respectively, in the release profiles shown in Figure 5.7.

5.4 Conclusion

Grinding soybean hulls to smaller pieces improved its enzymatic hydrolysis.

Hydrolysis of ground but unpretreated hulls using A. niger and T. reesei broths showed that glucose and xylose release almost linearly correlated with the cellulase and xylanase loadings (i.e., U/g hull); mannose was easily hydrolyzed by enzyme to almost 90% conversion even at relatively low enzyme loadings; the enzyme broth only slightly improved galactose release (from ~18% conversion in enzyme-free control to ~30% with enzyme); and arabinose release was almost undetectable (~5% conversion). The CO2-

H2O based pretreatment significantly improved the enzymatic hydrolysis outcomes. The study of pretreatment pressure and temperature effects on sugar release and inhibitory

102 degradation product formation concluded that the optimal condition is at or near 1250 psi and 130°C (for the fixed moisture content – 2 mL water per g hulls, and pretreatment time – 30 min). Using this pretreatment condition, the conversions were glucose – 25%, xylose – 54%, galactose – 63%, arabinose – 65% and mannose – 118%, after 72 h hydrolysis of 50 g/L hulls with 5 mL/g A. niger enzyme. Compared to the hydrolysis of unpretreated hulls with 2.5-fold more enzyme, the pretreatment improved glucose, xylose, galactose, arabinose and mannose release by 55%, 35%, 105%, 683% and 52%, respectively. Combined inhibitor (acetic acid, HMF, and furfural) formation by this pretreatment was at least one order of magnitude lower than that by a report on dilute acid pretreatment of soybean hulls. The inhibitor concentrations were negligible compared to the reported tolerable threshold. Very high conversions: 97% for glucose,

98% for xylose, 41% for galactose, 59% for arabinose, 87% for mannose and 89% for total reducing sugar, were demonstrated with hydrolysis of 200 g/L CO2-H2O pretreated hulls with Spezyme CP at 18 FPU/g loading. The CO2-H2O pretreatment followed by enzymatic hydrolysis was shown to be very effective in monomerizing all carbohydrate types in soybean hull with negligible acetic acid, HMF, and furfural formation, making soybean hull a highly feasible biorefinery feedstock.

Also, the vessel cost analysis shows that the vessel for the CO2-H2O based pretreatment at 750 psi is even significantly cheaper than the dilute acid pretreatment vessel, the latter requires more expensive corrosion-resistant material for construction.

Overall, the work clearly indicates that the CO2-H2O based pretreatment requires much lower pressures for treating biomass of low lignin contents like soybean hulls, compared to the pressures previously reported for treating other lignocellulosic biomass. The lower

103 pressure requirement coupled with the very chemically mild condition allow the use of cheaper reactors and make this new pretreatment method not only effective but also economical and practical.

104

CHAPTER VI

HIGH MONOMERIC SUGAR YIELDS FROM ENZYMATIC HYDROLYSIS OF

SOYBEAN MEAL AND EFFECTS OF MILD HEAT PRETREATMENTS WITH

CHELATORS

6.1 Introduction

Defatted soybean meal has high protein content (around 50%) and excellent amino acid profile [47] and is typically used in animal feed [31]. Soybean meal also has

30–35% carbohydrate [47], which is currently undervalued but has been proposed as biorefinery feedstock, after hydrolysis to fermentable sugars, to produce chemical and biofuels [134–136]. The carbohydrate comprises roughly equal amounts of non-structural and structural components [30,45]. The nonstructural carbohydrates are predominantly sucrose and galacto-oligosaccharides, with minimal amounts of monosaccharides and storage polysaccharides [42]. The structural carbohydrates, also known as non-starch polysaccharides (NSPs), include dietary fiber components such as cellulose, hemicellulose and pectin [137,138]. Hydrolyzing these carbohydrates enzymatically to fermentable sugars requires a complex enzyme system with at least pectinase, xylanase, cellulase, α-galactosidase and sucrase activities [91,104]. Trichoderma reesei and

Aspergillus niger are among the most extensively studied fungal species for production of these carbohydrases [91,139,140]. Li et al. [91] examined 15 Aspergillus species and T. reesei Rut C30 for production of these enzymes. Loman and Ju [92] developed models

105 for the effects of individual enzyme activities on hydrolytic conversions of soybean meal carbohydrate to soluble reducing sugar (by dinitrosalicylic acid, DNS, analysis) and carbohydrate (by phenol-sulfuric acid analysis). The enzymatic hydrolysis process will be more economical if all types of soybean carbohydrate can be hydrolyzed.

The cellulose content in soybean meal reported in the literature varied from 1-2%

[23] to 4.4-6.2% [45,46], presumably depending on processing methods (particularly the dehulling completeness) and different product requirements [141,142]. Typical A. niger fermentation broths had relatively low cellulase activities (~0.6 FPU/mL) [91,92]. This could limit glucose conversion from the soybean meals with higher cellulose content.

Also, soybean meal has rather high (around 8-10%) pectin content. While A. niger strains were screened in previous work for higher pectinase production [91], pectinase still tended to be a more limiting activity for soybean meal carbohydrate hydrolysis [92].

Also, pectin can be a protective layer covering cellulosic microfibrils that hinders the cellulase access [143]. Enzymatic hydrolysis of soybean meal carbohydrate can, therefore, benefit from improved pectin and cellulose hydrolysis.

Pretreatment has been shown effective to improve enzymatic hydrolysis of soybean meal [144,145], hulls [14,146], and fibers [147]. Fischer et al. [144] pretreated

(autoclaved) soybean meal at 125°C for 15 min, at two moisture levels. At the high moisture level, i.e., with 200 g meal in 1800 ml water, the heat pretreatment improved the enzymatic extractability (into hydrolysate) of carbohydrate to 85%, from 76% in the control without pretreatment. The improvement was, however, insignificant (78% versus

76%) with soybean meal containing only 15% moisture. The heat pretreatment in the presence of sufficient water was thought to loosen the intertwined structures between

106 protein and carbohydrate and, accordingly, increase the accessibility of carbohydrate to hydrolytic enzymes. Other pretreatment methods such as toasting and extrusion have also been investigated [145].

The objective of this study is to improve the sugar yields in enzymatic hydrolysis of soybean meal by different treatment methods. The carbohydrate composition of soybean meal used in this study was first analyzed using the standard National

Renewable Energy Laboratory (NREL) procedure [93]. An A. niger enzyme broth produced in this laboratory was used for the hydrolysis. For potential enhancements in enzymatic hydrolysis, three chelating agents, i.e., ethylenediaminetetraacetic acid

(EDTA), sodium hexametaphosphate (HMP), and citric acid were examined at a heated condition (90 °C, 2 h). Multivalent cations such as Ca2+ are known to be able to form ionic bonding/crosslinking with the polygalacturonate backbones of pectin [24,25]. Use of chelators (EDTA and HMP) has been shown to improve extraction of soybean okara pectin into aqueous media [26,27]. In addition to chelating, citric acid may act as a dilute acid pretreatment agent. Organic acids such as fumaric acid and maleic acid were found to have similar dilute acid pretreatment effects as sulfuric acid (all acids at 50 mM) on wheat straw (at 10% solid loading), giving comparable sugar (glucose and xylose) yields in the subsequent enzymatic hydrolysis [148]. Next, using a three factor-two level factorial design, the effects of heat treatment, citric acid, and cellulase supplementation on improving enzymatic hydrolysis were studied. Here, the rather mild heat treatment was done at 90 °C; at this temperature, sugar degradation and generation of fermentation inhibitors such as furfural and hydroxymethylfurfural (HMF) are expected to be very low

[149,150] and there is no need for use of expensive pressure vessels. Yields of total

107 reducing sugar and individual monomeric sugars were determined and compared to evaluate pre/treatment effects. This is the first report on citric acid-based biomass pretreatment, using its chelating and dilute acid effects, to improve enzymatic carbohydrate hydrolysis.

6.2 Materials and Methods

6.2.1 Materials

The soybean meal used in this study was provided by Archer Daniels Midland

Company (Decatur, IL). The enzyme broth used for enzymatic hydrolysis was produced in this laboratory by submerged A. niger NRRL 341 fermentation using soybean hull as carbon source [91]. The broth was clarified by centrifugation (12,000 × g for 15 min) and had the following measured enzyme activities: cellulase, 0.31 ± 0.02 FPU/mL; xylanase,

82.3 ± 4.2 U/mL; pectinase, 2.2 ± 0.5 U/mL; α-galactosidase, 4.1 ± 0.1 U/mL; and sucrase, 5.4 ± 0.1 U/mL. The commercial enzyme SPEZYME®CP (Genencor, Finland) was also used. It had the following measured activities: cellulase, 147 FPU/mL; xylanase,

349 U/mL; pectinase, 7.3 U/mL; and α-galactosidase, 2.2 U/mL. The chemicals used were all purchased from Fisher Scientific Inc.

6.2.2 Enzymatic hydrolysis

All enzymatic hydrolysis experiments were done with 250 mL Erlenmeyer flasks in a shaker (Thermo Scientific MaxQ 5000 Incubating/Refrigerating floor shaker,

Ashville, NC) at 50 °C and 250 rpm. In each flask 1.5 g soybean meal was added (at 50 g/L) to a 30 mL aqueous solution composed of 3 mL enzyme broth and 27 mL deionized

108 water (i.e., with an enzyme loading of 2 mL per g soybean meal). Initial pH was adjusted to 4.8 and sodium azide (0.05%) was added to prevent microbial contamination during the hydrolysis. Samples taken from the experiments were centrifuged at 10,000 rpm

(9300×g, Eppendorf 5415D) for 10 min to separately collect the wet solids and the supernatant for analyses.

6.2.3 Effect of soybean meal particle size on enzymatic hydrolysis

Soybean meal particles were separated by sieving into 4 groups of different particle sizes: 23.7-75 µm, 150-212 µm, 300-425 µm and 425-1180 µm. Duplicate systems for each particle size group were then subjected to 48-h enzymatic hydrolysis for comparison. Samples were taken from all systems at 0, 2, 4, 9, 24 and 48 h, and the hydrolysates (supernatants) collected by centrifugation were analyzed for reducing sugar concentrations by the standard dinitrosalicylic acid (DNS) method (described in the

Analytical methods section). All the subsequent studies were done using soybean meal powders of 23.7-75 µm sizes.

6.2.4 Effects of heat pretreatment with chelating agents EDTA, HMP and citric acid on enzymatic hydrolysis

EDTA, HMP and citric acid were added, respectively, at different concentrations

(2, 5 and 10 g/L) to the hydrolysis systems of 1.5 g soybean meal in 27 mL deionized water (without addition of the 3 mL enzyme broth). All systems were placed in a shaking water bath (ORS 200, Boekel Scientific Inc., Feasterville, PA) at 60 rpm, 90 °C for 2 h.

After being cooled to room temperature, each system was adjusted to pH 4.8 and then

109 added with 3 mL enzyme broth to start the enzymatic hydrolysis, following the same procedures described.

6.2.5 Factorial design for effects of heat and citric acid pretreatments and SPEZYME CP supplementation on enzymatic hydrolysis

Three factors were studied: heat pretreatment, citric acid pretreatment, and

SPEZYME CP supplementation, at two levels: with and without each pretreatment or supplementation. Following the 3-factor 2-level factorial design, 8 (= 23) systems, each in duplicate, were compared. These systems are summarized in Table 6.1. Heat pretreatment

(60 rpm, 90 °C for 2 h) and citric acid pretreatment (10 g/L, 52 mM) were done with procedures similar to those described in the previous section for studying the chelating agent effects, except that the two pretreatments could be applied independently or together. SPEZYME CP supplementation was done by adding 0.102 mL of the commercial enzyme, on top of the 3 mL A. niger enzyme broth, at the beginning of enzymatic hydrolysis. This supplementation raised the enzyme activities used in the hydrolysis, per g soybean meal, from 0.62 FPU cellulase, 165 U xylanase, 4.4 U pectinase and 8.2 U α-galactosidase to 10.6 FPU cellulase, 188 U xylanase, 4.9 U pectinase and 8.4 U α-galactosidase. The target of this supplementation was to raise the cellulase activity by 10 FPU/(g soybean meal) to improve the cellulose hydrolysis; therefore, the SPEZYME CP supplementation in Table 6.1 and later sections is often referred to as cellulase or FPU supplementation for brevity. (The supplementation also increased xylanase activity by 23 U/(g meal), which was however not as important because an earlier study already showed the high effectiveness of A. niger enzyme broth

110 in xylan conversion [92]). All procedures and conditions for enzymatic hydrolysis remained the same.

Table 6.1 Matrix for the factorial design for studying the effects of heat pretreatment, citric acid pretreatment, and FPU supplementation, and the code values of variables

System Heat Citric Acid FPU Control -1 -1 -1 Heat 1 -1 -1 Citric acid -1 1 -1 FPU -1 -1 1 Citric acid + Heat 1 1 -1 Heat + FPU 1 -1 1 Citric acid + FPU -1 1 1 Citric acid + Heat + FPU 1 1 1 Factors Lower level (-1) Upper level (1) Heat No heat treatment 90 °C, 2 h Citric acid No citric acid addition 10 g/L (52 mM) FPU No FPU supplementation Additional 10 FPU/g

6.2.6 Analytical methods

6.2.6.1 Carbohydrate composition analysis of soybean meal

Soluble and structural carbohydrates in soybean meal were determined using the standard NREL procedure [93]. Soybean meal was first extracted with water and the water extract collected was later analyzed for the soluble carbohydrate composition. After water extraction, the remaining solids were subjected to two steps of acid hydrolysis to determine the structural carbohydrate content. Loss of monomeric sugars due to acid degradation was considered. The procedures were done with triplicate soybean meal samples. Total reducing sugar concentrations were measured by the DNS method, and

111 individual sugar concentrations were measured by high performance liquid chromatography (HPLC), as described in the following section.

6.2.6.2 Sugar analysis

Total reducing sugar concentrations were measured by the DNS method [94].

DNS reagent (3 mL) was mixed with 1 mL sample in a test tube. The mixture was heated in a boiling water bath for 5 min and after that, added with water to a total volume of 25 mL. After being cooled to ambient temperature, the mixture was measured for absorbance at 550 nm by using a UV/Vis spectrophotometer (UV-1601, Shimadzu

Corporation, Columbia, MD). Glucose solutions were used as calibration standards for conversion of absorbance to total reducing sugar concentration.

The concentrations of individual sugars in liquid samples were measured by

HPLC (Shimadzu LC 10A) with refractive index (RI) and UV detectors. Samples were prepared with proper dilutions and filtered through 0.22 μm nylon filters. For stachyose, raffinose, sucrose, glucose, xylose, galactose, arabinose and fructose, a SUPELCOGEL

Pb column (30 cm × 7.8 mm) was used at 80C with the RI detector and a mobile phase of 0.5 mL/min HPLC grade water. The sample injection volume was 10 µL and total run time was 40 min. For galacturonic acid, an ion exchange column Aminex HPX-87H

(Bio-Rad, Hercules, CA) was used at 35C, with 0.6 mL/min 0.005N H2SO4 as the mobile phase and 20 µL sample injection. The total run time was also 40 min. Peak area of each sugar was converted to concentration (g/L) using the calibration established with respective sugar standards.

112

Sugar yields, Y (%), from hydrolysis were all calculated using the following equation:

퐶𝑖 푌𝑖 = × 100 50 × 퐶𝑖표

Here, C is the sugar concentration (g/L) in hydrolysate, C0 is the sugar content (g/g) in soybean meal, 50 (g/L) is the soybean meal loading used in hydrolysis, and subscript i denotes specific sugar, i.e., reducing sugar, glucose, xylose, arabinose, galactose, and fructose.

6.3 Results and Discussion

6.3.1 Sugar composition of soybean meal

The sugar composition of the soybean meal used in this study is given in Table

6.2. In addition to the expected stachyose, raffinose and sucrose, monosaccharides

(glucose, xylose, arabinose, galactose, and fructose) were also detected in the soluble carbohydrate portion collected during the NREL analysis procedure. Most of the monosaccharides are believed to come from some extents of hydrolysis of oligo- and poly-saccharides during the (Soxhlet) water extraction used in the NREL procedure.

When tallying the content of each monomeric sugar in soybean meal (the last column in

Table 6.2), the glucose, fructose and galactose contents in stachyose, raffinose and sucrose found in the soluble carbohydrate portion are calculated and added to those already detected as monosaccharides in the soluble and structural carbohydrates.

According to Table 6.2, the soybean meal used in this study had 30.2 ± 3.9% total carbohydrate (on a wet basis, with 9.38 ± 0.18% moisture), including 12.8 ± 0.6% soluble

113 carbohydrate and 17.4 ± 3.5% structural carbohydrate. These values are within the ranges reported in the literature for soybean meal: 27.9-34.2% total carbohydrate, 13-15% soluble carbohydrate, and 14.9-21.2% structural carbohydrate [23,45,46,151]. The variations might come from different soybean cultivars and growing conditions. The total reducing sugar content directly measured for this soybean meal is 26.1 ± 0.3%. This value is lower than the total carbohydrate content (30.2 ± 3.9%) given in Table 6.2, obtained from the HPLC analysis. The difference can be attributed to the use of a single sugar, glucose, as the calibration standard for the total reducing sugar analysis, while different sugars have different calibration coefficients when analyzed by the DNS method

[152].

As shown in Table 6.2, the total glucose content in this soybean meal is 9.4 ±

1.2% with 5.1 ± 0.9% from the structural carbohydrate. Structural glucose comes predominantly from cellulose, with a much smaller contribution from pectin [30,45]. The

5.1 ± 0.9% structural glucose obtained is reasonably consistent with the 6.2% cellulose content in soybean meal reported by Bach Knudsen and Hansen [45]. Some of the cellulose may be due to incomplete dehulling: soybean hulls contain 30-50% cellulose

[76,146], and up to about 7% crude hull fibers can be present in defatted soybean meals, particularly those with comparatively lower protein contents [141,142]. Soybean meal also has a considerable amount of structural galactose (3.9 ± 1.1%) which is found mainly in pectin. Fructose content is 4.5 ± 0.3%, all of which comes from the soluble carbohydrate including free fructose and the fructose contained in stachyose, raffinose, and sucrose. Galacturonic acid content is 3.3 ± 0.5%, which makes up the backbone of pectin. According to literature, arabinose is found in both hemicellulose and pectin, and

114 the content found in this study (2.9 ± 0.2%) is comparable to the reported values

[23,45,46]. Mannose content is 2.1 ± 0.6%, which comes from both pectin and hemicellulose in soybean meal [45,46] and residual soybean hulls [76,146].

Table 6.2 Sugar composition in soybean meal

Sugar in Soluble Total monomeric Sugar structural carbohydrate (%) sugar (%) carbohydrate (%) Stachyose 3.6 ± 0.2 - - Raffinose 0.68 ± 0.02 - - Sucrose 4.4 ± 0.2 - - Glucose 0.97 ± 0.06 5.1 ± 0.9 9.4 ± 1.2 Xylose 0.64 ± 0.06 1.3 ± 0.3 1.9 ± 0.4 Galactose 0.20 ± 0.07 3.9 ± 1.1 6.1 ± 1.3 Arabinose 0.14 ± 0.02 2.8 ± 0.2 2.9 ± 0.2 Fructose 1.2 ± 0.2 - 4.5 ± 0.3 Mannose - 2.1 ± 0.6 2.1 ± 0.6 Galacturonic 0.96 ± 0.08 2.3 ± 0.4 3.3 ± 0.5 acid Total 12.8 ± 0.6 17.4 ± 3.1 30.2 ± 3.9

6.3.2 Effect of soybean meal particle size on enzymatic hydrolysis

Figure 6.1 shows the release profiles of total reducing sugar in enzymatic hydrolysis of soybean meal for the following particle size ranges: 23.7-75 µm, 150-212

µm, 300-425 µm and 425-1180 µm. The final yields (at 48 h) were higher with smaller particles: 62.9 ± 0.4% for 23.7-75 µm particles, 63.3 ± 0.6% for 150-212 µm particles,

59.0 ± 1.3% for 300-425 µm particles, and 56.9 ± 0.1% for 425-1180 µm particles. Even larger differences were found in the earlier hydrolysis rates. For example, after 4 h the reducing sugar yield for 23.7-75 µm particles already reached 53.3 ± 1.7% whereas the

115 yields for larger particles were only in the range of 35.7-38.0%. Because of the clearly improved enzymatic hydrolysis, all of the soybean meal used in the subsequent studies had the particle size range of 23.7-75 µm. Also, since no significant improvement in reducing sugar yield was observed after 24 h for these small particles, all of the later enzymatic hydrolysis studies were conducted for 24 h.

23.7-75 µm 150-212 µm 300-425 µm 425-1180 µm 70%

60%

50%

40%

30%

20% Reducing sugar sugar Reducing (%)yield

10%

0% 0 6 12 18 24 30 36 42 48 Time (h)

Figure 6.1 Effect of particle size on total reducing sugar yield released by enzymatic

hydrolysis of soybean meal

6.3.3 Effects of EDTA, HMP and citric acid

The EDTA, HMP and citric acid concentrations studied, under heated condition

(90 °C for 2 h), were 2, 5 and 10 g/L, corresponding to 6.8, 17.1 and 34.2 mM for EDTA,

3.3, 8.2 and 16.3 mM for HMP, and 10.4, 26.0 and 52.0 mM for citric acid. Figure 6.2 shows the total reducing sugar yields after 24 h enzymatic hydrolysis of the chelator- pretreated soybean meal. For comparison, the yields from two levels of control systems 116 are indicated in Figure 6.2 as two horizontal lines: one at 62.3 ± 1.8% for the base control without either chelator or heat pretreatment, the other at 70.3 ± 1.3% for the control without chelator but with the associated heat pretreatment. While all chelator-pretreated systems had higher total reducing sugar yields than the base control did (62.3 ± 1.8%), only the systems of 5 and 10 g/L citric acid and 10 g/L HMP, respectively, gave higher total reducing sugar yields compared to the heat-pretreated, chelator-free control. The presence of EDTA at any of the 3 concentrations studied or HMP at the lowest 2 g/L concentration even reduced the apparent effectiveness of heat pretreatment in increasing total reducing sugar yield. Furthermore, the reducing sugar yield did not increase with increasing EDTA concentrations (p = 0.607) but increased with increasing HMP (p =

0.092) and citric acid (p = 0.043) concentrations.

The causes for insignificant concentration dependency of EDTA and, more importantly, the statistically significant negative effects of EDTA and dilute HMP in total reducing sugar yield are unclear. However, others have confirmed that treating soybean okara with EDTA and HMP [26,27] and orange peel with EDTA [153] under heated conditions would lead to higher pectin extraction into the aqueous media. The negative effects likely occurred during the enzymatic hydrolysis, not pretreatment; for example, by unfavorable interactions of EDTA and HMP with some enzyme components.

117

EDTA HMP Citric acid

80% Heat pretreatment 75% Control

70%

65%

60%

55% Totalreducing sugar yield(%) 50% 2 5 10 Chelating agent (g/L)

Figure 6.2 Effects of heated pretreatments (90 °C, 2 h) with different concentrations of

EDTA, HMP and citric acid, respectively, on total reducing sugar yield from enzymatic

hydrolysis of the pretreated soybean meal

Nevertheless, this set of experiments clearly established citric acid as the most effective pretreatment agent, among the 3 chelators evaluated, for improving the total reducing sugar yield from enzymatic hydrolysis of soybean meal. The highest yield obtained was 75.1 ± 0.9% with 10 g/L citric acid. Also, citric acid is much cheaper than

EDTA and HMP. Bulk chemical costs per metric ton of these chelators are: ETDA,

$1,800-$2,500; HMP, $970-$1,010; and citric acid, $650-$700 (from the www.alibaba.com website). Further, A. niger has been used to produce citric acid commercially [154,155]. If the presence of citric acid can be proven helpful to biomass hydrolysis by A. niger enzymes, it is possible to adjust the fermentation conditions or A. niger strains used for enzyme production to produce optimal mixtures of enzyme and citric acid for the applications.

118

6.3.4 Factorial design for effects of heat and citric acid pretreatments and SPEZYME CP supplementation

6.3.4.1 Total reducing sugar yields

The total reducing sugar yields from 24 h enzymatic hydrolysis of the 8 systems evaluated according to the 3-factor, 2-level factorial design are shown in Figure 6.3. The regression equation obtained is:

Reducing sugar yield (%) = 72.3 + 5.0 H + 2.3 CA + 4.5 FPU+ 1.5 (H × CA) + 0.10 (H

× FPU) + 0.83 (CA × FPU) + 0.61 (H × CA × FPU) (r2 = 0.95)

Where H stands for heat pretreatment (90 °C, 2h), CA for citric acid pretreatment (52 mM), and FPU for the SPEZYME CP supplementation that raises cellulase by 10 FPU/(g soybean meal). The bold-font terms have statistically significant effects (p < 0.05) on reducing sugar yield; others do not. All independent variables (H, CA and FPU) are in coded values in the above equation, with “-1” and “1” for without and with the pretreatment or supplementation.

All terms have positive coefficients, indicating that including any of these factors

(heat pretreatment, citric acid pretreatment, and SPEZYME CP supplementation) improved the total reducing sugar yield. The individual effects of single factors and the interaction effect between heat and citric acid pretreatments are significant (p < 0.0001 for H, = 0.001 for CA, < 0.0001 for FPU, and = 0.007 for H×CA). The other interaction effects are not significant: p = 0.858 for H×FPU, 0.092 for CA×FPU, and 0.193 for

H×CA×FPU. The significant H×CA interaction is not too surprising, as the higher temperature was reported to improve both the pectin extraction with chelating agents

119

[26,27] and the mild acid hydrolysis of biomass [108,148]. The insignificant H×CA×FPU interaction is less expected because acid pretreatment at high temperature is a common pretreatment method for improving enzymatic hydrolysis of cellulose [156].

The combined effects of all three factors improved the 24-h reducing sugar yield from 62.3% in the control (without any of these factors) to 87.1%, representing a 40% improvement. Heat pretreatment and cellulase supplementation contributed more; each increased the reducing sugar yield by about 8% (corresponding to a 13% improvement).

Citric acid alone increased the reducing sugar yield from 62.3% to 63.4%; it became a more effective factor when combined with the heat treatment, increasing the reducing sugar yield to 75.1%. It is important to note that this yield obtained with combined heat and citric acid was found similar to that with combined heat and cellulase (76.6%).

Considering the huge price difference between citric acid and cellulase, it helps to understand and maximize the citric acid effect.

120

100%

80%

60%

40%

Reducing sugar yield(%) 20%

Figure 6.3 Effects of heat (H) and citric acid (CA) pretreatments and cellulase

supplementation (FPU) on reducing sugar yield after 24 h enzymatic hydrolysis of

soybean meal

6.3.4.2 Monomeric sugar yields

Figure 6.4 shows the effects of these factors on glucose, xylose, galactose, arabinose and (fructose + mannose) yields. Fructose and mannose are shown together because their peaks in HPLC chromatograms could not be accurately separated (retention times for mannose and fructose were about 21.9 and 22.1, respectively). It is however important to note the following: nearly all fructose would come from soluble carbohydrates such as stachyose, raffinose and sucrose; even in the control, these soluble carbohydrates were completely hydrolyzed after 24 h enzymatic hydrolysis

(chromatogram not shown), i.e., fructose yield are expected to be essentially 100% in all systems; and, therefore, although reported here as (fructose + mannose) yields, the

<100% yields in these systems were due to incomplete mannose hydrolysis and the 121 effects of pretreatment/supplementation were on changing only the mannose yields. In addition to Figure 6.4, the regression equations for these monosaccharide yields (%) are given as follows:

Glucose yield (%) = 70.1 + 4.2 H + 3.2 CA + 7.1 FPU + 0.22 (H × CA) – 0.85 (H ×

FPU) + 2.0 (CA × FPU) + 0.22 (H × CA × FPU); (r2 = 0.89)

Xylose yield (%) = 94 + 0.47 H + 0.60 CA + 2.6 FPU – 0.20 (H × CA) – 0.60 (H ×

FPU) + 0.80 (CA × FPU) + 0.45 (H × CA × FPU); (r2 = 0.42)

Galactose yield (%) = 83.2 + 2.6 H + 1.7 CA + 2.0 FPU + 0.28 (H × CA) – 1.4 (H ×

FPU) – 2.0 (CA × FPU) + 1.1 (H × CA × FPU); (r2 = 0.60)

Arabinose yield (%) = 47 + 13.6 H + 8.6 CA + 11.9 FPU + 3.5 (H × CA) – 0.47 (H ×

FPU) + 0.50 (CA × FPU) – 0.97 (H × CA × FPU); (r2 = 0.95)

(Fructose + Mannose) yield (%) = 86.2 + 2.2 H – 0.49 CA + 2.7 FPU – 0.73 (H ×

CA) – 0.20 (H × FPU) – 0.06 (CA × FPU) + 1.7 (H × CA × FPU); (r2 = 0.62)

Again, the bold-font terms have statistically significant effects (p < 0.05) on the specific sugar yield; others do not. All independent variables (H, CA and FPU) are in coded values: “-1” or “1” for without or with the pretreatment or supplementation.

As shown in Figure 6.4, systems with any of the pretreatments and supplementation had higher yields for all of the above monosaccharides compared to the control (although some increases, especially for the xylose yield, were not statistically significant). The percentage yield improvements, = (new yield – old yield)/(old yield) ×

100%, were smaller for the sugars that already had high yields in the control; for example, xylose had a 90.0 ± 4.6% yield in the control and was increased by up to 10% by the additional pretreatment/supplementation factors. The most significant percentage

122 improvements were for arabinose, mannose (estimated by subtracting fructose, at 100% yield, from the combined yield of fructose and mannose), and glucose yields, by 382%,

113%, and 51%, respectively. These sugars were poorly hydrolyzed in the control by the

A. niger enzyme: 17.3 ± 3.7% for arabinose, 34.3 ± 1.1% for mannose, and 57.6 ± 2.6% for glucose. The system with all three pretreatment/supplementation factors gave the highest yields of all monosaccharides: glucose, 86.8 ± 5.2%; xylose, 98.1 ± 1.6%; galactose, 87.5 ± 5.2%; arabinose, 83.6 ± 1.6%; and mannose, 72.9 ± 1.0% (or fructose + mannose, 91.4 ± 3.1%).

The different increases in monosaccharide yields provide some insights into the pretreatment/supplementation effects on different structural carbohydrates: cellulose, hemicellulose, and pectin. As mentioned earlier, soluble oligosaccharides were practically all hydrolyzed in the control; therefore, the improved monosaccharide yields resulted from higher hydrolysis of structural carbohydrates. For monosaccharide release from soybean meal, xylose is essentially all from hemicellulose (xylan); fructose is from soluble carbohydrates (stachyose, raffinose, and sucrose); mannose and arabinose can come from both pectin and hemicellulose (for arabinose, approximately 0.9% from hemicellulose and 1.7% from pectin [45]; for mannose, no values reported separately from hemicellulose and pectin); and glucose and galactose are from both soluble carbohydrates (stachyose, raffinose and sucrose) and structural carbohydrates. For glucose, the structural carbohydrate source is predominantly cellulose, although pectin and hemicellulose also contain some (~ 1.2%) glucose [30]; for galactose, pectin is the main structural carbohydrate source with some possible contribution from hemicellulose

[30].

123

Xylan was already well hydrolyzed in the control to give a 90.0 ± 4.6% xylose yield; the pretreatments/supplementation could further xylan hydrolysis slightly. Very significant increases in glucose, arabinose and mannose yields indicate more substantial improvements in cellulose and pectin hydrolysis by the pretreatment/supplementation factors. This finding is reasonable because cellulase supplementation would enhance cellulose hydrolysis and citric acid and heat pretreatments could improve pectin structure loosening and extraction to promote pectin hydrolysis and to render cellulose more accessible to cellulase [25,27].

The regression models fitted well for glucose and arabinose (r2 = 0.89 for glucose and 0.95 for arabinose) but not for xylose, galactose and fructose + mannose yields (r2 =

0.42 for xylose, 0.60 for galactose and 0.62 for fructose + mannose). Xylose, galactose and fructose + mannose yields were already high in the control (90.0 ± 4.6%, 72.8 ±

4.1% and 79.1 ± 3.4% respectively); so, the remaining ranges were rather small for the improvements by pretreatment/supplementation to achieve statistical significance, given the inevitable experimental errors. For glucose yield, any of the 3 factors improved the glucose yield significantly (p < 0.05), but none of the interaction effects were significant.

Cellulase supplementation gave the strongest positive effect, with the largest beta coefficient (7.1) in the regression equation and p < 0.001 for FPU (compared to 0.006 for

H and 0.021 for CA). For arabinose yield, in addition to the significant (positive) effects of individual factors, the interaction between heat and citric acid was also significantly positive (p = 0.023). Mild acid hydrolysis might have occurred at the moderately high temperature (90 C) to effect some extents of arabinose release from hemicellulose and pectin during the pretreatment (prior to the enzyme hydrolysis).

124

(a) (b) 100% 100%

80% 80%

60% 60%

40% 40%

20% Xylose Yield (%) Glucose Glucose Yield (%) 20%

0% 0%

100% (c) 100% (d)

80% 80%

60% 60%

40% 40%

20%

20% Arabinose Yield (%) Galactose Galactose (%) Yield

0% 0%

100% (e)

80%

60%

40%

20%

0% Fructose Fructose Mannose + Yield (%)

Figure 6.4 Effects of heat (H) and citric acid (CA) pretreatments and SPEZYME

(primarily cellulase) supplementation (FPU) on monosaccharide yields after 24 h

enzymatic hydrolysis of soybean meal: (a) glucose, (b) xylose, (c) galactose, (d)

arabinose, and (e) fructose + mannose

125

The effects of heat and citric acid pretreatments are clearly very complex. To gain some more understanding, the effects of separate and combined citric acid and heat pretreatments on monosaccharide release from soybean meal are examined with more analyses to differentiate the releases occurring during the pretreatments from the releases during the enzymatic hydrolysis. Monosaccharide concentrations in these hydrolysates before (0 h) and after 24 h enzymatic hydrolysis by the A. niger enzymes (without

SPEZYME supplementation) are compared in Figure 6.5. Prior to enzymatic hydrolysis

(Figure 6.5(a)), xylose releases, compared to other monosaccharides, were already much higher: 37.8 ± 3.2% for the control, 55.9 ± 8.3% for the heat pretreated system, 64.9 ±

2.4% for the citric acid pretreated system, and 69.7 ± 5.4% for the system subjected to simultaneous heat and citric acid pretreatments. Citric acid alone was very effective in enhancing xylose release during the pretreatment, nearly as effective as the combined citric acid and heat pretreatments. On the other hand, neither citric acid nor heat alone caused more than 20% releases of any non-xylose sugars during the pretreatment. Only when citric acid and heat were combined could the pretreatment increase glucose and

(fructose + mannose) yields, but not the galactose and arabinose yields, more clearly, to

25.0 ± 1.8% and 45.1 ± 4.3%, respectively. These findings from Figure 6.5(a) suggest the following: (A) these pretreatments caused substantial hydrolysis of the hemicellulosic xylan during the pretreatments; (B) these pretreatments had relatively weak effects on pectin and galacto-oligosaccharide hydrolyses, although earlier work indicated improved pectin extraction [25,27]; and (C) heat pretreatment under the citric acid-created mildly acidic condition (pH measured to be 3.63) caused more sucrose hydrolysis (to glucose and fructose) during the pretreatment. As for the comparison of results at 0- and 24-h

126 enzymatic hydrolysis, the most important conclusion to draw is that enzymes, not the pretreatments, were primarily responsible for the high monosaccharide releases observed after 24 h enzymatic hydrolysis.

0 h 100% 80% 60% 40% Yield Yield (%) 20% 0% Glucose Xylose Galactose Arabinose Fructose+Mannose

Control Heat Citric acid Citric acid+Heat

24 h 100% 80% 60% 40% Yield Yield (%) 20% 0% Glucose Xylose Galactose Arabinose Fructose+Mannose

Control Heat Citric acid Citric acid+Heat

Figure 6.5 Monosaccharide releases from soybean meal observed after the pretreatments

(prior to enzymatic hydrolysis, 0 h), by separate and combined heat and citric acid

pretreatments, and after (24 h) enzymatic hydrolysis by A. niger enzymes (without

SPEZYME supplementation)

6.4 Conclusion

Using the standard NREL procedure, the soybean meal used in this stud was found to have total carbohydrate content of 30.2 ± 3.9% (on wet mass basis, with 9.4 ±

0.2% moisture) including 12.8 ± 0.6% soluble carbohydrate and 17.4 ± 3.5% structural

127 carbohydrate, with the following monomeric sugars: glucose, 6.4 ± 1.2%; xylose, 1.9 ±

0.4%; galactose, 6.1 ± 1.3%; arabinose, 2.9 ± 0.2%; fructose, 4.5 ± 0.3%; mannose, 2.1 ±

0.6% and galacturonic acid, 3.3 ± 0.5%. Having smaller soybean meal particle sizes moderately improved the final reducing sugar yield from (48 h) A. niger enzyme hydrolysis, e.g., 62.9 ± 0.4% for 23.7-75 µm particles versus 56.9 ± 0.1% for 425-1180

µm particles, and strongly increased the early hydrolysis rates, e.g., 53.3 ± 1.7% reducing sugar yield after only 4 h for 23.7-75 µm particles versus 35.7-38.0% for larger particles.

Additions of chelators EDTA, HMP and citric acid at 2, 5 and 10 g/L concentrations under 2-h 90 °C heat pretreatment gave mixed effects on enzymatic hydrolysis: 5 and 10 g/L citric acid and 10 g/L HMP improved reducing sugar yield over that by heat pretreatment alone, while 2 g/L HMP and EDTA at all 3 concentrations had even negative effects. The latter suggested possibility of negative interactions between chelators and enzyme components. Among the chelators studied, citric acid was clearly the most effective in increasing enzymatic hydrolysis of soybean meal. A subsequent study of the effects of heat (90 °C, 2 h) and citric acid (10 g/L) pretreatments and

SPEZYME (primarily cellulase) supplementation (10 FPU/g meal) by a 3-factor 2-level factorial design showed that including any of these factors increased yields of total reducing sugar and all monosaccharides over those in the control. For total reducing sugar yield, combining all 3 factors increased it from 62.3% (control) to 87.1%, representing a 40% improvement; also, combining heat and citric acid pretreatments increased it to 75.1%, similar to 76.6% from combining heat pretreatment and

SPEZYME FPU supplementation. The latter is important because of the large price difference between citric acid and cellulase. For monosaccharide yields, the largest

128 percentage improvements were for arabinose (382%), mannose (113%) and glucose

(51%). Heat (90°C, 2 h) and citric acid (10 g/L) pretreatments and cellulase supplementation (10 FPU/g) all increased reducing sugar and monosaccharide yields.

Together, they increased reducing sugar yield from 62.3% to 87.1% and yielded

86.8±5.2% glucose, 98.1±1.6% xylose, 87.5±5.2% galactose, 83.6±1.6% arabinose, and

91.4±3.1% (fructose + mannose). These high monosaccharide yields achieved in this study strongly promote the valuable use of soybean meal hydrolysate as fermentation feedstock.

129

CHAPTER VII

BETTER UNDERSTANDING OF ENZYMATIC PROCESSING OF SOYBEAN

FLOUR CARBOHYDRATE THROUGH MODELING MONOMERIC SUGAR

RELEASE

7.1 Introduction

Soybean carbohydrate comprises roughly equal amounts of non-structural and structural components [30,45]. The nonstructural carbohydrates are predominantly sucrose and galacto-oligosaccharides, with very small amounts of monosaccharides and storage polysaccharides [42]. The structural carbohydrates, also known as non-starch polysaccharides (NSPs), include dietary fiber components such as cellulose, hemicellulose, and pectin [137,138]. Hydrolyzing these carbohydrates enzymatically to fermentable sugars requires a complex enzyme system with at least pectinase, xylanase, cellulase, α-galactosidase and sucrase activities [91,104]. Loman and Ju [92] developed models for effects of individual enzyme activities on hydrolytic conversions of soybean meal carbohydrate to soluble reducing sugar (by dinitrosalicylic acid, DNS, analysis) and carbohydrate (by phenol-sulfuric acid analysis). The enzymatic hydrolysis process will be more economical if all types of soybean carbohydrate can be hydrolyzed.

A better understanding of how enzyme compositions affect the hydrolysis of different soybean flour carbohydrates (cellulose, hemicellulose, pectin, and galacto-

130 oligosaccharides) is important for further improvement. This has been investigated in this study by modeling the effect of enzyme composition on the kinetic release of individual monomeric sugars from different carbohydrate components. The models are valuable for understanding the hydrolysis mechanisms and be helpful for further reactor design, batch and continuous process design and optimization of overall economics.

7.2 Materials and Methods

7.2.1 Materials

The soybean flour used in this study was provided by Archer Daniels Midland Company

(Decatur, IL). It had 30.2 ± 3.9% total carbohydrate (on a wet basis, with 9.38 ± 0.18% moisture content), including 12.8 ± 0.6% soluble carbohydrate and 17.4 ± 3.5% structural carbohydrate. Individual monomeric sugar contents are, glucose, 9.4 ± 1.2%; xylose, 1.9

± 0.4%; galactose, 6.1 ± 1.3%; arabinose, 2.9 ± 0.2%; 4.5 ± 0.3%; mannose, 2.1 ± 0.6% and galacturonic acid, 3.3 ± 0.5% [44]. The enzyme broth used for enzymatic hydrolysis was produced in this laboratory by submerged A. niger NRRL 322 and A. foetidus NRRL

341 fermentation using soybean hull as a carbon source. The A. niger strains were obtained from the United States Department of Agriculture (USDA) Agricultural

Research Service (ARS) Culture Collection. The commercial enzyme SPEZYME®CP

(Genencor, Finland) was also used. It had the following measured activities: cellulase,

147 FPU/mL; xylanase, 349 U/mL; pectinase, 7.3 U/mL; α-galactosidase, 2.2 U/mL and sucrose, 0.2 U/mL. The chemicals used were all purchased from Fisher Scientific Inc.

7.2.2 Enzymatic hydrolysis of soybean flour

All enzymatic hydrolysis experiments were done with 250 mL Erlenmeyer flasks in a shaker (Thermo Scientific MaxQ 5000 Incubating/Refrigerating floor shaker, Ashville,

131

NC) at 50 °C, pH 4.8 and 250 rpm. All hydrolysis experiments were done with 50 g/L soybean flour (5 g soy flour in 100 mL enzyme solution) and 0.05% sodium azide for controlling microbial contamination. Different composition of cellulase, xylanase, pectinase, α-galactosidase, and sucrose were used to find out their effect on monomeric sugar release from enzyme hydrolysis by combining different A. niger enzyme broth and commercial cellulase enzyme. Enzyme loading (U/g soy flour) used for different systems were varied in wide ranges: cellulase, 0.3 – 21.4 FPU/g; xylanase, 23.7 – 871.7 U/g; pectinase, 0.5 – 34.7 U/g; α-galactosidase, 0.1 – 28.9 U/g and sucrose, 0.01 – 18.2 U/g.

Enzymatic hydrolysis was run for 48 h and samples were taken from the experiments at different time intervals which were centrifuged at 10,000 rpm (9300×g, Eppendorf

5415D) for 10 min to separately collect the supernatant for analyses. Individual sugar concentrations in hydrolysates, i.e. glucose, xylose, galactose, arabinose, and fructose+mannose were measured by HPLC which will be described in the analytical method section.

7.2.3 Analytical methods

7.2.3.1. Enzyme activities measurement

Cellulase, xylanase, pectinase, α-galactosidase, and sucrase enzyme activities were analyzed for different A. niger enzyme broths and commercial cellulase enzyme. The cellulase assay used was modified from that reported by Ghose [97]. The cellulase activity measured using filter paper as the substrate. The method reported by Bailey et al.

[98] was adopted for the xylanase assay, and beechwood xylan (Sigma Aldrich, St. Louis,

MO) was used as the substrate. The pectinase methods were established in this laboratory

[99], and the substrate was citrus pectin (Sigma Aldrich, St. Louis, MO). The sucrase

132 assay used in this study was modified from a method reported by Uma et al. [100] using sucrose as substrate. The -galactosidase activity measurement was by a method modified from Kumar et al. [101] in which p-nitrophenyl-α-D-galactopyranoside (Sigma

Aldrich, St. Louis, MO) was used as the substrate. The enzyme activities were calculated using the following equations:

glucose released (푚𝑔) 1 푚푚표푙 1000 휇푚표푙 Cellulase (퐹푃푈/푚퐿) = × × (60 푚𝑖푛 )(0.1 푚퐿 enzyme sample) 180 푚𝑔 1 푚푚표푙

= 0.925 × glucose released (푚𝑔).

푈 xylose released (푚𝑔) 1 푚푚표푙 1000 휇푚표푙 Xylanase ( ) = × × 푚퐿 (5 푚𝑖푛 )(0.1 푚퐿 enzyme sample) 150.13 푚𝑔 1 푚푚표푙

= 13.32 × xylose released (푚𝑔).

푈 Pectinase ( ) = 1.57 × galacturonic acid released (푚𝑔) 푚퐿

푈 Sucrase ( ) = 2.78 × glucose released (푚𝑔). 푚퐿

푈 α − Galactosidase ( ) = 7.19 × 푝 − nitrophenol released (푚𝑔) 푚퐿

7.2.3.2. Monomeric sugar analysis

The concentrations of individual sugars in liquid samples were measured by HPLC

(Shimadzu LC 10A) with refractive index (RI) and UV detectors. Samples were prepared with proper dilutions and filtered through 0.22 μm nylon filters. For glucose, xylose, galactose, arabinose and fructose+mannose, a SUPELCOGEL Pb column (30 cm × 7.8 mm) was used at 70C with the RI detector and a mobile phase of 0.5 mL/min HPLC

133 grade water. The sample injection volume was 25 µL and total run time was 40 min.

Glucose, xylose, galactose, and arabinose concentrations were determined with a total run time of 40 min and their corresponding retention times were 17.2, 18.2, 19.5, and 20.8 respectively. Fructose and mannose are shown together because their peaks in HPLC chromatograms could not be accurately separated (retention times for mannose and fructose were about 23.9 and 24.1, respectively). Peak area of each sugar was converted to concentration (g/L) using the calibration established with respective sugar standards.

Sugar yields, Y (%), from hydrolysis were all calculated using the following equation:

퐶𝑖 푌𝑖 = × 100 50 × 퐶𝑖표

Here, C is the sugar concentration (g/L) in hydrolysate, C0 is the sugar content (g/g) in soybean meal, 50 (g/L) is the soybean meal loading used in hydrolysis, and subscript i denotes specific sugar, i.e., glucose, xylose, arabinose, galactose, and fructose+mannose.

7.3. Results and discussion

7.3.1 Effect of enzyme loading monomeric sugar yield in enzyme hydrolysis: a Modeling approach

A saturation-type model was used to describe the final individual monomeric sugar yield achieved after 48 h hydrolysis at the optimized reaction conditions (pH 4.8, temperature

50 °C). The assumptions for different sources of individual sugars and the enzyme responsible for their hydrolysis are described here. Xylose is mostly all from hemicellulose (xylan); fructose is from soluble carbohydrates (stachyose, raffinose, and sucrose); mannose and arabinose can come from both pectin and hemicellulose [45]; and

134 glucose and galactose are from both soluble carbohydrates (stachyose, raffinose, and sucrose) and structural carbohydrates. Structural glucose (5.1 ± 0.9%) are predominantly from cellulose; although hemicellulose and pectin also contain glucose [30]. For structural galactose, pectin is the main source of some possible contribution from hemicellulose [30]. Cellulase, xylanase, pectinase are responsible for cellulose, hemicellulose and pectin hydrolysis. Sucrase enzyme is responsible for hydrolyzing sucrose and the sucrose portion of stachyose and raffinose with different affinity to the substrate. α-Galactosidase is responsible for the release of galactose (by cleaving α-(1,6)- bond) sugar from stachyose and raffinose.

Figure 7.1 shows the effect of enzyme loading of different enzymes on the yield of different monomeric sugar in enzyme hydrolysis at 48 h considering all of these factors.

In all the figures, the secondary y-axis is the corresponding enzyme activities in the broth of particular enzyme in the enzyme loading shown in x-axis; for example, in Figure

7.1(b), primary y-axis shows the change of sugar yield with the change of pectinase loading (U/g) and secondary y-axis shows the cellulase loading (FPU/g) in corresponding pectinase loading which will be helpful in describing the effect of both enzyme on the sugar yield.

Xylose release increased with the increase of xylanase enzyme and reached to more than

90% yield after 400 U/g xylanase enzyme loading (Figure 7.1(a)). However, arabinose release was only 73% at 412 U/g xylanase with 16.8 U/g pectinase which increased to

82.1% with 19.5 U/g pectinase activity. Arabinose release depends on both xylanase and pectinase since arabinose is present in both hemicellulose and pectin. Change of glucose, arabinose and galactose yields with the change of pectinase loading are shown in Figure

135

7.1(b). When the pectinase activity is lower, glucose yield is very low even though cellulase enzyme is higher. However, when the pectinase activity increased, lower cellulase enzyme may become effective hydrolyzing cellulose which contributed more total glucose release. For example, when the pectinase activity was 1.0 U/g, the glucose yield was 22.2% despite having higher cellulase activity (20 FPU/g); but the glucose yield increased to 84.0% when the pectinase activity was 16.8 U/g with only 0.7 FPU/g.

However, glucose yield increased further to 94.4% with the increase of cellulase enzyme

(5.7 FPU/g cellulase and 17.1 U/g pectinase). Therefore, glucose release may depend on the extent of pectin hydrolysis by pectinase. Fructose+mannose release increased with the increase of sucrase and pectinase activity.

The combined effect of different enzymes on total monomeric sugar release is therefore very difficult to describe/conclude by figure shown here, and for that reason, a modeling approach was taken to describe their interaction effect. For different sugar yield equations, experimental observations have also been considered to describe the interaction of different enzyme and substrate, i.e. dependency of cellulose hydrolysis on the extent of pectin hydrolysis.

136

(a) 40 (b) 25 100% 100% 35 20 80% 30 80%

25 15 60% 60%

20 Yield (%) Yield Yield (%) Yield 10

40% 15 40%

Pectinase (U/g) Pectinase Cellulase (FPU/g) Cellulase 10 20% 20% 5 5

0% 0 0% 0 0 500 1000 0 10 20 30 40 Xylanase (U/g) Pectinase (U/g) Xylose Arabinose Pectinase (U/g) Glucose Galactose Arabinose Cellulase

80% 30 80% 30 25 25 60% 60%

20 20

Yield (%) Yield Yield (%) Yield

40% 15 40% 15

Pectinase (U/g) Pectinase Pectinase (U/g) Pectinase 10 10 20% 20% 5 5

0% 0 0% 0 0 10 20 30 40 0 5 10 15 20 α-Galactosidase (U/g) Sucrase (U/g) Glucose Galactose Fructose+Mannose Pectinase (U/g) Glucose Fructose+Mannose Pectinase (U/g)

Figure 7.1. Effect of different enzyme loading (U/g) on monomeric sugar yield in

enzymatic hydrolysis of soybean flour

137

7.3.1.1 Yield Model

The model equations used for maximum yields (%) of individual sugars are given below:

퐸푠 퐸푠 퐸푐 퐸푝 1 푆 푆 푆 푆 푌퐺푙푢푐표푠푒(%) = [훼푔,0 + 훼푔,푠 ( 퐸푠 ) + 훼푔,표푙𝑖푔표 퐸푠 + 훼푐 ( 퐸푐 ) ( 퐸푝 ) + 퐶푔푙푢 퐾푠1+ 퐾푠2+ 퐾푐+ 퐾 + 푆 푆 푆 푝 푆

퐸푥 퐸푝 푆 푆 훼푔,ℎ ( 퐸푥 ) + 훼푔,푝 ( 퐸푝 )]; 퐾푥+ 퐾 + 푆 푝 푆

(1)

퐸푥 1 푆 푌푋푦푙표푠푒(%) = [훼푥푦푙,0 + 훼푔,ℎ ( 퐸푥 )]; 퐶푥푦푙 퐾 + 푥 푆

(2)

퐸푥 퐸푝 퐸훼 1 푆 푆 푆 푌퐺푎푙푎푐푡표푠푒(%) = [훼푔푎푙,0 + 훼푔푎푙,ℎ ( 퐸푥 ) + 훼푔푎푙,푝 ( 퐸푝 ) + 훼푔푎푙,표푙𝑖푔표 ( 퐸훼 )]; 퐶푔푎푙 퐾푥+ 퐾 + 퐾훼+ 푆 푝 푆 푆

(3)

퐸푥 퐸푝 1 푆 푆 푌퐴푟푎푏𝑖푛표푠푒(%) = [훼푎푟,0 + 훼푎푟,ℎ ( 퐸푥 ) + 훼푎푟,푝 ( 퐸푝 )]; 퐶푎푟 퐾푥+ 퐾 + 푆 푝 푆

(4)

퐸푠 퐸푠 1 푆 푆 푌퐹푟푢푐푡표푠푒+푀푎푛푛표푠푒(%) = [훼푓푚,0 + 훼푓푚,푠 ( 퐸푠 ) + 훼푓푚,표푙𝑖푔표 퐸푠 + 퐶푓푚 퐾 + 퐾 + 푠1 푆 푠2 푆

퐸푥 퐸푝 푆 푆 훼푓푚,ℎ ( 퐸푥 ) + 훼푓푚,푝 ( 퐸푝 )]; 퐾푥+ 퐾 + 푆 푝 푆

(5)

th Here, Ci is the total content of i sugar in soybean flour; αi,j is the sugar content in soybean flour where “i” denotes the individual sugar (glucose, xylose, galactose,

138 arabinose and fructose+mannose) and “j “denotes the carbohydrate source, i.e. pectin, hemicellulose, sucrose, and oligosaccharides). Ek is the total enzyme loading (U/L) where

“k” denotes the enzyme type, and S is total soybean flour loading (i.e., 50 g/L in this study). Kc, Kx, Kp, Ks, and Kα are respective half-maximum constants for cellulase, xylanase, pectinase, sucrase, and α-galactosidase.

7.3.1.2. Best-fit model parameters for monomeric sugar yield at 48 h

Model-predicted yields for monomeric sugar with best-fit parameters are plotted against experimental yields in Figure 7.2. The model equations describe the experimentally measured monomeric sugar yield well as shown in Figure 7.2. R2 values for glucose, xylose, galactose, and fructose+mannose are > 0.95 and 0.937 for arabinose.

139

Glucose Xylose 100% 100%

80% 80%

60% y = 0.9468x 60% R² = 0.9567 y = 0.9939x 40% 40% R² = 0.9633

20% 20%

Model Predicted Yield (%) Yield Predicted Model Model Predicted Yield (%) Yield Predicted Model

0% 0% 0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100% Experimental Yield (%) Experimental Yield (%) Galactose Arabinose 100% 100%

80% 80%

60% 60% y = 0.9167x y = 0.9907x 40% R² = 0.937 40% R² = 0.9505 20%

20% Model Predicted Yield (%) Yield Predicted Model 0% (%) Yield Predicted Model 0% 0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100% Experimental Yield (%) Experimental Yield (%)

Fructose+Mannose 100%

80%

60% y = 0.941x R² = 0.9661 40%

20% Model Predicted Yield (%) Yield Predicted Model 0% 0% 20% 40% 60% 80% 100% Experimental Yield (%)

Figure 7.2 Comparison of model-predicted and experimentally measured yields of

glucose, xylose, galactose, arabinose and fructose+mannose in hydrolysate after 48 h

hydrolysis.

The carbohydrate composition was evaluated from the best-fit model parameters and are shown in Figure 7.3. Accordingly, pectin is the largest structural carbohydrate component

140 and requires higher pectinase activity for hydrolysis (Kp = 6.83 U/g). Half-maximum constant (enzymes loading for 50% yield) was highest for xylanase (43.5 U/g) meaning higher xylanase enzyme is needed for complete hemicellulose hydrolysis. Hemicellulose content from the best-fit model was 17.1% of which 25.2% is xylan. Therefore, there shouldn’t be a higher requirement for xylanase for complete hydrolysis since activity by definition means the release of 1 µmol of xylose released from substrate per min. The reason might be the complexity of soybean flour hemicellulose substrate since it has other sugar, i.e. arabinose (11.0%), glucose (25.7%), galactose (37.7%) and fructose+mannose (0.39%) which might be harder to hydrolyze compared to pure xylan substrate (beechwood xylan) used for xylanase activity measurement. However, the xylanase enzyme in A. niger enzyme broth is also higher (>100 U/mL) which were able to give higher hemicellulose hydrolysis yield at 48 h. Cellulose content is 9.6% (due to incomplete dehulling), requiring cellulase with Kc = 0.467 FPU/g (corresponds to 16.1/g- cellulose); cellulase is, however, more limited in A. niger enzymes. Cellulose and hemicellulose hydrolysis were found to depend strongly on pectin hydrolysis. Half- maximum constants of sucrase for sucrose and sucrose unit in the stachyose+raffinose substrate, Ks1 and Ks2 are 0.364 and 0.451 respectively which means the affinity of sucrase enzyme on sucrose is higher than on the sucrose unit in stachyose+raffinose. In addition, Kα for α-galactosidase to release galactose from stachyose+raffinose is 1.22 U/g.

141

Half-maximum constants (U/g SF)

Pectin, 34.3% Soluble Sugar, 39.1% Kc 0.467

Kx 43.5

Cellulose, 9.6% Hemicellulose, Kp 6.83 17.1% Ks1 0.364

Ks2 0.451 Soybean flour Carbohydrate Kα 1.22

Galactose Xylose, Galacturonic , 22.8% acid, 26.9% 25.2% Galactose , 37.7% Arabinose Fructose+ , 26.1% Mannose, Glucose, 24.3% 25.7% Arabinose , 11.0%

Fructose+Mannose, 0.39% Pectin Hemicellulose

Figure 7.3 Carbohydrate composition of soybean flour from model parameters

7.3.2 Kinetic profiles of monomeric sugar during enzyme hydrolysis

Enzyme hydrolysis was done for 48 h and samples were taken at 2, 4, 7, 12, 24 and 48 h to evaluate the kinetic release of monomeric sugar during enzyme hydrolysis.

Figure 7.4 shows the kinetic release of these sugar at four different enzyme composition.

Figure 7.4(a) shows the effect of commercial cellulase enzyme (SPEZYME, 10 FPU/g-

SF) effect where Figure 7.4(b) and 4(c) shows the effect of A. niger of different enzyme composition. Figure 7.4(d) shows the combination of A. niger and commercial cellulase

142 enzymes. The glucose release in Figure 7.4(b), (c) and (d) is higher from the very beginning compared to Figure 7.4(a) (10 FPU/g-SF SPEZYME only system) because of having other necessary enzymes in higher than the SPEZYME. However, their release rate decreased over time for Figure 7.4(b) and (c). When the pectin-containing sugar hydrolysis is increased, the glucose release increased further with the increase of cellulase enzyme. Structure-wise, cellulose is in the primary cell wall, and pectin are at the surface of the carbohydrate matrix giving protective layer, therefore, improving the hydrolysis of pectin would increase the cellulose hydrolysis. The hydrolysis rate of arabinose is the slowest among other sugars. The pectin containing arabinose are in the core matrix of pectin requiring other part of the pectin to get hydrolyzed first. In Figure

7.4(d), all the sugar release rates were very fast because of having very high enzymes concentrations. Complex interferences exist among different carbohydrates in soybean flour and the modeling approach can be successful in describing them and helpful to an understanding of enzyme hydrolysis mechanism.

143

Glucose (b) 100% Xylose (a) 100% Galactose Arabinose 80% Fructose+Mannose 80%

60% 60%

Yield (%) Yield Yield (%) Yield 40% 40% Glucose Xylose 20% 20% Galactose Arabinose Fructose+Mannose 0% 0% 0 12 24 36 48 0 12 24 36 48 Time (h) Time (h)

100% (c) 100%

80% 80%

60% 60% (d) Yield (%) Yield 40% (%) Yield Glucose 40% Xylose Glucose Galactose Xylose 20% Arabinose 20% Galactose Fructose+Mannose Arabinose Fructose+Mannose 0% 0% 0 12 24 36 48 0 12 24 36 48 Time (h) Time (h)

Figure 7.4. Monomeric sugar release profiles by (a) SPEZYME; with, per g SF, 10 FPU

cellulase, 23.7 U xylanase, 0.5 U pectinase, 0.1 U α-galactosidase, and 0.01 U sucrase;

(b) A. niger enzyme; with, per g SF, 0.5 FPU cellulase, 156.1 U xylanase, 10.3 U

pectinase, 11.4 U α-galactosidase, and 2.9 U sucrase; (c) 0.7 FPU cellulase, 412.1 U xylanase, 16.8 U pectinase, 14.3 U α-galactosidase, and 5.3 U sucrase; and (d) 11.1 FPU

cellulase, 538.5 U xylanase, 19.5 U pectinase, 12.7 U α-galactosidase, and 18.2 U

sucrase.

144

7.3.2.1 Kinetic Model

For describing the time-dependent hydrolysis performance for monomeric sugars release in enzyme hydrolysis, different empirical models have been used. During the initial stage, the rate is almost linear; however, at later stages, it decreases continuously and ceases over time. In this study the empirical model described by Walseth [157] is chosen as the basis:

푌 = 퐴 + 퐵푡푛

Y is the yield (%), A is the percentage of readily soluble carbohydrate, t is time (h), and B and n are empirical constants, where n = 0.5 is typically used [158]. B has been proposed as a function of the initial enzyme-to-substrate ratio (E/S):

퐸 푚 퐵 = 푘 ( ) 푆

Where k and m are empirical constants. The above model, however, fails at long reaction time; as the hydrolysis rate decreases, the parameter n deviates further from 0.5 with time. Accordingly, the following modified empirical model [159] can be used:

푡 (0.5−√ ) 푌 = 퐴 + 퐵푡 휏푑

Where τd represents a characteristic time which describes how fast the hydrolysis deviates from the initial kinetics (due to substrate change and enzyme deactivation). This model was shown to describe the long-term hydrolysis well (before t is so large that n becomes negative). Considering all the previous assumptions used for yield model, the kinetic model equations for individual sugar release are given below:

145

푡 푚 푡 푚푝 1 퐸 푐 (0.5−√ ) 퐸푝 (0.5−√ ) 푐 휏푑푐 휏푑푝 푌퐺푙푢푐표푠푒(%) = [훼푔,0 + 푘푐 ( ) 푡 × 푘푝 ( ) 푡 + 퐶푔푙푢 푆푐 푆푝

푡 푚 푡 푚푝 푚 푡 퐸 ℎ (0.5−√ ) 퐸푝 (0.5−√ ) 퐸 푠 (0.5−√ ) 푥 휏푑ℎ 휏푑푝 푠 휏푑푠 훽푔,ℎ푘ℎ ( ) 푡 + 훽푔,푝푘푝 ( ) 푡 + 훽푔,푠푘푠1 ( ) 푡 + 푆ℎ 푆푝 푆푠

푚 푡 퐸 푠 (0.5−√ ) 푠 휏푑푠 훽푔,표푙𝑖푔표푘푠2 ( ) 푡 ]; (6) 푆표푙𝑖푔표

푡 1 퐸 푚ℎ (0.5−√ ) 푥 휏푑ℎ 푌푋푦푙표푠푒(%) = [훼푥푦푙,0 + 훽푥푦푙,ℎ푘ℎ ( ) 푡 ]; (7) 퐶푥푦푙 푆ℎ

푡 1 퐸 푚ℎ (0.5−√ ) 푥 휏푑ℎ 푌퐺푎푙푎푐푡표푠푒(%) = [훼푔푎푙,0 + 훽푔푎푙,ℎ푘ℎ ( ) 푡 + 퐶푔푎푙 푆ℎ

푡 푚푝 푚 푡 퐸푝 (0.5−√ ) 퐸 훼 (0.5−√ ) 휏푑푝 훼 휏푑훼 훽푔푎푙,푝푘푝 ( ) 푡 + 훽푔푎푙,표푙𝑖푔표푘훼 ( ) 푡 ]; (8) 푆푝 푆훼

푡 푚 푡 푚푝 1 퐸 ℎ (0.5−√ ) 퐸푝 (0.5−√ ) 푥 휏푑ℎ 휏푑푝 푌퐴푟푎푏𝑖푛표푠푒(%) = [훼푎푟,0 + 훽푎푟,ℎ푘ℎ ( ) 푡 + 훽푎푟,푝푘푝 ( ) 푡 ]; 퐶푎푟 푆ℎ 푆푝

(9)

푡 1 퐸 푚ℎ (0.5−√ ) 푥 휏푑ℎ 푌퐹푟푢푐푡표푠푒+푀푎푛푛표푠푒(%) = [훼푓푚,0 + 훽푓푚,ℎ푘ℎ ( ) 푡 + 퐶푓푚 푆ℎ

푡 푚푝 푚 푡 퐸푝 (0.5−√ ) 퐸 푠 (0.5−√ ) 휏푑푝 푠 휏푑푠 훽푓푚,푝푘푝 ( ) 푡 + 훽푓푚,푠푘푠1 ( ) 푡 + 푆푝 푆푠

푚 푡 퐸 푠 (0.5−√ ) 푠 휏푑푠 훽푓푚,표푙𝑖푔표푘푠2 ( ) 푡 ]; (10) 푆표푙𝑖푔표

146

7.3.2.2 Best-fit model parameters for kinetic performances

The best-fit model parameters to describe the kinetic performances are shown in Table

7.1 and Figure 7.5 shows the comparison of model predicted yield and experimental yield at a different time from the kinetic model. Characteristic time parameter τd can give the insight on enzyme stability or the substrate stability based on the structural changes or availability over time. τd values for xylanase (1884 h) and pectinase (1216 h) is higher compared to sucrase (447 h) and α-galactosidase (288 h). The lower value for sucrase and

α-galactosidase could mean the stability of these enzymes are lower or there is a limitation on the substrate availability over time (i.e. substrate or product inhibition).

Cellulase has the lowest τd value (7 h). Although k values depend on units of different enzyme per substrate ratio, however, almost 8 times higher ks1 (0.07592) value compared to ks2 (0.00932) for sucrase enzyme shows the decreased rate of hydrolysis of the sucrose unit in stachyose+raffinose than sucrose sugar by sucrase enzyme. Highest m value was found for α-galactosidase and the lowest value was for xylanase.

Table 7.1 Best-fit kinetic model parameters

Kinetic Model Parameters

kc 0.04023 mc 0.40 τdc 7.05

kx 0.00110 mx 0.29 τdx 1884.1

kp 0.00061 mp 0.70 τdp 1216.5

kα 0.00008 mα 0.95 τdα 288.1

ks1 0.07592 ms 0.31 τds 446.7

ks2 0.00932

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Glucose Xylose 100% 100%

80% 80%

60% 60% y = 0.9977x y = 0.994x R² = 0.9416 R² = 0.9359

40% 40% Model Predicted Yield (%) Yield Predicted Model Model Predicted Yield (%) Yield Predicted Model 20% 20%

0% 0% 0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100% Experimental Yield (%) Experimental Yield (%)

Galactose Arabinose

100% 100%

80% 80%

60% 60% y = 0.9883x y = 1.0017x R² = 0.9059 R² = 0.9455

40% 40% Model Predicted Yield (%) Yield Predicted Model Model Predicted Yield (%) Yield Predicted Model 20% 20%

0% 0% 0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100% Experimental Yield (%) Experimental Yield (%)

Fructose+Mannose

100%

80%

60%

40% y = 0.9859x R² = 0.9659 20%

0% Model Predicted Yield (%) Yield Predicted Model 0% 20% 40% 60% 80% 100% Experimental Yield (%)

Figure 7.5 Comparison of model predicted and experimental yield of monomeric sugar

release from the kinetic model.

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7.4 Conclusion

The models are beneficial for the understanding of interfering interactions among carbohydrates on their enzyme hydrolysis and optimization of enzyme activities and reactor/process design for soy carbohydrate hydrolysis. The enzyme process would produce soy protein products with high protein contents and hydrolysate with fermentable, monomerized sugars, adding value to the major protein and carbohydrate components in soybeans.

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CHAPTER VIII

EFFECTS OF IONIC STRENGTH, PROTEASE AND HEAT TREATMENT ON

PROTEINACEOUS RELEASE FROM SOY FLOUR DURING ENZYME

PROCESSING

8.1 Introduction

The soy protein is particularly valuable, with all essential amino acids [29]. In typical soy processing, the protein ends up in the powdered soy flour or meal after oil extraction. Containing about 80% of the original dry bean weight, soy flour comprises approximately 50% protein, 30-35% carbohydrate and other minor components [30,160].

The high protein content and good amino acid profile and digestibility of soy flour [31] make it an ideal protein source for feed and food [29,161]. Soy flour can be further processed to make products with enriched protein content, i.e., soy protein concentrate

(SPC) and soy protein isolate (SPI). SPC has 60-68% protein content, made by removing the soluble carbohydrate in soy flour by processes like dilute acid wash or alcohol (20-

80%) wash at pH around the isoelectric point of soy protein [33,34]. SPI has about 90% protein content and is generally produced by a serial steps of dissolving protein at high pH, separating the solution from remaining solids, and then re-precipitating protein from the solution at the isoelectric point [33]. The series of SPI production steps typically result in relatively low protein recovery (40%-70%) [33]. On the other hand, the high contents of non-starch polysaccharides (NSPs) and galacto-oligosaccharides in soy

150 flour/meal and SPC can cause indigestibility concerns to monogastric animals such as fish, pig and poultry [35,36,162]. Economics of the abovementioned enzymatic processing of soy flour depends strongly on the extent of protein loss, which was a common concern in some previous enzyme-based soy processing studies due to the proteolytic activity of enzymes used [163,164]. For example, different carbohydrases and proteases were studied for enzyme-assisted aqueous oil extraction from full-fat soybeans

[163–165]. Although satisfactory oil extraction could be achieved, the high extent of protein hydrolysis caused low protein recovery (30% by isoelectric precipitation and 70-

74% by ultrafiltration) in these processes [164]. Besides protease-catalyzed proteolysis, protein loss due to dissolution into the aqueous hydrolysate may be affected by other factors such as hydrolysate ionic strength (by salting-in and salting-out effects) and pH

(by being different from isoelectric points of some of the proteins) [166,167]. Apparent protein loss due to dissolution can also be affected by the properties of the starting soy flour. The process conditions and procedures used for soy flour production can generate different contents of amino acids, peptides and lower-molecular-weight proteins and affect the protein conformation. To minimize protein loss for higher protein recovery and better economics, these factors deserve more investigations.

As part of this study, dry heating as a pretreatment (and sterilization) method was also investigated for effects on the enzyme hydrolysis of carbohydrate and recovery of protein. Heat treatment on soy flour can cause the Maillard reaction initiated by glycosylation of the amino groups of amino acids, peptides and proteins with reducing sugars, which leads to increased molecular weights, reduced solubility and even crosslinking of these proteinaceous substances depending on reaction conditions

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[168,169]. In addition, high temperature can cause protein denaturation to expose more hydrophobic groups and reduce protein solubility [167,170].

The objective of this study was to understand and minimize protein loss associated with the new enzyme-based soy flour processing. Possible factors were considered thoroughly, as described above, and investigated in this study. A. niger enzymes with different protease activity levels were used to evaluate the extent of protein loss due to proteolytic effects, in addition to the effects of other non-enzymatic factors.

Different commercially heat-processed soy flour/meal products were compared for the performance of enzyme processing regarding both carbohydrate hydrolysis and protein loss. Finally, the effects of controlled heat treatment applied in this laboratory on soy flour without previous commercial toasting were studied at different enzyme-processing stages, i.e., before enzyme hydrolysis, solid (SPC) and a liquid fraction (SPI and sugar containing hydrolysate), final SPI and hydrolysate by comparing the overall material balance.

8.2 Materials and Methods

8.2.1. Materials

Different grades of soy products including the 7B-grade defatted soy flour (SF),

Toasted Nutri Soy (TN) and Bakers Soy (BS) were provided by the Archer Daniels

Midland Company (Decatur, IL, USA). The enzymes used in this study were produced in this laboratory by submerged A. niger NRRL 341 fermentations made at different operating conditions. Four fermentations, in 3-L vessels with 1 L working volume, were made to obtain enzyme broths EB1, EB2, EB3 and EB4. The EB1 broth was produced

152 with a medium containing ground soy hulls, 20 g/L; proteose peptone, 1 g/L; urea, 0.3 g/L; (NH4)2SO4, 1.4 g/L; K2HPO4, 2 g/L; CaCl2·2H2O, 0.4 g/L; MgSO4·7H2O, 0.3 g/L;

Tween 80, 0.2 g/L; CoCl2·6H2O, 0.02 g/L; FeSO4·7H2O, 5 mg/L; MnSO4·4H2O, 1.6 mg/L; and ZnSO4·7H2O, 1.4 mg/L. The medium for EB2 had similar composition except that 3.8 g/L soy flour was added and the soy hull amount was almost doubled at 38 g/L.

The media for making EB3 and EB4 both had 2x concentrations of nitrogen sources

(proteose peptone, urea and (NH4)2SO4) and 2x soy hull (40 g/L) compared to the medium for EB1. Earlier studies had indicated that different enzymes had different optimal production pH [171]. Two pH control schemes were used here. For EB1 production, pH was maintained at 6 in the first 2 days and then allowed to drop naturally to 5 before controlled at pH 5 in the following 3 days. For EB2, EB3 and EB4 production, pH was controlled to drop linearly from 7 to 6 in the first 3 days, maintained at 6 in day 4, and then dropped linearly to 5 during day 5. EB3 and EB4 fermentations differed in the agitation speed used. The agitation was by a 6-blade turbine at 350 rpm for

EB1, EB2, and EB3 fermentations while the agitation speed for EB4 production was higher at 400 rpm. All fermentations were harvested after 5 days. Dissolved oxygen concentration (DO) was maintained at above 20% (air saturation) by automatic supplementation of pure oxygen. Cell-free enzyme broths were collected by 15-min centrifugation at 12,000 × g. The 6 relevant enzyme activities in these enzyme broths and their ionic strengths (estimated by calculation from the initial salt concentrations in media and the average amounts of NaOH and HCl added for pH control) are given in Table 8.1.

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Table 8.1 Enzyme activities measured for different enzyme broths (EBs) used in this study and their ionic strengths

Enzyme EB1 EB2 EB3 EB4

Cellulase (FPU/mL) 0.53 ± 0.03 0.50 ± 0.01 0.52 ± 0.01 0.65 ± 0.01

Xylanase (U/mL) 159.6 ± 6.5 171.7 ± 14.5 126.0 ± 5.8 301.9 ± 8.6

Pectinase (U/mL) 4.18 ± 0.26 5.50 ± 0.11 6.32 ± 0.29 6.06 ± 0.50

α-Galactosidase (U/mL) 5.22 ± 0.09 8.02 ± 0.31 7.02 ± 0.10 7.80 ± 0.24

Sucrase (U/mL) 3.46 ± 0.07 5.48 ± 0.01 3.57 ± 0.08 4.91 ± 0.08

Protease (BAEE U/mL) 72.8 ± 1.8 122.6 ± 6.2 139.3 ± 11.2 ND

Ionic strength (M) 0.156 0.156 0.188 0.188

Note: ND - Not determined; FPU - Filter Paper Unit; and BAEE - N-benzoyl-L-arginine ethyl ester (substrate for protease assay)

8.2.2 Common methods used for enzymatic processing of soy flour

In this study the enzymatic processing experiments, as described separately in the following sections, were all carried out for 48 h with 250 mL Erlenmeyer flasks in a shaker (Thermo Scientific MaxQ 5000 Incubating/Refrigerating floor shaker, Ashville,

NC) operating at 50 °C and 250 rpm shaking speed. A fixed solid loading of 250 g/L was used, with 10 g soy flour in 40 mL liquid (EBs plus water). Initial pH was adjusted to 4.8 with 3 N HCl and 0.05% sodium azide was added to control microbial contamination.

After hydrolysis, the wet solids (SPC) and supernatant were separated by 10-min centrifugation at 10,000 rpm (9300×g, Eppendorf 5415D). The supernatant was heated for 30 min in a boiling water bath and after cooled to room temperature, centrifuged

154 again to collect the heat-precipitated SPI from remaining liquid hydrolysate. The molecular weight profiles of proteins in the collected SPC, SPI and hydrolysate, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the protease effect study. Concentrations of proteinaceous substances in the hydrolysates before and after SPI collection were measured by the Kjeldahl method. Hydrolysates were also measured for concentrations of total reducing sugar and individual monomeric sugars. Analytical methods are described in a later section.

8.2.3 Ionic strength effect on protein loss

The enzyme broths produced by fermentations contained mineral ions provided in the fermentation media. Medium ionic strength is known to change soy protein solubility

[166] and, consequently, can affect protein loss during the enzymatic processing. To separate the effect of ionic strength from that of enzymatic proteolysis, enzyme broths

EB1 and EB3 were heated in a boiling water bath for 30 min to deactivate the enzymes.

These broths were then added at different loading to have different ionic strengths: 0

(control, only DI water), 0.0389, 0.0469, 0.0938, 0.1557 and 0.1875 M, and then contacted with the 7B-grade soy flour following the same enzyme processing procedures as described above. The ionic strengths of original broths are given in Table 8.1. The

SPC, SPI and hydrolysate samples collected were analyzed by the Kjeldahl method.

8.2.4 Protease effect on protein loss

For evaluating the effect of protease level on soy proteins during the enzyme processing, a set of experiments were made with the following enzyme loadings: 1 mL

EB1, 4 mL EB2 and 4 mL EB3 per g soy flour, to give significantly varied protease

155 loadings: correspondingly, 73 U, 490 U and 557 U per g soy flour. The 7B-grade soy flour (without heat treatment) was used in these experiments. Enzyme-free control systems were also included for comparison. The SPC, SPI and hydrolysate samples collected were analyzed by the SDS-PAGE and Kjeldahl methods.

8.2.5 Heat treatment effects on protein loss and carbohydrate hydrolysis

The heat treatment effects were studied with two sets of experiments. In the first set, enzymatic processing was done with different commercially heat-treated soy flour products (all from ADM): Toasted Nutria Soy (TN), Bakers Soy (BS) and 7B-grade soy flour (SF). Among them, TN was subjected to the highest extent of heat treatment, BS was only minimally heat treated, and the 7B-grade SF was not heat treated (from the website www.adm.com). For comparison they were all hydrolyzed at 1 mL EB2 per g soy flour. Corresponding enzyme-free control systems were also studied. These SF products were first determined for total carbohydrate contents following the NREL method (two steps of acid hydrolysis) [93] with phenol sulfuric acid assay. The total carbohydrate contents were used to determine the percentage releases of reducing sugar by the enzymatic processing. The second set of experiments were done with the 7B-grade soy flours with and without a 2-h dry-heat treatment at 160°C applied in this laboratory.

Enzymatic processing of these soy flours was made with 1 mL EB4 per g soy flour.

8.2.6 Analytical methods

The reducing sugar concentration was measured with the dinitrosalicylic (DNS) acid method [94]. 3 mL DNS reagent and 1 mL test sample were mixed in a test tube and after that, heated in a boiling water bath for 5 min. Deionized water was added to increase

156 the total volume to 25 ml. After being cooled down to the room temperature, the diluted mixture was measured for absorbance at 550 nm in a UV/Vis spectrophotometer (UV-

1601, Shimadzu Corporation, Columbia, MD). The total carbohydrate concentration was measured using the phenol sulfuric acid colorimetric method [172]. First, 1 mL sample was mixed with 1 mL aqueous phenol solution (5% v/v) in a test tube and then 5 mL concentrated sulfuric acid was added. After 10 min reaction, the tube content was vortexed for 30 s and allowed to cool to room temperature (in approximately 10 min).

Then the absorbance at 490 nm was measured, against a reference solution prepared in an identical manner but with 1 mL deionized water in place of sample. Calibration curves for both reducing sugar and total carbohydrate analyses were established using standards of pure glucose solutions that were processed by the same procedures.

The concentrations of individual sugars released into the hydrolysate were measured by high-performance liquid chromatography (HPLC) using a Shimadzu machine equipped with a pump (LC-10AT), column oven (CTO-20A), refractive index detector (RID-10A) and a system controller (SCL-10A). A SUPELCOGEL Pb column

(30 cm × 7.8 mm, sulfonated polystyrene divinylbenzene packing material) with its guard column was used at 80 °C. The mobile phase was HPLC grade water at a flow rate of 0.5 mL/min, and the sample injection volume was 10 µL. Samples were properly diluted and filtered through 0.22 μm nylon filters (Sigma-Aldrich, St. Louis, MO, USA).Glucose, xylose, galactose, arabinose, and fructose concentrations were determined with a total run time of 40 min and their corresponding retention times were 17.4, 18.5, 19.5, 20.8 and

22.2 min, respectively. The peak area of each sugar was converted to concentration using the calibration established with pure standards of respective sugar.

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SDS–PAGE was done for separating proteins by molecular weight according to the modified method of Laemmli [173]. Protein samples were prepared using SDS, β- mercaptoethanol (thiol reducing agent). SDS-PAGE was applied to analyze the SPC, SPI and hydrolysate with enzyme treatment and without enzyme treatment. The sample preparation and running conditions were followed the protocol of the Mini-

PROTEAN® TGX™ Precast Gels (Bio-Rad, CAT #4569035). 5 μL sample (with proper dilution) was mixed with 4.75 μL Laemmli (SDS-PAGE) sample buffer (Bio-Rad, CAT

#161-0737), and 0.25 μl β-mercaptoethanol (Bio-Rad, CAT #161-0710). Then the mixing solution was heated at 95°C for 5 min. After heating, the samples were loaded to the gels to run. Finally, the gels were stained by Simple Blue Safe Stain (Thermo Fisher,

CAT# LC6065).The Kjeldahl method [95] was used to measure the nitrogen (N) contents of samples. A 50 mL sample containing 10 to 200 mg/L proteinaceous substances was added to a flask and digested with 10 mL reagent containing 134 mL/L concentrated sulfuric acid, 134 g/L potassium sulfate, and 7.3 g/L cupric sulfate. The digestion was carried out to completion until the reaction mixture became a clear solution. Then 30 mL water and 10 mL of a distillation reagent containing 500 g/L NaOH and 25 g/L

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8.3 Results and Discussion

8.3.1 Ionic strength (IS) effect

Proteinaceous concentrations in supernatants collected after 48 h contact of 7B- grade soy flour with the heat-deactivated enzyme broths (EBs), after being diluted to different ionic strengths (IS), are compared in Table 8.2. Note that the soy flour used here was not heat treated. The concentrations are given for both supernatants before and after the SPI collection step. Results from the enzyme-free control with negligible IS are also included. Clearly, significant percentages of proteinaceous substances originally in the soy flour ended up in the supernatants without any actions of enzymes. For the control system, about 28% and 20% proteinaceous releases were found in the supernatants before and after SPI collection, respectively; i.e., about 8% proteins/peptides could be collected as SPI. The proteinaceous release into supernatants before SPI collection increased substantially with increasing IS: from about 28% in the control of negligible IS to almost

40% in the system of undiluted (4 mL/g-SF), heat-deactivated EB3 with IS of 0.188 (M).

The corresponding proteinaceous releases found in supernatants after SPI collection also increased with increasing IS but only slightly, approximately from 20% to 22%. This finding indicated that predominant majority of the IS-effected additional releases were proteins/peptides that could be recovered in the SPI collection step.

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Table 8.2 Effect of ionic strength of enzyme broth on proteinaceous release during enzymatic processing of soy flour

Ionic Enzyme Enzyme Protein in supernatant % Proteinaceous release strength (M) broth loading (g/L) (mL/g- w/o SPI after SPI w/o SPI after SPI SF) collection collection collection collection

~0 Control -- 35.1 ± 1.2 25.1 ± 0.3 28.1 ± 1.0% 20.1 ± 0.2%

0.0389 EB1 1 38.1 ± 0.8 25.7 ± 0.7 30.5 ± 0.6% 20.5 ± 0.6%

0.0469 EB3 1 39.8 ± 0.3 26.0 ± 1.1 31.9 ± 0.2% 20.8 ± 0.9%

0.0938 EB3 2 43.3 ± 2.1 26.4 ± 1.7 34.6 ± 1.7% 21.1 ± 1.4%

0.1557 EB1 4 47.0 ± 0.9 27.3 ± 0.1 37.6 ± 0.7% 21.8 ± 0.1%

0.1875 EB3 4 49.4 ± 1.6 28.2 ± 1.6 39.5 ± 1.3% 22.5 ± 1.3%

Notes: 1. Enzyme broths (EBs) used were deactivated by boiling for 30 min. 2. Hydrolysates were from contacting 250 g/L 7B-grade soy flour with deionized water-diluted EBs for 48 h at pH 4.8 and 50°C. 3. Estimated protein concentrations also included amino acids and peptides.

The IS effect-contributed releases can be quantitated by subtracting the proteinaceous concentrations in supernatants from the systems of heat-deactivated EBs by those from the control (of negligible IS). The largest IS-contributed proteinaceous releases (with undiluted deactivated EB3) were 14.3 (= 49.4 – 35.1) and 3.1 (= 28.2 – 25.1) g/L in hydrolysates before and after SPI collection, respectively. Thus calculated contributions due to only IS effect are shown in Figure 8.1. Within the range investigated, the IS contributions to increased proteinaceous releases (g/L) showed good linear correlations

160 with the IS (M) in deactivated EBs. For the supernatants before SPI collection, the correlation equation is:

IS contribution to proteinaceous release = 78.527 × IS (R² = 0.985)

For the supernatants after SPI collection, the correlation equation is:

IS contribution to proteinaceous release = 15.156 × IS (R² = 0.982)

As mentioned above, the IS effect was more prominent in supernatants before SPI collection, giving a much larger slope of increase. Note that the IS of EBs came mainly from (NH4)2SO4 and K2HPO4 in the fermentation medium. Future studies to minimize their concentrations, without negatively affecting enzyme production, are warranted to reduce proteinaceous release.

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16 y = 78.527x 14 R² = 0.9852

12 Before SPI collection 10 After SPI collection 8

6 y = 15.156x 4 R² = 0.9818

Ionic strength contribution Ionicstrength (g/L) 2

0 0 0.05 0.1 0.15 0.2 Ionic strength (M)

Figure 8.1 Contributions of ionic strength to increased proteinaceous release in supernatants before and after SPI separation (same experimental procedures as for Table

8.2)

8.3.2 Protease effect on soy flour protein during enzymatic processing

The proteinaceous concentrations in hydrolysates before and after SPI collection are summarized in Table 8.3 for processing of 7B-grade soy flour (SF) with enzyme solutions of 3 protease levels: 73, 490 and 557 BAEE U/(g SF) and the enzyme-free control. Owing to the proteolysis, even higher proteinaceous releases than those found with heat-deactivated EBs occurred. For hydrolysates before SPI collection, the release increased from about 28% in the control (enzyme-free, negligible IS) to 63-64% in the systems of undiluted EB1 and EB3. The increased proteinaceous releases (e.g., for EB3,

= 64% - 28% = 36%) came from both effects of increasing IS and protease. The IS contributions were already quantitated in the previous section (e.g., from Table 8.2 for 162

EB3, = 39.5% - 28.1% = 11.4%); the remaining were calculated as the protease- responsible releases (e.g., for EB3, = 36% - 11.4% = 24.6%). Thus, estimated contributions from these two effects were given in Table 8.3 for both supernatants before and after SPI collection. The A. niger protease indeed caused more proteinaceous releases, and the protease-responsible effect appeared to level off at about 25% in hydrolysates before SPI collection, with protease loading of about 500 BAEE U/(g SF).

Predominant majority of protease released proteinaceous materials could still be recovered by the SPI collection step; e.g., out of the about 25% protease-responsible releases by EB1 and EB3, about 16-18% were recovered in the SPI collection step leaving only 7-9% in the final hydrolysates.

Table 8.3 Protease effect on proteinaceous releases in hydrolysates before and after SPI collection

Protein in % Proteinaceous Ionic strength effected Protease effected % Protease Ionic hydrolysate (g/L) release % proteinaceous release Proteinaceous release (BAEE strength U/g SF) (M) w/o SPI w/ SPI w/o SPI w/ SPI w/o SPI w/ SPI w/o SPI w/ SPI collection collection collection collection collection collection collection collection

0 0 35.1 ± 1.2 25.1 ± 0.3 28.1 ± 1.0 20.1 ± 0.2 - - - -

73 0.0389 52.8 ± 2.6 29.6 ± 0.1 40.6 ± 2.1 23.7 ± 0.1 2.4% 0.4% 10.2% 3.1%

490 0.1557 78.8 ± 0.9 35.9 ± 1.3 63.0 ± 0.7 28.7 ± 1.0 9.5% 1.7% 25.4% 6.9%

557 0.1875 80.1 ± 2.2 39.2 ± 0.6 64.1 ± 1.8 31.4 ± 0.5 11.4% 2.4% 24.6% 8.8%

Notes: 1. Hydrolysates from the enzymatic processing of 250 g/L 7B-grade soy flour (SF) with 1 mL EB1, 4 mL EB2 and 4 mL EB3 per g SF, giving the corresponding protease loadings of 73, 490 and 557 BAEE U/g SF. 2. Estimated protein concentrations could include amino acids and peptides in addition to proteins.

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SDS-PAGE results are shown in Figures 8.2 (A) and 8.2 (B) for SPC (lanes 1-4 of

Figure 8.2 (A)), SPI (lanes 5-8 of Figure 8.2 (A)) and final hydrolysates (lanes 10, 11 of

Figure 8.2 (A) and lanes 1-4 of Figure 8.2 (B)) collected from the systems described above in Table 8.3. Glycinin (11S) and β-conglycinin (7S) are the major proteins, constituting about 52% and 35%, respectively, of proteins in soybeans [174]. β-

Conglycinin (150-180 kDa) is composed of subunits α, α, and β with respective molecular weights of about 72 kDa, 68 kDa and 52 kDa [175]. Glycinin (360 kDa), with a compact quaternary structure stabilized by disulfide, electrostatic and hydrophobic bonds [174,175], is composed of acidic (A) subunits of 34-43 kDa and 10-15 kDa and a basic (B) subunit of 18-25 kDa [163,164,176]. All these 7S and 11S subunits are present in the SPC and SPI (lanes 1 and 5 of Figure 8.2 (A)) from the control system. However, some subunits, most notably α’ and α of 7S and the acidic subunit of 37 kDa of 11S, disappeared in the SPC and SPI from systems with higher protease activities, i.e., undiluted EB1 (lanes 4 and 8) and EB3 (lanes 3 and 7). New bands, presumably proteolytic products, also appeared in these lanes, e.g., some at 50-65 kDa and 20-25 kDa. SPI from these systems (lanes 7 and 8 of Figure 8.2 (A)) also had much broader and darker bands at around 18 and 30 kDa, compared to the SPI from the control (lane 5).

Degradation by the A. niger protease was however not apparent for subunits 7S-β, 11S-B, and 11S-A of 10-15 kDa. Hydrolysates from these systems also had higher amounts of smaller peptides, below 15 kDa and especially below 10 kDa (Figure 8.2 (B)).

Degradation was less apparent in the SDS-PAGE results for the system of 4-fold diluted

EB1 (but some protein loss also occurred in this system as described above with results shown in Table 8.3).

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Although high ionic strengths and protease activities in the enzyme broths could cause substantial proteinaceous release, the significant release occurred even in the enzyme-free control: 35.1 ± 1.2 and 25.1 ± 0.3 g/L, respectively, in hydrolysates before and after the SPI collection. Soy flour pretreatment before the enzyme processing may be desirable for reducing proteinaceous release. Results of heat pretreatment are described in the following sections.

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(A) 1 2 3 4 5 6 7 8 9 10 11

250kDa 150 100 7S-α’, α 75

7S-β 50

11S-A 37

25 11S-B 20

11S-A 15

(B)

250kDa 150 100 75

25 20

1 2 3 4 5

Figure 8.2 SDS-PAGE results for SPC, SPI and final hydrolysates collected from the enzyme-free control (Ct) and 3 systems with A. niger enzyme broths (labeled with their protease loadings: 73, 557 and 490 BAEE U/g SF, as given in Table 3): (A) Lanes 1-4,

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SPC (Ct, 73, 557 and 490 BAEE U/g SF); Lanes 5-8, SPI (Ct, 73, 557 and 490 BAEE

U/g SF); Lane 9, marker; Lanes 10-11, hydrolysate H (Ct and 73 BAEE U/g SF), (B)

Lane 2, 73 BAEE U/g SF (DF 40); Lane 3, 557 BAEE U/g SF (DF 4); Lane 4, 490 BAEE

U/g SF (DF 4); and Lane 5, marker Sample dilution factors (DF) used for the analysis are also given in the lane labels.

8.3.3 Proteinaceous release and carbohydrate hydrolysis of commercial soy flours of different toasting extents

Potential heat treatment effects were first investigated with 7B-grade soy flour

(SF), Bakers Soy (BS) and Toasted Nutria Soy (TN) with increasing extents of commercial toasting. Results are compared in Figure 8.3 for (a) the unrecovered proteinaceous concentrations in the final hydrolysate after SPI collection and (b) the reducing sugar conversions achieved by enzymatic carbohydrate hydrolysis. The conversion is calculated as the percentage of reducing sugar in hydrolysate out of the total carbohydrate introduced with the starting soy flour. The total carbohydrate contents of TN, BS and SF were measured to be (25.5 ± 0.5)%, (25.8 ± 0.2)% and (26.1 ± 0.3)%, respectively. Results from the corresponding enzyme-free controls are also shown in

Figure 8.3.

As shown in Figure 8.3(a), for all 3 soy flours the predominant portion of proteinaceous release occurred in the enzyme-free controls, and the unrecovered proteinaceous concentrations in these controls decreased with increasing heat treatment extents: 20.1% from 7B SF, 13.2% from BS, and 7.6% from TN. The additional proteinaceous releases due to the enzyme broth were comparatively small and similar:

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(3.6 ± 0.2)%, or 3.7%, 3.8% and 3.4%, respectively, for the 3 soy flours. On the other hand, as shown in Figure 8.3(b), the enzyme broth was predominantly responsible for carbohydrate hydrolysis. Heat treatment did not show any negative effect on the reducing sugar conversions achieved with the enzyme broth (p = .458); rather, the conversions seemed to be higher for soy flours with more heat treatment: (74.4 ± 1.5)% for TN, (71.7

± 1.8)% for BS, and (70.4 ± 2.6)% for 7B-grade SF. The positive effect of heat treatment on the enzyme-effected conversion, after subtracting the conversions in corresponding enzyme-free controls, was statistically significant (p = .018 from one-way ANOVA):

70.7% (= 74.4% - 3.7%) for TN, 64.8% (= 71.7% - 6.9%) for BS, and 54.5% (= 70.4% -

15.9%).

35 Control Enzyme processing Control Enzyme processing 80% 30

25 60% 20

15 40%

inhydrolysate (g/L) 20%

5 Proteinaceous Proteinaceous concentration 0% 0 sugar Reducing (%) conversion TN BS SF TN BS SF (b) (a)

Figure 8.3 (a) Unrecovered proteinaceous concentrations (g/L) in final hydrolysates after

SPI collection and (b) reducing sugar conversions observed with commercial soy flours

of different extents of toasting (TN > BS > 7B-grade SF); processing was done without

(enzyme-free controls) and with 1 mL EB2 per g soy flour.

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Accordingly, heat treatment of soy flour, within the extents investigated in this study, can significantly reduce proteinaceous release without negatively affecting carbohydrate hydrolysis. In the enzyme-free controls, higher heat treatment extents gave lower releases of both proteinaceous materials and soluble carbohydrate. This observation may be attributed to the occurrence of Maillard reaction between reducing sugars and proteinaceous materials during the heat treatment, leading to covalent bonding and even crosslinking of soy flour constituents [168,177].

8.3.4 Effects of heat treatment on enzymatic processing of 7B-grade soy flour

While the order of toasting extent was known for the above soy flours, actual processing conditions were unclear. Heat treatment effects were evaluated in more detail with the 7B-grade soy flours with and without the well-defined 2-h dry-heat treatment at

160°C in the laboratory. Corresponding enzyme-free controls were also evaluated.

Moisture and ash contents of soy flours and SPCs, wet SPC weights, hydrolysate volumes before and after SPI collection, monosaccharide and proteinaceous concentrations in final hydrolysates are measured for all systems.

8.3.4.1 Proteinaceous release and distribution among SPC, SPI, and hydrolysate

Proteinaceous concentrations and % releases in final hydrolysates after SPI collection are summarized in Table 8.4. With or without the A. niger enzymes, the heat- treated soy flour gave far lower proteinaceous concentrations in the final hydrolysate,

~40% of those from soy flour without the heat treatment. The enzyme-contributed increases in the proteinaceous release were again comparatively low: 2.1 and 3.7 g/L for soy flour with and without heat treatment. The total solid (dry) recovery and

169 proteinaceous contents of SPCs and the percentage proteinaceous distributions among

SPC, SPI and hydrolysates are also given in Table 8.4. The protein content of SPC was calculated as (dry solid - carbohydrate remained in SPC - ash)/dry solid × 100%, where the carbohydrate remained in SPC was calculated as (initial soy flour carbohydrate - sugar in hydrolysate before SPI collection). Heat treatment was shown to increase the protein content of SPC (in both control and enzyme systems) slightly (by ~0.9-1.2%) but increase the SPC solid recovery significantly (e.g., from 54.4% to 70.5% in the enzyme system), resulting in much higher protein recovery in SPC (91.2% in the enzyme system, compared to 75.3% from non-heat-treated soy flour). Together with the protein recovery in SPI, heat treatment enabled recovery of 95-96% of the original proteinaceous materials in soy flour, with or without A. niger enzymes in the aqueous processing solution; only 4-

5% unrecovered proteinaceous release occurred. Without heat treatment, the combined protein recovery in SPC and SPI decreased to 83-85%, and about 6-7% of recovery relied on the SPI collection step. On the other hand, in systems with heat-treated soy flour, the

SPI collection step contributed only 3.5-3.6% proteinaceous recovery. Depending on the economic analysis, the SPI collection step may be eliminated for enzyme processing of heat-treated soy flour to lower the overall processing cost.

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Table 8.4 Effects of heat treatment (160 °C, 2h) on proteinaceous release, solid recovery

in SPC, protein distribution among SPC, SPI and hydrolysate, and monomeric sugar

concentrations in hydrolysate for systems with 250 g/L soy flour loading

Enzyme system Control (1 mL EB4/g-SF)

No heat Heat No heat Heat treatment treatment treatment treatment

Protein concentration in hydrolysate 25.6 ± 0.4 10.0 ± 0.4 29.3 ± 0.6 12.1 ± 0.5 (g/L)

% Proteinaceous release in hydrolysate 20.4 ± 0.3 8.0 ± 0.3 23.5 ± 0.5 9.7 ± 0.4

Solid recovery in SPC (% of SF) 63.5 ± 0.9 78.6 ± 1.1 54.4 ± 1.5 70.5 ± 0.5

Protein content in SPC (%) 62.8 63.7 69.2 70.4

Protein distribution among SPC, SPI and hydrolysate (% of total protein)

SPC 79.7 92.2 75.3 91.2 SPI 5.6 3.5 7.4 3.6 Hydrolysate 14.7 4.3 17.3 5.2

Soluble sugar concentrations (g/L)

Stachyose 9.0 ± 0.4 9.5 ± 0.2 0 0 Raffinose 2.8 ± 0.3 2.4 ± 0.2 0 0 Sucrose 11.0 ± 0.1 12.4 ± 0.3 0 0 Glucose 2.6 ± 0.1 0 12.9 ± 0.2 13.7 ± 0.4

Xylose 2.5 ± 0.5 2.1 ± 0.2 3.2 ± 0.1 3.2 ± 0.1

Galactose 5.3 ± 0.2 2.2 ± 0.3 12.1 ± 0.3 15.9 ± 0.5

Arabinose 0 0 2.5 ± 0.1 5.1 ± 0.1

Fructose 3.0 ± 0.6 0.4 ± 0.1 14.0 ± 0.4 14.7 ± 0.3

Other sugars 4.7 ± 0.9 7.6 ± 1.2 11.0 ± 0.5 14.0 ± 0.3

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8.3.4.2 Sugar release

Concentrations of monomeric sugars in hydrolysates are also given in Table 8.4.

Here, the “other sugars” were estimated from some “unknown peaks” observed in the

HPLC chromatogram and their concentration was estimated using an average calibration coefficient from other sugar standards (which did not differ much; e.g., 7.64, 7.68, 8.29,

7.89 and 8.49 (× area/106) for glucose, xylose, galactose, arabinose and fructose, respectively). The unknown peaks appeared at retention times around 16.1 min, between glucose (17.4 min) and sucrose (15.0 min); they might be galacturonic acid-containing sugars (monomers and dimers). Stachyose and raffinose concentrations in the control were found to have no significant effect according to one-way ANOVA (p = 0.380 and

0.383 respectively). For the enzyme-free controls, heat treatment was shown (Table 8.4) to reduce the glucose and galactose concentrations significantly (p = .001 and .013; .051 for apparently less fructose also), plausibly due to the Maillard reaction between sugar and protein. On the contrary, sucrose concentration was found higher in heat treated control (12.4 ± 0.3 g/L, compared to 11.0 ± 0.1 g/L in no-treatment control), which shows heat treatment has an effect on the increase of sucrose (p = 0.047). However, considering the moisture content (1.4% in heat treated SF and 9.3% in original SF), the effect is not significant (p = 0.294 for sucrose, also 0.615 for Stachyose and 0.232 for raffinose). Heat treatment improved monosaccharide yields in the systems with A. niger enzyme broth. The improvements were statistically significant for galactose, arabinose and “other sugars” (p = .023, .003 and 0.036, respectively), suggesting heat treatment was particularly helpful for pectin hydrolysis because these sugars are all components of

172 pectin [30]. The summed sugar concentration, from enzyme processing of 250 g/L SF, increased from 55.7 g/L to 66.6 g/L with the heat treatment.

8.3.4.3 Material balances

Material balances are shown in Figure 8.4 (control, without A. niger enzyme broth) and Figure 8.5 (with enzyme broth) to compare the effects of heat treatment of 7B- grade soy flour (SF). For easy illustration, the basis is taken as 100 g starting SF. For systems without heat treatment, the protein content in original SF was 50%, carbohydrate content was 30.2% including 12.8% soluble carbohydrate (according to the NREL analysis), and ash content was 6.7%. Owing to the different moisture contents, i.e., 9.3% and 1.4% in SF without and with heat treatment, the same basis of 100 g SF corresponded to slightly larger protein, carbohydrate and ash amounts in the heat-treated SF. Material balances clearly showed that the protein amounts remained in SPCs were considerably higher in heat-treated systems (50.2 g in control and 49.6 g in enzyme system) compared to those without heat treatment (39.9 g in control and 37.7 g in enzyme system). For enzyme-processed systems, although the summed monomeric sugar concentration in hydrolysate from heat-treated SF was higher (66.6 g/L) than that from non-treated SF

(55.7 g/L), the amounts in final hydrolysates were not as different (15.7 g versus 16.5 g) because of the smaller volume of hydrolysate collected in the heat-treated system. The carbohydrate remaining in the wet SPC from enzyme-processed systems differed significantly in the extent of monomerization. In the heat-treated system, out of the 16.9 g remaining carbohydrate in SPC, 13.3 g was already monomerized, and only 3.5 g was not; in the non-heat-treated system, a smaller amount (13.2 g) of carbohydrate remained in SPC but only 7.4 g was monomerized, and 5.9 g was not. If desirable to increase SPC

173 protein content, the entrapped sugar, already hydrolyzed and soluble, can be removed, e.g., by ethanol-water wash of SPC [103], while the insoluble non-hydrolyzed carbohydrate cannot. Also, having a larger amount of hydrolyzed monomeric sugars in

SPC can potentially improve its metabolizable energy value for feed and food uses and reduce the indigestibility concern associated with non-hydrolyzed carbohydrate [31].

Considering the amount of unconverted/insoluble sugar in SPC, the carbohydrate conversion achieved by heat treatment improved from 80.6% to 89.2%. Overall, heat treatment is conclusively shown to have very important positive effects on the enzymatic soy flour processing in reducing protein loss and increasing hydrolysis of insoluble carbohydrate.

174

Control

No treatment Heat treatment (160°C, 2 h)

100 g SF 100 g SF Protein = 50 g Enzyme Hydrolysis Protein = 54.4 g Carbohydrate = 30.2 g Water = 400 mL Carbohydrate = 32.9 g Moisture = 9.4 g (pH 4.8, 50°C, 250 rpm) Moisture = 1.4 g Ash = 6.7 g Ash = 7 g Others = 3.7 g Others = 4.3 g

Solid fraction (SPC) = 255 g Solid fraction (SPC) = 196 g

(Moisture content = 67.6%) (Moisture content = 69.1%) Protein = 50.2 g Protein = 39.9 g Centrifugation Carbohydrate = 20.6 g Carbohydrate = 24.5 g 9300 × g, 10 min (soluble = 6.1 g, insoluble = (soluble= 4.2 g, insoluble =

16.4 g) 18.4 g) Moisture = 176.1 g Moisture = 132.5 g Ash = 4.1 g Ash = 3 g

Liquid fraction = 300 mL Liquid fraction = 240 mL Protein = 10.1 g Protein = 4.2 g Carbohydrate = 9.6 g Carbohydrate = 8.4 g Ash = 3.7 g Ash = 2.9 g

Solid fraction (SPI) = 10.8 g SPI Collection Solid fraction (SPI) = 7.1 g Protein = 2.8 g 1. Heat precipitation Protein = 1.9 g Carbohydrate = 0.2 g (100 °C, 30 min) Carbohydrate = 0.2 g Moisture = 7.8 g 2. Centrifugation Moisture = 5 g Ash = 0.1 g (9300 × g, 10 min) Ash = 0.1 g

Liquid fraction (H) = 292 mL Liquid fraction (H) = 235 mL Protein = 7.3 g Protein = 2.4 g Carbohydrate = 9.4 g Carbohydrate = 8.2 g Ash = 3.6 g Ash = 2.8 g

Figure 8.4 Material balances compared for controls (without A. niger enzyme broth) with

and without heat treatment (160 °C, 2h) of 7B-grade soy flour

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Enzyme System

No treatment Heat treatment (160°C, 2 h)

100 g SF 100 g SF Protein = 50 g Enzyme Hydrolysis Protein = 54.4 g Carbohydrate = 30.2 g Enzyme = 100 mL Carbohydrate = 32.9 g Moisture = 9.4 g Water = 300 mL Moisture = 1.4 g Ash = 6.7 g (pH 4.8, 50°C, 250 rpm) Ash = 7 g Others = 3.7 g Others = 4.3 g

Solid fraction (SPC) = 187 g Solid fraction (SPC) = 271 g (Moisture content = 70.8%) (Moisture content = 73.9%) Protein = 37.7 g Protein = 49.6 g Carbohydrate = 13.2 g Centrifugation Carbohydrate = 16.9 g (soluble = 7.4 g, insoluble = 9300 × g, 10 min (soluble = 13.3 g, insoluble = 5.9 g) 3.5 g) Moisture = 132 g Moisture = 200 g Ash = 3.5 g Ash = 4.0 g

Liquid fraction = 305 mL Liquid fraction = 240 mL Protein = 12.3 g Protein = 4.8 g Carbohydrate = 17.0 g Carbohydrate = 16.0 g Ash = 3.2 g Ash = 3.0 g

Solid fraction (SPI) = 13.5 g SPI Collection Solid fraction (SPI) = 7.3 g Protein = 3.7 g 1. Heat precipitation Protein = 2.0 g Carbohydrate = 0.5 g (100 °C, 30 min) Carbohydrate = 0.3 g Moisture = 9.3 g 2. Centrifugation Moisture = 5 g Ash = 0.1 g (9300 × g, 10 min) Ash = 0.1 g

Liquid fraction (H) = 296 mL Liquid fraction (H) = 235 mL Protein = 8.7 g Protein = 2.8 g Carbohydrate = 16.5 g Carbohydrate = 15.7 g Ash = 3.1 g Ash = 2.9 g

Figure 8.5 Material balances compared for enzyme processing (1 mL EB4/g-SF) of 7B-

grade soy flour with and without heat treatment (160 °C, 2h)

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8.4 Conclusion

Effect of broth ionic strengths on proteinaceous release was investigated after deactivating enzyme broths and diluting them to IS of 0-0.1875 M. Increasing IS linearly increased proteinaceous release. For 7B-grade SF of no/minimal toasting, with the highest IS (0.1875 M) from undiluted EB3, IS contributed 11.4% and 2.4% proteinaceous release before and after SPI collection, respectively. Predominant majority of the proteinaceous materials released due to IS effect could be recovered in the SPI collection step. Enzyme processing of soy flour is typically carried out with 4-fold diluted enzyme broths. At the lower IS (0.039 and 0.047 M for EB1 and EB3), the IS effect was negligible (0.4-0.7% after SPI collection). Protease effect was significantly stronger than the IS effect. SDS-PAGE showed the A. niger enzymes mostly degraded α’ and α subunits of -conglycinin (7S) and the acidic 37-kDa subunit of glycinin (11S). However, proteolysis was not extensive, and the majority of the released proteins could still be recovered in SPI. New bands appeared at 50-65 kDa, and 20-25 kDa and broader/darker bands at ~18 and 30 kDa in the SPI collected from systems of undiluted enzyme broths.

Protease-caused releases in the final hydrolysates after SPI collection were up to 8.8% with undiluted EB3 and only ~3% with 4-fold diluted EB1. Majority of the proteinaceous release from 7B SF occurred in water without IS or protease effect: 28.1% and 20.1%, before and after SPI collection. Heat treatment of soy flour was found to be very effective for reducing proteinaceous release, first by comparing soy flours of different toasting extents (TN > BS > SF, but actual conditions unknown) and then more thoroughly by 7B

SF without and with 2-h heat treatment at 160 °C. The latter heat treatment reduced proteinaceous loss (in the final hydrolysate) from 17.3% (no treatment) to 5.2% and

177 increased sugar conversion from 80.6% to 89.2%. The SPC had ~70% protein and, with heat treatment, nearly 80% of the carbohydrate remaining in SPC was monomeric sugars

(in hydrolysate trapped in wet SPC without a wash). Soy protein and soluble sugars obtained in this process can greatly increase the value of soybeans for feed/food, and fermentation feedstock uses.

178

CHAPTER IX

IMPROVED CARBOHYDRASE PRODUCTION BY ASPERGILLUS NIGER

FERMENTATION FOR SOYBEAN MEAL CARBOHYDRATE HYDROLYSIS FOR

USE AS FERMENTATION FEEDSTOCK

9.1 Introduction

While enzymatic processing of soybean meal can be an effective way of adding value to the soybean industry, it can be highly economically advantageous to produce an effective enzyme mixture in a single fermentation, over the use of blended enzymes produced from multiple fermentation processes. Development of soy-based biorefinery requires a complex mixture of enzymes to degrade all types of carbohydrate present in the soybean meal. It was later found that complete hydrolysis of soybean meal carbohydrate would require enzymes containing at least cellulase, xylanase, pectinase, α- galactosidase and sucrase [92]. Soybean hull is a by-product of soybean industry and is a very cheap carbon source for use in fermentation medium for enzyme production.

However, the enzyme produced by A. niger NRRL 322 was not high enough to completely hydrolyze all the carbohydrate. The activities of the enzyme produced by this strain are, pectinase 6.36 ± 0.32 U/mL; α-galactosidase, 4.50 ± 0.03 U/mL; cellulase,

0.31 ± 0.01 FPU/mL, xylanase, 101.7 ± 1.5 U/mL and sucrase, 3.35 ± 0.14 U/mL. The

179 half-maximum constants Km for these enzymes were estimated at 27.5 FPU/(g meal) for cellulase, 3.15 U/(g meal) for xylanase, 14.7 U/(g meal) for pectinase, and 1.16 U/(g meal) for α-galactosidase has been found by modeling the enzyme hydrolysis of soybean meal carbohydrate. [92]. Xylanase enzyme produced by this strain is already very high compared to Km-value for xylanase. However, for effective and complete hydrolysis of soybean meal carbohydrate requires higher activities of pectinase and α-galactosidase, along with cellulase and sucrase [92].

The objective of this study is to produce enzymes with improved carbohydrase production using a single A. niger fermentation. Enzyme production by Aspergillus species is sensitive to the nutrient and environmental conditions. In this study, different soybean hull loading and different starting pH in fermentation was evaluated. In the previous study, 20 g/L soybean hull was used, while in this current study higher solid loading (up to 100 g/L) was studied at two different pH level. Effect of fed-batch soybean addition was also evaluated to continual production of the enzyme. Since, in Aspergillus niger fermentation, the different enzyme can be produced at different pH range, to evaluate that dependency, difference pH gradient (with different pH drop rate per h) was studied to maximize all the all production.

9.2 Materials and methods

9.2.1 Materials

Aspergillus niger strain NRRL 322 used in this study was obtained from the

United States Department of Agriculture (USDA) Agricultural Research Service (ARS)

Culture Collection. The culture was maintained on slants of potato dextrose agar (30 g/L,

180

Sigma, P2182) at 4 °C. Soybean hull for this study was provided by Archer Daniels

Midland Company (Decatur, IL, USA). Total reducing sugar content of soybean hull was

64 ± 2% by weight and the monomeric sugar contents were: glucose, 35.7 ± 1.3%; xylose, 13.2 ± 0.8%; galactose, 5.9 ± 0.5%; arabinose, 6.5 ± 0.2%; and mannose, 5.7 ±

1.4% [146]. The main equipment used included a UV-visible spectrophotometer

(Shimadzu UV-1601, Colombia, MD); a shaker (Thermo Scientific MaxQ 5000

Incubating/Refrigerating floor shaker, Ashville, NC); 2 fermentors with controls for pH, dissolved oxygen concentration (DO), agitation and temperature (BioFlo 110,

NewBruswick Scientific, Edison, NJ); a water bath (Boekel Scientific ORS-200); and a micro centrifuge (Eppendorf Centrifuge 5415D). Unless otherwise specified, chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA). Proteose peptone was from

Remel Microbial Products (division of Thermo Fisher Scientific).

9.2.2 Preculture and fermentation

The inoculum was prepared by aseptically adding three loops of cells from a mature slant to 150 mL preculture medium in 500 mL shake flask, and incubating for 48 h at 25 °C in a shaker (Thermo Scientific MaxQ 5000 Incubating/Refrigerating floor shaker, Ashville, NC) at 200 rpm. The medium composition was: 1.4 g/L (NH4)2SO4, 2.0 g/L KH2PO4, 0.3 g/L MgSO4·7H2O, 0.4 g/L CaCl2·2H2O, 0.3 g/L urea, 1.0 g/L proteose peptone, 0.2 g/L Tween 80, 20 g/L soybean hull, 0.005 g/L FeSO4·7H2O, 0.0016 g/L

MnSO4·H2O, 0.0014 g/L ZnSO4·7H2O, and 0.002 g/L CoCl2·2H2O. The preculture was used to inoculate the stirred tank fermentors at 10% (v/v). The fermentors were operated with 1-1.5 L working volume under controls of DO, pH, agitation, temperature and foaming, the last by automatic addition of Trans-278 (Trans-Chemco, Inc., Bristol, WI).

181

The production medium was differed by carbon and nitrogen content than the seed culture medium which was described in the following sections. The DO was maintained at above 20% air saturation by automatic oxygen addition in all of the fermentation experiments. The agitation rate was maintained at 350-450 rpm. At a later stage of all the fermentation, DO started to increase and all the fermentation were harvested when the

DO was between 70-80%. Daily samples were taken for enzyme analysis. Samples were centrifuged at 10,000 g for 10 min to remove the solid biomass, and the supernatants collected were stored at -20 C prior to analysis.

9.2.3 Batch fermentation: pH and solid loading study

Enzyme production studies were done both in batch and fed-batch fermentation process with different solid loading and starting pH. Fermentation F1 and F2 were done with 2x (40 g/L) soybean hull and 2x (2.7 g/L) nitrogen-containing components

((NH4)2SO4, urea, and proteose peptone) at two different starting pH. After autoclaving, pH was adjusted by 1N HCl and 1N NaOH and the starting pH for F1 and F2 were 6 and

7. The pH was allowed to vary naturally: decreasing initially due to, e.g., production of organic acids and consumption of ammonia, and increasing later due to, e.g., ammonia release from endogenous metabolism. For both F1 and F2, fermentations were harvested after 120 h. Effect of higher solid loading was studied in fermentation F3 and F4.

Soybean hull concentration in both F3 and F4 was 5x (100 g/L) and nitrogen loading in

F3 was 2x while in F4, nitrogen loading was 5x. The starting pH of F3 and F4 were 6 and

7 and the harvest time was 171 h and 137 h respectively.

182

9.2.4 Fed-batch fermentation

Fed-batch soybean hull addition was studied to induce more enzyme production.

F5 fermentation was done with 3x soybean hull additional, 2x nitrogen addition keeping other nutrients same. 2x of soybean hull was added initially and fermentation was started at pH 6. Another 1x dry heat sterilized (3h at 160 °C) was added after 72 h of fermentation. The fermentation was done for 120 h. F6 fermentation was done with total

5x soybean hull started at pH 6. 2x soybean hull (40 g/L) was added initially, then another 3x (60 g/L) was added in 24 h interval, 1x each time, after 48 h of fermentation.

Fermentation was continued for 190 h when the DO increase to 72%.

9.2.5 pH gradient study

Three fermentations were done at different pH gradient to improve all the enzyme production. For all the fermentations, 5x soybean hull and 2x nitrogen were used along with other same nutrients, and the starting pH and temperature were also same (pH = 7 and T = 25 °C). In F7 fermentation, pH was dropped by the automatic control at a rate of

0.0156 pH per h up to 96 h, whereas, the pH dropping rate in F8 fermentation was 0.0292 pH per h up to 96 h and in F9 fermentation, the pH dropping rate was 0.0357 pH per h up to 84 h. After that, there was no control on the pH was maintained. In F8, another 1x fed- batch nitrogen addition was done at 0.25x in every 12 h after 24 h of fermentation.

9.2.6 Enzyme analysis

Five groups of extracellular enzymes were analyzed: cellulase, xylanase, pectinase, -galactosidase, and sucrase. Tests were all done in triplicate and results

183 reported as the average values with standard deviations. By definition, one unit of enzyme activity corresponds to the activity that gives the target product at a rate of 1

µmol/min. In this study, the target product concentration was determined by the non- specific 3,5-dinitrosalicylic acid (DNS) test method using different reducing sugars as standards, except for the α- galactosidase. The cellulase assay used was modified from that reported by Ghose [97]. It was found to be the most suitable for samples with cellulase activities in the range of 0.05 FPU/mL to 3 FPU/mL. The analysis procedure was as follows: (1) Cut Whatman No. 1 filter paper into pieces of 6  1 cm, ~ 50 mg/piece. Roll and insert a piece (1 cm in height) into a 25 mL tube. Add 1.4 mL 0.05 M sodium citrate buffer (pH 4.8) and 100 µL sample under analysis to the tube. The filter paper should be completely immersed in the solution. (2) Prepare the blank in the same way but without the filter paper. (3) Incubate the samples and blanks in a water bath at

50C for 1 h. (4) Add 3 mL regular DNS solution that consisted of 10 g/L 3,5- dinitrosalicylic acid, 16 g/L sodium hydroxide (NaOH) and 300 g/L sodium-potassium tartrate to each sample and blanks to stop the enzyme reaction. (5) Incubate the DNS- added tubes in boiling water (100C) for 10 min. (6) Add deionized water to make the total volume 25 mL, mix, and then measure the absorbance of reaction supernatant at 540 nm with a spectrophotometer. Cellulase activity was calculated using the following equation by determining the amount (mg) of reducing sugar released, using the pre- establish calibration with pure glucose solutions of different concentrations as standards.

glucose released (푚𝑔) 1 푚푚표푙 1000 휇푚표푙 Cellulase (퐹푃푈/푚퐿) = × × (60 푚𝑖푛 )(0.1 푚퐿 enzyme sample) 180 푚𝑔 1 푚푚표푙

= 0.925 × glucose released (푚𝑔).

184

The method reported by Bailey et al. [98] was adopted for the xylanase assay.

µL test sample and 900 µL xylan substrate mixture to a 25 mL test tube. (3) Prepare the

DNS analysis was then done to determine the amount (mg) of reducing sugar released, using D-xylose solutions as standards. The xylanase activity was calculated using the following equation:

푈 xylose released (푚𝑔) 1 푚푚표푙 1000 휇푚표푙 Xylanase ( ) = × × 푚퐿 (5 푚𝑖푛 )(0.1 푚퐿 enzyme sample) 150.13 푚𝑔 1 푚푚표푙

= 13.32 × xylose released (푚𝑔).

The pectinase methods were established in this laboratory [99]. The procedure was however similar to that described above for the xylanase assay, with four differences.

First, the substrate solution/suspension was prepared by mixing 0.5 g citrus pectin (Sigma

Aldrich, St. Louis, MO) in 100 mL 0.1 M sodium citrate buffer and then adjusting the pH to 4.8. Second, the samples and blanks were incubated at 50C for 30 min (instead of 5

185 min as in the xylanase assay). Third, the DNS solution used did not contain sodium- potassium tartrate to prevent precipitation of residual substrate. Fourth, the calibration for determining the released amount of reaction product(s) is made with standard solutions of

D-galacturonic acid (monohydrate). The activity was calculated according to the following equation:

푈 Pectinase ( ) = 1.57 × galacturonic acid released (푚𝑔) 푚퐿

The sucrase assay used in this study was modified from a method reported by

Uma et al. [100]. The method was best for samples with sucrase activities in the range of

0.2 - 2.0 U/mL. The procedure was very similar to that for the pectinase assay, with the following differences: (1) sucrose was used for preparing the substrate solution; (2) the enzyme reaction at 50C was allowed for 20 min, and (3) the regular (tartrate-containing)

DNS solution was used. Glucose standards were used for DNS analysis calibration. The sucrase activity was calculated as:

푈 Sucrase ( ) = 2.78 × glucose released (푚𝑔). 푚퐿

The -galactosidase activity measurement was by a method modified from

M sodium carbonate (pH 9.8) to each sample, and blank to stop the reaction and develop the color from released p-nitrophenol; (6) add 100 µL test sample to the blank; and (7) measure the absorbance at 405 nm. Calibration established with pure p-nitrophenol standards was used for quantitation of the enzyme-released p-nitrophenol. The - galactosidase activity was calculated by the following equation:

푈 α − Galactosidase ( ) = 7.19 × 푝 − nitrophenol released (푚𝑔) 푚퐿

9.3 Results and discussion

9.3.1 Comparison of enzyme production at different fermentation condition

Figure 9.1(a) shows the pectinase activities of all the fermentation runs at the end of fermentation. Lowest pectinase activity was found in F1 where 2x C and 2x N was used with starting pH 6, and the activity was 7.6 ± 0.3 U/mL. All other fermentation gave

9% - 152% improvement in pectinase production compared to F1. Fed-batch process improved the pectinase activity by 34.3% (3x C) and 58.1% (5x C) respectively while the highest improvement was found in F8 and F9 (117.9% in F8 and 151.5% in F9 and both with 5x C) which were run with pH gradient of 0.0292 and 0.0357 pH drop per h respectively. Highest pectinase activity was 19.1 ± 0.4 U/mL. Highest α-galactosidase and cellulase activities were also found in F9, and the corresponding activities are 15.7 ±

0.4 U/mL and 0.88 ± 0.06 FPU/mL respectively. Slower pH drop was not found effective as others for improved enzyme production. Significant improvement in cellulase activities was observed when the pH drop was 0.0292 – 0.0357 per h. The more insight

187 into the experimental results at different experimental conditions is described in the following sections.

25 (a) Pectinase 20

5 Final Activity Activity (U/mL)Final

0 F1 F2 F3 F4 F5 F6 F7 F8 F9 20 (b) α-Galactosidase 15

5 Final Activity (U/mL) 0 F1 F2 F3 F4 F5 F6 F7 F8 F9

1.0 (c) Cellulase

0.5 Final Activity Activity (FPU/mL)Final 0.0 F1 F2 F3 F4 F5 F6 F7 F8 F9

Figure 9.1 Comparison of pectinase, α-galactosidase and cellulase production in different fermentations; F1 = 2x C,2x N, pH 6; F2 = 2x C, 2x N, pH 7; F3 = 5x C, 2x N, pH 6; F4

= 5x C, 5x N, pH 7; F5 = 3x C, 2x N, pH 6, Fed batch; F6 = 5x C, 2x N, pH 6, Fed batch;

F7 = 5x C, 2x N, pH gradient = 0.0156 pH drop per h; F8 = 5x C, 2x N, pH gradient =

0.0292 pH drop per h; and F9 = 5x C, 2x N, pH gradient = 0.0357 pH drop per h.

188

9.3.2 Effect of solid loading

Effect of different solid loading on enzyme production was studied at two pH level. Figure 9.2 shows the enzyme production profile at different solid loading; F1 and

F3 were done at starting pH 6 with 2x C – 2x N and 5x C – 2x N respectively, where F2 and F4 were done at starting pH 6 with 2x C – 2x N and 5x C – 5x N respectively.

Maximum cellulase, pectinase and α-galactosidase production in F3 in higher compared to F1 (cellulase, 0.55 FPU/mL; pectinase, 11.4 U/mL and α-galactosidase, 11.4 U/mL in

F3 compared to cellulase, 0.44 FPU/mL; pectinase, 7.6 U/mL and α-galactosidase, 7.8

U/mL in F1). Pectinase productivity is higher in earlier stages of fermentation (0.063 –

0.067 U/mL-h for F1 and F2 at 24) compared to other enzymes (0.006 – 0.01 U/mL-h for

α-galactosidase and up to 0.0008 FPU/mL-h for cellulase). The highest rate of pectinase and α-galactosidase production occurred at 48 h for F1 and F2 (0.11 – 0.13 U/mL-h for pectinase, 0.15 – 0.16 U/mL-h for α-galactosidase) whereas, highest cellulase production

(~0.005 FPU/mL-h) occurred in later stages (at 72 h) of fermentation. All the enzyme production depends on the consumption of available sugar hydrolyzed from soybean hull.

Since pectin is in outside of soybean carbohydrate, hydrolysis of pectin occurred initially and that is why the increase of pectinase production was observed earlier than other enzymes. α-Galactosidase production occurred with the consumption of available oligomeric sugars present in the fermentation system. Moreover, at the later stage, cellulase production increased with the consumption of monomeric sugar in soybean hull.

There was some lag in pectinase production observed in F3 and F4 (also the pH decreased slower than F1). Although in F3, there was a higher amount of soybean hull

(100 g/L) than F1 (40 g/L), however, the available enzyme/soybean hull is lower with

189 poorer mixing in F3 made the hydrolysis of soybean hull slower. Improved enzyme production wasn’t found in F4 compared to F2, which is probably higher nitrogen availability of nitrogen (5x N) from protease peptone, which means the enzyme production depends on the growth of A. niger from the hydrolysis of soybean hull, jot from other available carbon or nitrogen source to support their growth. Pectinase production in F2 and F4 decreased in the later stage of fermentation which showed degradation of pectinase either by protease enzyme or instability of pectinase at lower pH. Total volumetric productivity of pectinase in F1, F2 F3 and F4 are 0.059, 0.075,

0.057 and 0.062 U/mL-h respectively, while for α-galactosidase, 0.065, 0.059, 0.066 and

0.044 U/mL-h respectively and ~ 0.003 FPU/mL-h for cellulase in all fermentations.

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12 1.0 12 1.0 Pectinase Pectinase α-galactosidase α-galactosidase 10 10 pH Sucrase 0.8 0.8 Cellulase pH 8 8 0.6 0.6 6 6 0.4 0.4

4 4

pH/Activity (U/mL)pH/Activity (FPU/mL)Cellulase

pH/Activity (U/mL) pH/Activity Cellulase (FPU/mL)Cellulase

0.2 0.2 2 2

0 0.0 0 0.0 0 24 48 72 96 120 144 0 24 48 72 96 120 144 F1 F2 Time (h) Time (h)

12 1.0 12 1.0 Pectinase Pectinase α-galactosidase α-galactosidase 10 Sucrase 10 Sucrase pH 0.8 0.8 Cellulase pH Cellulase 8 8 0.6 0.6 6 6 0.4 0.4

4 pH/Activity (U/mL) pH/Activity

Cellulase (FPU/mL)Cellulase 4

pH/Activity (U/mL)pH/Activity (FPU/mL)Cellulase

0.2 0.2 2 2

0 0.0 0 0.0 0 24 48 72 96 120 144 168 0 24 48 72 96 120 144 F3 Time (h) F4 Time (h)

Figure 9.2 Effect of solid loading in enzyme production; F1 = 2x C, 2x N, pH 6; F2 = 2x

C, 2x N, pH 7; F3 = 5x C, 2x N, pH 6; and F4 = 5x C, 5x N, pH 7.

9.3.3 Effect of fed-batch addition

In all the previous fermentations, the rate of pectinase production started decreasing after 72 h and in later stages either pectinase production stopped or decreased.

Substrate coming from soybean hull hydrolysis which is responsible for inducing

191 pectinase production might get depleted. Therefore, fed-batch addition of soybean hull was studied at same starting pH to increase pectinase production. Figure 9.3 shows the effect fed-batch addition of soybean hull on pectinase production and the corresponding other enzyme production profile. The clear improvement in continual pectinase production was found in F5 in Figure 9.3 compared to F1 in Figure 9.2. Bothe the fermentation was done at same starting pH (6), same initial soybean hull loading (2x) and same initial nitrogen content (2x). The difference was the fed-batch soybean hull addition

(another 1x at 72 h). In F1 (Figure 9.2), pectinase productivity decreased from 0.116

U/mL-h at 48 h to 0.086 U/mL-h at 72 h and then 0.025 U/mL-h at 96 h; while in F5

(Figure 9.3), pectinase productivity was 0.126 U/mL-h at 48 h, then decreased to 0.064

U/mL-h at 72 h, but again increased to 0.106 U/mL-h at 96 h. In F6, similar continual production of pectinase occurred after each time of soybean hull addition, however, the rate is not as same as F5 (0.070 – 0.047 U/mL-h), probably due to the mixing limitation at higher solid loading at later stages. Final pectinase activities in F5 and F6 were 10.2 ±

0.2 U/mL and 12.0 ± 0.4 U/mL respectively with total volumetric productivity of 0.080

U/mL-h and 0.060 U/mL-h respectively compared to 7.6 ± 0.3 U/mL with 0.059 U/mL-h total productivity. Cellulase production also increased by very little amount with fed- batch soybean hull addition. Cellulase activity in F5 and F6 was 0.52 ± 0.02 FPU/mL and

0.58 ± 0.01 U/mL respectively compared to 0.44 ± 0.01 FPU/mL. Fed-batch soybean hull addition had no or minimal impact on α-galactosidase production.

192

12 1.0 14 1.0 Pectinase Pectinase α-galactosidase α-galactosidase 12 10 pH Sucrase 0.8 0.8 Cellulase pH 10 Cellulase 8 0.6 0.6 8 6 6

0.4 0.4 pH/Activity (U/mL)pH/Activity

4 (FPU/mL) Cellulase

pH/Activity (U/mL) Cellulase Cellulase (FPU/mL) 4 0.2 0.2 2 2

0 0.0 0 0.0 0 24 48 72 96 120 144 0 24 48 72 96 120 144 168 192 F5 Time (h) F6 Time (h)

Figure 9.3 Effect of fed-batch soybean hull addition in enzyme production; F5 = 3x C, 2x

N, pH 6, Fed batch (2x C initially + 1x C at 72 h); and F6 = 5x C, 2x N, pH 6, Fed batch

(2x C initially + 1x C at 48 h + 1x C at 72 h + 1x C at 96 h).

9.3.4 Effect of pH gradient

In all the previous fermentation, pH was allowed to drop naturally during the fermentation. Cell growth and pectinase production were found better at higher pH, and other enzymes production was found higher in relatively lower pH (probably around the optimum hydrolysis pH 4.8). Effect of different rate of pH drop was studied to maximize the enzyme production of all the enzymes during fermentation. Figure 9.4 shows the effect of pH gradient in enzyme production; 0.0156 pH drop/h (96 h) for F7, 0.0292 pH drop/h (96 h) for F8 and 0.0357 pH drop/h (84 h) for F9. In all the fermentation, 5x (100 g/L) soybean hull and 2x N were added in the batch process except 3x N was added in

F8. Enzyme production for all the enzyme increased with the increase of pH drop per h.

193

Highest enzyme production occurred in F9 in which pH gradient was 0.0357 pH drop/h and highest enzyme activities are – pectinase, 19.1 ± 0.04 U/mL; α-galactosidase, 15.7 ±

0.4 U/mL; cellulase, 0.88 ± 0.06 FPU/mL and sucrase, 5.8 ± 0.1 U/mL.

24 1.0 24 1.0 Pectinase Pectinase α-galactosidase α-galactosidase 20 Sucrase 20 Sucrase pH 0.8 0.8 pH Cellulase 16 16 0.6 0.6

12 12

0.4 0.4

pH/Activity (U/mL)pH/Activity (FPU/mL)Cellulase pH/Activity (U/mL) pH/Activity 8 (FPU/mL)Cellulase 8

0.2 0.2 4 4

0 0.0 0 0.0 0 24 48 72 96 120 144 F7 0 24 48 72 96 120 144 F8 Time (h) Time (h)

24 1.0 Pectinase α-galactosidase 20 Sucrase 0.8 pH 16 Cellulase 0.6 12 0.4

pH/Activity (U/mL)pH/Activity Cellulase (FPU/mL)Cellulase

0.2 4

0 0.0 0 24 48 72 96 120 144 F9 Time (h)

Figure 9.4 Effect of pH gradient in enzyme production; F7 = 5x C, 2x N, pH gradient =

0.0156 pH drop per h; F8 = 5x C, 2x N, pH gradient = 0.0292 pH drop per h; and F9 = 5x

C, 2x N, pH gradient = 0.0357 pH drop per h.

194

9.3.5 Sucrase production

Figure 9.5 shows the sucrase production at the end of fermentation. Among all the fermentation, highest improvement (~90%) compared to F1 (with 2x C and 2x N) has been found in F4 fermentation which is batch fermentation with highest starting soybean hull, and highest nitrogen (5x for both) and the highest activity was 10.1 ± 0.1 U/mL. The lowest sucrase activity was found in F3 which was run in batch process with 5x C, and 2x

N and the final activity was 2.4 ± 0.1 U/mL.

Sucrase 12 10 8 6 4

Final Activity Activity (U/mL)Final 2 0 F1 F3 F4 F6 F7 F8 F9

Figure 9.5. Comparison of sucrase production in different fermentation

9.4 Conclusion

Different factors have been evaluated in this study to increase the enzyme production in A. niger fermentation. With 40 g/L (2x) soybean hull and at starting pH 6, the enzyme activities were – pectinase, 7.6 ± 0.3 U/mL; α-galactosidase, 7.9 ± 0.2 U/mL; cellulase, 0.44 ± 0.01 FPU/mL and 5.3 ± 0.1 U/mL. Effect of higher loading of soybean hull was studied from 40 (2x C) – 100 (5x C) g/L at two pH level (pH 6 and 7) and was found to have improved enzyme production. Highest sucrase activities (90%

195 improvement and the activity was 10.1 ± 0.1 U/mL) was found in highest soybean hull

(5x) and nitrogen addition (5x) systems. Pectinase, α-galactosidase and cellulase activities increased by 35%, 45%, and 26%. However, the increase of enzyme production was not proportional to substrate concentration. Effect of fed-batch soybean hull addition was also studied with total 3x (60 g/L) and 5x (100 g/L) soybean hull addition to improving pectinase production. Pectinase productivity and final activity (12.1 ± 0.4

U/mL) increased in the fed batch process. To better utilize the different pH dependency of the different enzyme production, three different pH gradient profiles were studied

(0.0156, 0.0292 and 0.0357 pH drop per h at 5x C soybean hull loading and starting pH

7). All the enzyme production, particularly, pectinase, α-galactosidase, and cellulase increased significantly with the increase of pH drop rate. Highest pectinase, α- galactosidase and cellulase activities were found in 0.0357 pH drop per h and activities were pectinase, 19.1 ± 0.04 U/mL; α-galactosidase, 15.7 ± 0.4 U/mL; cellulase, 0.88 ±

0.06 FPU/mL. In this current study, very high enzyme activities have been achieved compared to the previous studies which will make this process very potential to use in soybean meal as well as other lignocellulosic biomass processing to produce high monomerized fermentation feedstock for valuable fermentation-based product formation.

196

CHAPTER X

CONCLUSIONS AND RECOMMENDATIONS

10.1 Conclusions

In this study, we investigated in improving process parameters for different aspects of biorefinery, i.e., pretreatment, enzyme hydrolysis and enzyme production for different types of biomass. The conclusions obtained in this research projects are described in the following sections.

1. The CO2-H2O based pretreatment was proven to be effective for minimizing fermentation inhibitor generation while enabling high sugar conversion in subsequent enzyme hydrolysis. Furfural and HMF generation and, correspondingly, sugar loss/degradation were particularly lower than those by other pretreatment methods.

Pretreatment pressure had less effect on inhibitor generation than the pretreatment temperature and duration. Kluyveromyces marxianus fermentation showed complete growth and ethanol production inhibition at ≥ 14 g/L combined inhibitors. Among the conditions examined, 180 °C, 1800 psi, and 30 min was considered optimal, giving a high reducing sugar conversion (82.8 ± 2.8% reducing sugar, 74.8 ± 4.8% cellulose and

88.5 ± 6.9% hemicellulose) and low inhibitor formation: 1.8 mg HMF, 6.3 mg furfural and 76 mg acetic acid, per g reducing sugar released.

197

2. Hydrolysis of unpretreated hulls using A. niger and T. reesei broths showed that glucose and xylose release almost linearly correlated with the cellulase and xylanase loadings (i.e., U/g hull); mannose was easily hydrolyzed by enzyme to almost 90% conversion even at relatively low enzyme loadings; the enzyme broth only slightly improved galactose release (from ~18% conversion in enzyme-free control to ~30% with enzyme); and arabinose release was almost undetectable (~5% conversion). The CO2-

H2O based pretreatment significantly improved the enzymatic hydrolysis outcomes. The study of pretreatment pressure and temperature effects on sugar release and inhibitory degradation product formation concluded that the optimal condition is at or near 1250 psi and 130°C (for the fixed moisture content – 2 mL water per g hulls, and pretreatment time – 30 min). Compared to the hydrolysis of unpretreated hulls with 2.5-fold more enzyme, the pretreatment improved glucose, xylose, galactose, arabinose and mannose release by 55%, 35%, 105%, 683% and 52%, respectively. Combined inhibitor (acetic acid, HMF, and furfural) formation by this pretreatment was at least one order of magnitude lower than that by a report on dilute acid pretreatment of soybean hulls.

3. Also, the vessel cost analysis shows that the vessel for the CO2-H2O based pretreatment at 750 psi is even significantly cheaper than the dilute acid pretreatment vessel, the latter requires more expensive corrosion-resistant material for construction.

198 cheaper reactors and make this pretreatment method not only effective but also economical and practical.

4. Among soybean carbohydrates, pectin and glucan are more recalcitrant to hydrolyze. Under mild heat pretreatment, additions of chelators EDTA, HMP, and citric acid gave mixed effects on enzymatic hydrolysis: citric acid was the most positive while

EDTA was negative possibly due to enzyme interferences. Heat (90 °C, 2 h) and citric acid (10 g/L) pretreatments and cellulase supplementation (10 FPU/g) all increased reducing sugar and monosaccharide yields. Together, they increased reducing sugar yield from 62.3% to 87.1% and yielded 86.8 ± 5.2% glucose, 98.1 ± 1.6% xylose, 87.5 ± 5.2% galactose, 83.6 ± 1.6% arabinose, and 91.4 ± 3.1% (fructose+mannose).

5. Kinetic modeling of enzyme hydrolysis of soybean meal shows the pectin hydrolysis dependency of cellulose hydrolysis by cellulase enzyme. Pectinase and α- galactosidase enzymes were found to be limited by our fungal fermentation to completely monomerization of carbohydrates by only enzyme hydrolysis. The enzyme stability and/or substrate stability is lower for α-galactosidase and sucrase enzyme.

6. The proteinaceous loss occurred during enzymatic processing of soy flour (SF).

Here, effects of broth ionic strength (IS) and protease activity and SF toasting were examined and their individual contributions to proteinaceous release quantitated.

Increasing IS (up to 0.19 M) linearly increased proteinaceous release, which was however mostly recoverable in heat-precipitated SPI and responsible for negligible final loss (0.4-

0.7%) in typical enzyme-processing conditions. Protease effect was stronger, most apparently on β-conglycinin α’/α, and glycinin 37-kDa subunits. Majority of protease- released peptides were also recovered as SPI, leaving ~3% additional loss in typical

199 processing. For untoasted SF, the loss primarily (~20%) occurred in water without IS or protease effect. Toasting was very effective for reducing proteinaceous release, down to

5.2% by 2-h dry-heat treatment at 160°C. Results are useful to improve enzyme- processing performance and soybean value.

7. Different factors have been evaluated for increasing carbohydrase enzyme production by A. niger fermentation, i.e. higher substrate loading, fed-batch substrate addition, and pH gradient/profile. Highest sucrase activities (90% improvement and the activity was 10.1 ± 0.1 U/mL) was found in highest soybean hull (5x) and nitrogen addition (5x) systems. Pectinase productivity and final activity (12.1 ± 0.4 U/mL) increased in the fed bath process. All the enzyme production, particularly, pectinase, α- galactosidase, and cellulase increased significantly with the increase of pH drop rate.

Highest pectinase, α-galactosidase and cellulase activities were found in 0.0357 pH drop per h and activities were pectinase, 19.1 ± 0.04 U/mL; α-galactosidase, 15.7 ± 0.4 U/mL; cellulase, 0.88 ± 0.06 FPU/mL.

In this study, we investigated different aspects of biorefinery i.e. pretreatment, enzyme hydrolysis and enzyme production for different types of biomass. The summary of the whole research work is presented in Figure 10.1. This work will allow us to differentiate the requirement of the severity of pretreatment types and condition and enzyme composition regarding biomass composition. The work to model the enzyme hydrolysis of soybean meal and very high enzyme production along with the reduction of protein loss could be major steps forward for soybean bioprocessing.

200

Figure 10.1 Summary of the research

10.2 Recommendations

In this current study, we have shown the effectiveness of A. niger fermentation based enzyme for hydrolyzing soybean meal and soybean hull. The future recommendations of the developed process are summarized in Figure 10.2. In soy processing industries, other significant by-products are soy molasses and soy okara.

While soybean okara contains mainly cellulose and pectin, soy molasses has mainly sucrose and galacto-oligosaccharides. Optimal enzyme for hydrolysis of these specific streams of mixed substrates and it's effective, economic production is essential. We have developed the process for very high pectinase and α-galactosidase production, hence this enzyme can be used for effective soy okara and molasses hydrolysis.

201

On a dry basis, molasses has no insoluble polymeric carbohydrate and much more oligosaccharides, particularly stachyose and sucrose. It may require a different optimal enzyme. The enzyme production process can be modified to specifically heighten the activities of sucrase and -galactosidase. Using molasses as the inducing substrate can improve these specific enzyme productions. Also, because molasses is a liquid that can be easily pumped for controlled addition (unlike the solid substrate), enzyme titers can be raised a few folds effectively by maintaining inducers at optimal concentration ranges, i.e., high enough for potent induction but below thresholds for feed-back repression. This will cut down the enzyme cost significantly.

The enzyme production and enzyme hydrolysis in this work has the huge potential to use in soy protein nanoparticles production for rubber filler application too. Petroleum- based carbon black (CB) is the most commonly used filler in elastomer compounds. For sustainability, biofillers are under active development, with cost and performance as the critical issues to address. Soy protein (SP) biofiller can be developed with simple enzyme processing and knowledge of soy biomass structure and composition. Soybeans comprise cylindrical (30 μm x 70 μm) cells enclosed in the cell wall of complex poly-carbohydrate

(cellulose, hemicellulose, and pectin; 30% of bean weight) and filled inside with protein bodies (50%, 2-10 μm) and oil bodies (20%, 0.2-0.5 μm). In our lab, we have developed simple enzyme processing to separately collect the oil and protein bodies freed from cell wall confinement by the enzymes. SP has the scale and price to support meaningful filler development. Compared to CB (density: 1.8-2.1 g/cm3), SP (1.3 g/cm3) is much lighter, an important advantage for many applications. Current commercial SPI is very expensive because the processing, designed for oil production, causes SP denaturation and some

202 crosslinking. The protein bodies from enzyme processing, on the other hand, are weakly membrane-enclosed aggregates of 2 main globular proteins of 6-10 nm: β-conglycinin

(MW ~ 150 kDa) and glycinin (MW ~ 360 kDa). Similarly, CB is aggregates of 10-30 nm particles. Enzyme processing allows collection of all soybean components (oil, protein and hydrolyzed carbohydrate) for value-added uses. This can further improve the economics of protein filler co-production.

The knowledge of soy processing in this project can also be used for nanocellulose production from soybean hull. Soy hull has very high carbohydrate content

(70-80%) and 40-50% cellulose content. Removing other carbohydrate content will yield nanocellulose which will be ideal for filler application because of its high aspect ratio, high tensile strength and Young modulus and lower density. Nanocellulose can be produced from the enzymatic process. Aspergillus produces higher pectinase, xylanase and α-galactosidase activities and less cellulase activity which has the capability of removing hemicellulose, pectin content and retaining cellulose content of the soy hull.

Solubilized sugar can be used as feedstock for other fermentation-based product formation. Nanocellulose can also be produced directly from A. niger fermentation and enzyme can be coproduced by this process. Enzyme processed nanocellulose from soy hull will be more environment friendly, biodegradable, will minimize or diminishes the use of petroleum-based sources.

203

Figure 10.2 Future recommendations of the developed process.

204

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