N-Butanol Fermentation and Integrated Recovery Process: Adsorption, Gas

Stripping and Pervaporation

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Fangfang Liu, B.S.

Graduate Program in Chemical Engineering

The Ohio State University

2014

Dissertation Committee:

Professor Shang-Tian Yang, Advisor

Professor Aravind Asthagiri

Professor David Wood

Fangfang Liu

2014

Abstract

As a second generation biofuel, butanol has attracted increasing attention during the last decade. Biobutanol can be produced through traditional ABE fermentation.

However, fermentative butanol production is not yet economically competitive with petrochemical process, mainly due to high substrate cost, low product yield and concentration and high recovery cost. Many efforts have been made to improve fermentative butanol production.

Typical batch ABE fermentation usually gives a final butanol titer of 12-14 g/L.

Butanol recovery from this dilute solution by distillation is very energy-intensive. Many alternative separation techniques have been developed. Among them, adsorption is a promising technique for its simple operation. In order to selectively recover butanol and release the product inhibition effect, four commercial materials were identified as potential adsorbents for butanol separation. These four adsorbents, including activated carbon Norit ROW 0.8, zeolite CBV901, polymeric resin Dowex Optipore L-493 and

SD-2, showed high specific loading and adsorbent-aqueous partitioning coefficients for butanol. Adsorption isotherms and their regressions with Langmiur model were further studied for these adsorbents, which provided the theoretical basis for predicting the amount of butanol adsorbed on these adsorbents. In batch fermentation with in situ

adsorption without pH control, activated carbon showed the best performance with 21.9 g/L total butanol production, and 71.3 g/L glucose consumption. The total butanol production with activated carbon increased by 87.2%, 51.0%, 44.1% and 90.4%, respectively, compared to the control (without adsorbent), L-493, SD-2 and CBV901.

The integration of adsorption by activated carbon, with both free and immobilized cell fermentation, was demonstrated to be successful. The control free cell fermentation produced 18.3 g/L butanol in 54 h with a butanol productivity of 0.34 g/L·h, while free cell fermentation with adsorption produced >31.6 g/L butanol in 106 h with a butanol productivity of >0.30 g/L·h, offering a >70% increase in butanol titer. The control immobilized cell fermentation produced 16.4 g/L butanol in 47 h with a butanol productivity of 0.35 g/L·h, while immobilized cell fermentation with adsorption produced

~54.6 g/L butanol in 122 h with a butanol productivity of ~0.45 g/L·h, an increase of ~30% and ~200% in butanol productivity and butanol titer, respectively, compared to the control experiments. Furthermore, ~150 g/L of butanol in the condensate could be recovered from desorption of adsorbents, which was easily concentrated to ~640 g/L after simple and naturally occurring phase separation. Therefore, based on the estimation on energy consumption of other separation technology (typically >10 kJ/g), our highly- designed in situ product recovery (ISPR) process with activated carbon only required

~4.8 kJ/g butanol, with greater energy saving, showing its potential economical value for product recovery and integration with butanol fermentation to simultaneously remove inhibitory products.

iii

In order to reduce the substrate cost, butanol has been produced from lignocellulosic biomass and thus called second generation biofuel. There are many different lignocellulosic biomass that can be explored for this purpose. Efforts have also been devoted to improve butanol production. Besides metabolic engineering, butanol production can be boosted by external driving forces which can redirect the electron and carbon flow towards butanol synthesis. In this work, engineered mutant strain

Clostridium tyrobutyricum overexpressing adhE2 and ack knock out CtΔack-adhE2 was used. When provided with external driving forces, butanol production with high yields

(>0.30 g/g) was achieved in bioreactor. Fed-batch butanol fermentation from different carbon sources in a fibrous-bed bioreactor integrated with gas stripping was studied.

Final butanol titer, yield and productivity of 12.1 g/L, 0.25 g/g and 0.12 g/L∙h, respectively, were obtained from soybean hull hydrolysate (SHH). A glucose and xylose mixture mimicking sugar composition in SHH was also used to produce butanol, which resulted in a total butanol production of 24.7 g/L. This work was the first study to produce butanol from soybean hull hydrolysate integrated with gas stripping. This study demonstrated the feasibility of butanol fermentation from soybean hull and sugarcane bagasse hydrolysate integrated with butanol recovery by gas stripping.

Besides previously mentioned adsorption and gas stripping, pervaporation is also an effective way for butanol recovery from dilute solutions with high selectivity. In this work, high performance polydimethylsiloxane (PDMS) membranes and zeolite filled

PDMS mixed matrix membranes (MMMs) were developed to recover butanol from model solutions. The effects of membrane filler zeolite, feed butanol concentration, and

operating (feed) temperature on pervaporation performance of PDMS membranes and

PDMS MMMs was studied. With the feed solution of 1.5 wt% butanol at 47°C, the

PDMS MMM filled with 40 wt% zeolite was found to have the highest butanol separation factor of 77 with a butanol and total flux of 62 and 118 g/m2·h, respectively.

For both PDMS membranes and PDMS MMMs, the separation factor can be further increased by elevating operating temperature, and permeation fluxes can be further boosted by reducing membrane thickness, increasing feed butanol concentration, and/or elevating operating temperature. The apparent activation energies of butanol permeation in PDMS membrane and zeolite filled (40 wt%) PDMS MMM were evaluated and found to be 34.3 and 44.2 kJ/mol, respectively. Compared to other studies reported in the literature, this work demonstrated higher butanol separation performance by pervaporation and showed huge energy saving compared to traditional distillation.

This project demonstrated efficient butanol recovery by adsorption, gas stripping and pervaporation from both model solution and fermentation broth. In situ product recovery largely improved fermentative butanol production.

Dedication

Dedicated to my parents and sisters

Acknowledgements

First of all, I would like to give my sincere thanks to my advisor, Dr. Shang-Tian

Yang, without whom I could have achieved nothing in the past four years. Before I joined

Dr. Yang’s research group, I was just an ignorant person with a Bachelor’s Degree. I am eternally thankful and grateful for Dr. Yang’s guidance, encouragement and full support throughout my Ph.D. study. Dr. Yang is nice and easy-going with admirable personality.

I have benefited tremendously from him, both in academia and in life.

I would also like to thank Dr. David Wood and Dr. Aravind Asthagiri for taking out time to be on my committee, as well as their valuable suggestions and recommendations on my research project.

I am very thankful for Dr. Chuang Xue for his guidance at the beginning of my

Ph.D. study. He taught me all the hand-on techniques to operate anaerobic fermentation and different butanol recovery processes. I would also like to thank all the previous and current group members, especially Dr. Jingbo Zhao, Dr. Congcong Lu, Dr. Jianxin Sun,

Dr. Wei-Lun Chang, Dr. Mingrui Yu, Dr. Ru Zang, Dr. Kun Zhang, Dr. Yipin Zhou, Dr.

Chih-Chin Chen, Dr. Ying Jin, Dr. Zhongqiang Wang, Dr. Meimei Liu, Dr. Ehab Ammar,

Dr. Yinming Du, Jie Dong and Wenyan Jiang.

vii

Financial support from ARPA-E Electrobiofuel Program for this research project is greatly appreciated.

Finally, I would like to thank my parents Mr. Linhan Liu and Mrs. Songdan Wang and sisters for their unconditional love and support.

viii

Vita

June 2006………………………………………………………………Jinyun Senior High

2006-2010……………………………………………………B.S. Chemical Engineering,

Zhejiang University

2010-2014…………………………………………………Graduate Research Associate,

The Ohio State University

Publications

Li ZP, Liu BH, Liu FF, and Xu D. 2011. A composite of borohydride and super

absorbent polymer for hydrogen generation. J Power Sources 196(8):3863-3867.

Xue C, Zhao J, Liu F, Lu C, Yang ST, and Bai FW. 2013. Two-stage in situ gas stripping

for enhanced butanol fermentation and energy-saving product recovery. Bioresour

Technol 135:396-402.

Fields of Study

Major Field: Chemical Engineering

Table of Contents

Abstract ...... ii

Dedication ...... vi

Acknowledgements ...... vii

Vita ...... ix

Table of Contents ...... x

List of Tables ...... xvi

List of Figures ...... xix

Chapter 1: Introduction ...... 1

1.1 Research objective and tasks ...... 4

1.2 References ...... 6

1.3 Figures ...... 9

Chapter 2: Literature Review ...... 11

2.1 N-butanol properties, applications and production ...... 11

2.2 Butanol production from fermentation ...... 12

2.2.1 Acetone-Butanol-Ethanol (ABE) fermentation ...... 12

2.2.2 Strain development and metabolic engineering for improved ABE

fermentation ...... 15

2.2.3 Butanol production from sugars and lignocellulosic biomass ...... 18

2.2.4 External driving forces for butanol production ...... 20

2.3 Advanced butanol recovery techniques ...... 21

2.3.1 Adsorption...... 23

2.3.2 Gas stripping ...... 26

2.3.3 Pervaporation ...... 29

2.3.4 Other separation techniques ...... 32

2.4 References ...... 33

2.5 Tables and figures ...... 47

Chapter 3: Butanol Production in Fed-batch Fermentation with In Situ Product

Recovery by Adsorption ...... 67

3.1 Introduction ...... 68

3.2 Materials and methods ...... 71

3.2.1 Screening adsorbents for butanol adsorption ...... 71

3.2.2 Determination of adsorption isotherm ...... 72

3.2.3 Simulation and predictions ...... 72

3.2.4 Culture and medium ...... 73

3.2.5 Batch fermentation with in situ adsorption ...... 73

3.2.6 Adsorption of broth components on selected adsorbent ...... 74

3.2.7 Fed-batch fermentation with in situ adsorption with activated carbon 75

3.2.8 Desorption and product recovery ...... 76

3.2.9 Analytical methods ...... 77

3.3 Results and discussion ...... 77

3.3.1 Screening adsorbents for butanol adsorption ...... 77

3.3.2 Adsorption isotherm and prediction ...... 78

3.3.3 Batch fermentation with in situ adsorption in serum bottles ...... 79

3.3.4 Adsorption of broth components on selected adsorbent ...... 81

3.3.5 Fed-batch fermentation with adsorption by activated carbon ...... 82

3.3.6 Studies on desorption and butanol recovery ...... 85

3.3.7 Comparison to other studies...... 86

3.4 Conclusions ...... 90

3.5 References ...... 91

3.6 Tables and figures ...... 94

xii

Chapter 4: Fed-batch Butanol Fermentation by Engineered Clostridium tyrobutyricum with External Driving Forces in a Fibrous-bed Bioreactor Integrated with Gas Stripping

...... 106

4.1 Introduction ...... 107

4.2 Materials and methods ...... 109

4.2.1 Pretreatment and enzymatic hydrolysis of lignocellulosic biomass .. 109

4.2.2 Culture and medium ...... 110

4.2.3 Serum bottle fermentation ...... 111

4.2.4 Immobilized cell fermentation in a fibrous-bed bioreactor ...... 112

4.2.5 Fed-batch fermentation in a fibrous-bed bioreactor integrated with gas

stripping ...... 113

4.2.6 Analytical methods ...... 113

4.3 Results and discussion ...... 114

4.3.1 Serum bottle fermentation from glucose, xylose and soybean hull

hydrolysate ...... 114

4.3.2 Immobilized cell fermentation in a fibrous-bed bioreactor ...... 114

4.3.3 Fed-batch immobilized cell fermentation in a fibrous-bed bioreactor

integrated with gas stripping ...... 115

4.4 Conclusions ...... 117

4.5 References ...... 118

xiii

4.6 Tables and figures ...... 121

Chapter 5: High Performance PDMS (Mixed Matrix) Membrane for Butanol Recovery from Aqueous Solution by Pervaporation ...... 132

5.1 Introduction ...... 133

5.2 Experimental ...... 136

5.2.1 Materials ...... 136

5.2.2 Flat sheet membrane fabrication ...... 136

5.2.3 Experimental setup ...... 137

5.2.4 Analytical methods ...... 137

5.3 Results and discussion ...... 138

5.3.1 Membrane fabrication and characterization ...... 138

5.3.2 Effect of membrane filler on membrane separation ...... 138

5.3.3 Effect of feed concentration on membrane separation ...... 139

5.3.4 Effect of membrane thickness on membrane separation ...... 141

5.3.5 Effect of feed temperature on membrane separation ...... 142

5.3.6 Energy consumption analysis in pervaporation ...... 143

5.3.7 Membrane separation performance compared with literature ...... 145

5.4 Conclusions ...... 145

5.5 References ...... 146

xiv

5.6 Tables and figures ...... 149

Chapter 6: Conclusions and Recommendations ...... 160

6.1 Conclusions ...... 160

6.1.1 Butanol production integrated with adsorption...... 160

6.1.2 Butanol production from lignocellulosic biomass integrated with gas

stripping ...... 161

6.1.3 Butanol recovery by pervaporation using PDMS membranes ...... 162

6.2 Recommendations ...... 164

6.2.1 Butanol production integrated with adsorption...... 164

6.2.2 Butanol production from lignocellulosic biomass integrated with gas

stripping ...... 164

6.2.3 Butanol recovery by pervaporation ...... 165

6.3 References ...... 165

Bibliography ...... 166

List of Tables

Table 2.1 Properties of butanol and some other fuels ...... 48

Table 2.2 Butanol production from solventogenic Clostridia (substrates, pH, temperature

and products) ...... 49

Table 2.3 ABE fermentation by solventogenic clostridia from renewable biomass (AFEX,

ammonia fiber explosion; N/A, not available) ...... 51

Table 2.4 Comparison of advanced butanol separation techniques ...... 53

Table 2.5 Performances of different adsorbents for butanol recovery by adsorption (N/A,

not available) ...... 54

Table 2.6 Performance of different resins used for in situ butanol recovery coupled with

fermentation (a Calculated based on butanol; DVB, divinylbenzene; MV, methyl

viologen; N/A, not available) ...... 56

Table 2.7 Solvent selectivities of processes integrated with gas stripping for butanol

recovery (* L/min: liter gas per minute; L/(L∙min): liter gas per liter broth per

minute) ...... 57

Table 2.8 Integrated fermentation-gas stripping process for ABE production by Clostridia

from various substrates (* WPH, wood pulp hydrolysate) ...... 58

Table 2.9 Butanol separation performance of different membranes by pervaporation

(PDMS: polydimethylsiloxane; PAN: polyacrylonitrile; PMS: poly(methoxy xvi

siloxane); PTMSP: poly(1-trimethylsilyl-1-propyne); PEBA: poly(ether block

amide); ZIF: zeolitic imidazolate framework; EPDM: ethylene propylene diene

rubber; SBR: styrene butadiene rubber; TOA: trioctylamine; PVDF:

poly(vinylidene difluoride); PUR: polyurethane) ...... 59

Table 2.10 Integrated fermentation-pervaporation process for ABE production ...... 61

Table 2.11 Integrated fermentation-recovery process for ABE production (liquid-liquid

extraction and perstraction) ...... 62

Table 3.1 Comparison of n-butanol adsorption capacity of various adsorbents (Butanol

model solution was used except for the one by Maddox (1982)) ...... 95

Table 3.2 Langmuir parameters from least-squares regression ...... 96

Table 3.3 Comparison of n-butanol production in ABE fermentation with in situ butanol

adsorption by various adsorbents (a: Total butanol production estimated from the

final concentration in the fermentation broth and the adsorption isotherms) ...... 97

Table 3.4 Specific loading of components in fermentation broth (All the model solutions

initially contained 18.9 g/L glucose, 19.4 g/L acetone, 4.3 g/L ethanol, 38.7 g/L

butanol, 10.6 g/L acetic acid, and 4.0 g/L butyric acid) ...... 98

Table 3.5 Fermentation performance with in-situ butanol adsorption by activated carbon

(a: The first value is calculated directly based on the actual amount of butanol in

the broth and recovered from desorption, while the second value is based on a 80%

recovery rate as not all the butanol was recovered during desorption; b: Assuming

that butanol yield remained the same as the control fermentation) ...... 99

xvii

Table 3.6 Comparison of energy requirement for butanol recovery by different separation

methods ...... 100

Table 4.1 Composition of soybean hull and sugarcane bagasse hydrolysate (before rotary

evaporation) ...... 122

Table 4.2 Effect of different nitrogen and carbon sources on fermentation of mutant strain

CtΔack-adhE2 in serum bottles supplemented with 250 µM methyl viologen (MV)

(* YE, yeast extract) ...... 123

Table 4.3 Results of immobilized cell fermentation in a fibrous-bed bioreactor from

different carbon sources ...... 124

Table 5.1 Apparent activation energies (Ea) of butanol and water permeation in PDMS

membrane and zeolite filled (40 wt%) PDMS MMM (* MMM: mixed matrix

membrane) ...... 150

Table 5.2 Pervaporation performance of different membranes for butanol recovery from

aqueous solutions (PDMS: polydimethylsiloxane; PAN: polyacrylonitrile;

PTMSP: poly(1-trimethylsilyl-1-propyne); PEBA: poly(ether block amide); ZIF:

zeolitic imidazolate framework; EPDM: ethylene propylene diene rubber; SBR:

styrene butadiene rubber; TOA: trioctylamine; PVDF: poly(vinylidene difluoride);

PUR: polyurethane) ...... 151

xviii

List of Figures

Figure 1.1 Overview of research objective and tasks in this study ...... 10

Figure 2.1 Metabolic pathways in Clostridium acetobutylicum during both acidogenesis

(dotted arrows) and solventogenesis (solid arrows) ...... 63

Figure 2.2 Effect of feed butanol concentration on specific energy requirement for

butanol recovery by distillation ((XB)F: butanol mass fraction in the feed solution)

...... 64

Figure 2.3 Schematic diagram of alternative butanol recovery techniques. A. Adsorption,

B. Gas stripping, C. Pervaporation ...... 65

Figure 2.4 Mass transfer at a gas/liquid interface ...... 66

Figure 3.1 Bioreactor system with an external packed column for butanol adsorption . 101

Figure 3.2 Equilibrium isotherms of n-butanol with Norit ROW 0.8, CBV901, Dowex L-

493 and Dowex SD-2 A. at 37 °C; B. at 60 °C ...... 102

Figure 3.3 Kinetics of ABE fermentation of C. acetobutylicum JB200 at 37 oC, pH 5 A.

Batch free cell fermentation without adsorption (control); B. Fed-batch free cell

fermentation with adsorption by activated carbon; C. Repeated batch immobilized

cell fermentation without adsorption (control); D. Fed-batch immobilized cell

fermentation with adsorption by activated carbon ...... 103

xix

Figure 3.4 Desorption of n-butanol, water, and ABE mixture from activated carbon

determined thermogravimetrically ...... 105

Figure 4.1 Effect of different nitrogen and carbon sources on fermentation kinetics of

mutant strain CtΔack-adhE2 in serum bottles supplemented with 250 µM methyl

viologen (MV) (A: glucose; B: xylose; C: O.D.600; D: ethanol; E: butanol; F:

acetic acid; G: butyric acid) (Figure legend: 1, glucose + yeast extract + tryptone;

2, glucose + corn steep liquor; 3, xylose + yeast extract + tryptone; 4, xylose +

corn steep liquor; 5, soybean hull hydrolysate + yeast extract + tryptone; 6,

soybean hull hydrolysate + corn steep liquor) ...... 125

Figure 4.2 Fermentation kinetics of immobilized cell fermentation in a fibrous-bed

bioreactor from mixture of glucose and xylose supplemented with 250 µM methyl

viologen (MV) ...... 127

Figure 4.3 Fermentation kinetics of immobilized cell fermentation in a fibrous-bed

bioreactor from xylose supplemented with 250 µM methyl viologen (MV) ...... 128

Figure 4.4 Fed-batch immobilized cell fermentation in a fibrous-bed bioreactor integrated

with gas stripping from soybean hull hydrolysate (SHH) ...... 129

Figure 4.5 Fed-batch immobilized cell fermentation in a fibrous-bed bioreactor integrated

with gas stripping from sugarcane bagasse hydrolysate (SBH) ...... 130

Figure 4.6 Fed-batch immobilized cell fermentation in a fibrous-bed bioreactor integrated

with gas stripping from mixture of glucose and xylose mimicking soybean hull

hydrolysate (SHH) ...... 131

Figure 5.1 Pervaporation system used in this study ...... 153

Figure 5.2 Surface and cross-sectional scanning electron microscope (SEM) images of

PDMS membrane and zeolite filled PDMS MMM (A and B: surface of cross-

sectional image of PDMS membrane; C and D: surface and cross-sectional image

of zeolite filled PDMS MMM) ...... 154

Figure 5.3 Effect of ZSM-5 zeolite CBV28014 in PDMS membrane on butanol

separation by pervaporation (47 °C, 1.5 wt% butanol feed solution, ~100 µm in

thickness) ...... 155

Figure 5.4 Effect of feed butanol concentration on pervaporation performance of PDMS

membrane (47 °C, ~100 µm in thickness) ...... 156

Figure 5.5 Effect of membrane thickness on the performance of PDMS membranes: A,

butanol, water and total flux; B, butanol flux and separation factor (47 ºC, 1.5 wt%

butanol feed solution) ...... 157

Figure 5.6 Arrhenius plots of butanol and water fluxed for PDMS membrane and 40 wt%

zeolite filled PDMS MMM (y axis in log scale, 1.5 wt% butanol solution, ~100

µm in thickness) ...... 158

Figure 5.7 Effect of temperature on total flux and separation factor of PDMS membrane

and 40 wt% zeolite filled PDMS membranes (α represents separation factor, 1.5

wt% butanol solution, ~100 µm in thickness) ...... 159

xxi

Chapter 1: Introduction

Biofuel production is attracting more and more interests due to concerns on the eventual depletion of fossil fuels and global warming due to greenhouse gas emissions

(Durre 2007; Xue et al. 2013b). There are three generations of biofuels that are categorized by their source and type: first generation biofuel derived from seeds, grains or sugars; second generation (also referred to as advanced biofuel) derived from lignocellulosic biomass; third generation derived from algae or sea weeds (Nigam and

Singh 2011; Patil et al. 2008). The agricultural lignocellulosic biomass used for biofuel production is non-edible residues or non-edible whole plant biomass, which does not have issues about competing for food with animals (Naik et al. 2010; Nigam and Singh

2011). This renewable and sustainable feedstock has been targeted for future biofuel production (Ragauskas et al. 2006).

Currently, bioethanol and biodiesel are the two major biofuels. As an alternative fuel substitute, biobutanol is superior to ethanol in many aspects: higher energy content, lower volatility and lower water absorption (Durre 2007; Lee et al. 2008). Butanol has market estimated to be $247 billion by 2020 (Green 2011). Butanol can be produced through traditional acetone-butanol-ethanol (ABE) fermentation by solventogenic clostridia usually with a solvent ratio of A:B:E=3:6:1 (Jones and Woods 1986). ABE 1

fermentation once was the second most important large-scale industrial fermentation process during the first half of 20th century but gradually declined during 1950s due to more efficient and economical petrochemical process (Jones and Woods 1986). Recently, biological butanol production regained attention as a renewable and sustainable process.

However, fermentative butanol production still has several challenges that remained unsolved. Traditional acetone-butanol-ethanol (ABE) fermentation usually has high feedstock cost, low butanol titer, yield, and productivity, costly product recovery and high waste water (Green 2011; Gu et al. 2011). Tremendous efforts have been devoted to improve butanol fermentation performance to tackle with those issues.

Typical batch ABE fermentation produces 10-13 g/L butanol and 15-18 g/L ABE

(Durre 1998; Ezeji et al. 2004). The low butanol titer is caused by the product (butanol) inhibition on cells (Qureshi and Ezeji 2008). Mutagenesis and metabolic engineering have been used to develop strains with higher butanol tolerance, production and/or yield.

Dong reported two mutants Clostridium. acetobutylicum EA2018 with an improved butanol ratio of 70% (vs. 60%) among solvents and Rh9 with a butanol tolerance of 19 g/L (Dong 2012). Recently, a mutant strain C. acetobutylicum JB200 with high butanol production derived from C. acetobutylicum ATCC 55025 was obtained by spontaneous mutation in a fibrous-bed bioreactor (FBB) which can produce up to ~25 g/L butanol

(Zhao et al. 2009). Many mutant strains of C. acetobutylicum ATCC 824 have been engineered to produce more butanol/solvents (Huang et al. 2010; Papoutsakis 2008). For example, Jiang knocked out the acetoacetate decarboxylase gene (adc) and the mutant had an increased butanol/acetone ratio (Jiang et al. 2009). Besides native solventogenic

clostridia, metabolic engineering also made butanol production in nonnative hosts possible. Escherichia coli is a well studied microbe and has been engineered to produce butanol. Atsumi constructed a recombinant E. coli for butanol production but the titer was low (< 1 g/L) (Atsumi et al. 2008). Dellomonaco also engineered E. coli to produce butanol with a much higer titer of ~14 g/L (Dellomonaco et al. 2011). Shen also reported that ~30 g/L butanol was produced with high yield in engineered E. coli driven by external driving forces (Shen et al. 2011). Another successfully engineered strain was obtained from C. tyrobutyricum by Yu (Yu et al. 2012; Yu et al. 2011). Originally C. tyrobutyricum ATCC 25755 is a butyric acid producing bacterium and Yu achieved butanol production in this heterologous host by overexpressing aldehyde/alcohol dehydrogenase 2 (adhE2), which converts butyryl-CoA to butanol (Yu et al. 2011).

Besides, higher butanol titer was observed by knocking out the acetate kinase (ack) gene in the host bacterium (Yu et al. 2011). This engineered C. tyrobutyricum (Δack-adhE2) can produce 10-16 g/L butanol depending on the carbon source used in the fermentation

(Yu et al. 2011).

Instead of traditional starchy feedstock and molasses, many studies have been focused on butanol production from lignocellulosic biomass to reduce the substrate cost, including wood pulp (Lu et al. 2013), corn fiber (Qureshi et al. 2008a), corn stover

(Qureshi et al. 2010b), corn straw (Lin et al. 2011), wheat straw (Qureshi et al. 2007;

Qureshi et al. 2008b; Wang et al. 2013), wheat bran (Liu et al. 2010), barley straw

(Qureshi et al. 2010a), switchgrass (Qureshi et al. 2010b), rice straw (Amiri et al. 2014;

Gottumukkala et al. 2013; Moradi et al. 2013), willow stem and bark (Han et al. 2013).

Besides carbon sources, alternative nitrogen sources have also been explore. Corn steep liquor is a by-product of corn wet-milling and has been used to replace the original nitrogen sources in P2 medium (Qureshi et al. 2004).

In order to recover butanol from dilute fermentation broth, alternative separation technologies other than conventional distillation are developed, such as gas stripping

(Ezeji et al. 2003; Lu et al. 2012; Xue et al. 2013a), adsorption (Lin et al. 2012; Nielsen and Prather 2009; Qureshi et al. 2005), pervaporation (Dong et al. 2014; Li et al. 2010), and liquid-liquid extraction (Dhamole et al. 2012). If these separation techniques integrated with fermentation process, they can not only efficiently recover butanol but also release the product inhibition effect and therefore boost butanol production.

1.1 Research objective and tasks

The overall research objective is to develop integrated processes for n-butanol fermentation and in situ product recovery by different alternative butanol separation techniques including adsorption, gas stripping and pervaporation. Besides, butanol production from lignocellulosic biomass will also be explored. Figure 1.1 provides an overview of this project. Specific tasks are given below.

Task 1: butanol production integrated with adsorption using activated carbon and other adsorbents

Activated carbon and several other adsorbent materials were used to selectively recover butanol in an integrated fermentation-recovery system. A hyper-butanol 4

producing mutant strain C. acetobutylicum JB 200 was used in this study. Details are given in Chapter 3.

Task 2: butanol production in the co-existence glucose and xylose with external driving forces integrated with gas stripping

Butanol production by mutant strain C. tyrobutyricum in a fibrous bed bioreactor with external driving forces integrated with gas stripping was studied. Three different carbon sources were investigated: the mixture of glucose and xylose, two lignocellulosic biomass hydrolysates including soybean hull and sugarcane bagasse hydrolysates. Details are given in Chapter 4.

Task 3: butanol recovery from aqueous model solution by pervaporation using polydimethylsiloxane (PDMS) membranes

Butanol recovery from aqueous model solution by pervaporation was studied.

Polydimethylsiloxane (PDMS) membrane and PDMS mixed matrix membrane incorporated with zeolite were studied. Details are given in Chapter 5.

1.2 References

Amiri H, Karimi K, and Zilouei H. 2014. Organosolv pretreatment of rice straw for efficient acetone, butanol, and ethanol production. Bioresource Technol 152:450- 456.

Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, Chou KJY, Hanai T, and Liao JC. 2008. Metabolic engineering of Escherichia coli for 1- butanol production. Metab Eng 10(6):305-311.

Dellomonaco C, Clomburg JM, Miller EN, and Gonzalez R. 2011. Engineered reversal of the beta-oxidation cycle for the synthesis of fuels and chemicals. Nature 476(7360):355-U131.

Dhamole PB, Wang ZL, Liu YQ, Wang B, and Feng H. 2012. Extractive fermentation with non-ionic surfactants to enhance butanol production. Biomass Bioenerg 40:112-119.

Dong HJT, W. W. Dai, Z. J. . 2012. Biobutanol. Advances in biochemical engineering/biotechnology 128:85-100.

Dong ZY, Liu GP, Liu SN, Liu ZK, and Jin WQ. 2014. High performance ceramic hollow fiber supported PDMS composite pervaporation membrane for bio-butanol recovery. J Membrane Sci 450:38-47.

Durre P. 1998. New insights and novel developments in clostridial acetone/butanol/isopropanol fermentation. Appl Microbiol Biot 49(6):639-648.

Durre P. 2007. Biobutanol: an attractive biofuel. Biotechnol J 2(12):1525-1534.

Ezeji TC, Qureshi N, and Blaschek HP. 2003. Production of acetone, butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping. World J Microb Biot 19:595-603.

Ezeji TC, Qureshi N, and Blaschek HP. 2004. Butanol fermentation research: Upstream and downstream manipulations. Chem Rec 4(5):305-314.

Gottumukkala LD, Parameswaran B, Valappil SK, Mathiyazhakan K, Pandey A, and Sukumaran RK. 2013. Biobutanol production from rice straw by a non acetone producing Clostridium sporogenes BE01. Bioresource Technol 145:182-187.

Green EM. 2011. Fermentative production of butanol - the industrial perspective. Curr Opin Biotech 22(3):337-343.

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1.3 Figures

Research Objective To develop integrated processes for n-butanol fermentation and recovery by adsorption, gas stripping and pervaporation

Task 1 Task 2 Task 3 Butanol production Butanol production in Butanol recovery integrated with the co-existence of from aqueous model

adsorption using glucose and xylose solution by activated carbon and with external driving pervaporation using other adsorbents forces integrated with polydimethylsiloxane (Chapter 3) gas stripping (PDMS) membranes

(Chapter 4) (Chapter 5)

Figure 1.1 Overview of research objective and tasks in this study

Chapter 2: Literature Review

2.1 N-butanol properties, applications and production

N-butanol or 1-butanol or normal butanol or n-butyl alcohol (hereafter ‘butanol’) is a 4-carbon primary alcohol with the molecular formula C4H9OH. It is naturally produced by genus Clostridia species (Jones and Woods 1986; Kumar and Gayen 2011).

It has a molecular weight of 74.12 g mol-1, a density of 0.81 g cm-3, a melting point of -

89.3 °C and a boiling point of 117.7 °C (Green 2011; Lee et al. 2008). Pure butanol is a colorless liquid with a distinct odor and partial miscible with water (solubility in water is

73 g L-1 at 25 °C) but completely miscible with organic solvents.

Butanol is used as an industrial intermediate in the synthesis of many chemicals, such as butyl acrylate and a wide variety of butyl esters. It is also used as a solvent in coating, paints and many other chemical applications. With concerns about fossil fuel depletion and green gas emission, biobutanol stands out as a renewable and environmental friendly biofuel (Yu et al. 2011). Currently, ethanol is blended into gasoline at different percentages (Durre 2007). Compared to ethanol, butanol is a superior alternative fuel/ fuel additive, with higher energy content, lower volatility (thus less explosive and safer) and lower water absorption (thus does not pick up water moisture,

which causes corrosion) (Durre 2007; Lee et al. 2008). Besides, butanol can be more easily blended with gasoline or even directly used in existing internal combustion engines

(Lee et al. 2008; Nigam and Singh 2011). Table 2.1 summarizes butanol properties in comparison with ethanol and gasoline (Lee et al. 2008).

Currently, butanol is almost exclusively produced via petroleum refinery and catalyzed thermochemical reactions: the OXO process, in which petrochemical propylene is hydroformylated to butyraldehyde and then further hydrogenated to produce butanol

(Yang et al. 2013). With increasing concerns over global warming and the depletion of fossil fuels, biological production of n-butanol has regained many researchers’ attention.

Butanol is naturally produced by many Clostridia species. Many efforts are made to improve fermentative butanol production.

2.2 Butanol production from fermentation

2.2.1 Acetone-Butanol-Ethanol (ABE) fermentation

Biological butanol production has a long history. Fermentative butanol production was first reported by Pasteur in 1861(Gabriel 1928; Gabriel and Crawford 1930). In the following years, Albert Fitz obtained butanol production from glycerol and designated his culture Bacillus butylicus (Fitz 1876; Fitz 1878). Martinus Beijerinck also isolated two butanol-producing microorganisms named Granulobacter butylicus and Granulobacter saccharobutyricum (Beijerinck 1893). Later in the first decade of the 20th century, the coproduction of acetone and isopropanol was discovered (Schardinger 1905; Schardinger

1907). This fermentative acetone production played an important role in the World War I and was one of the largest industrial fermentation processes in the world in the early 20th century (Durre 1998; Jones and Woods 1986; Lee et al. 2008). However, fermentative butanol production gradually declined by the 1960s due to increased substrate cost and cannot compete with the more efficient and economical petrochemical process (Groot and Luyben 1986). Concerned with depleting fossil fuels and environmental issues, there is a renewed interest in fermentative butanol production during the past few decades

(Yang et al. 2013). More details on the history of acetone-butanol-ethanol (ABE) fermentation can be found in the excellent review article by Jones and Woods (Jones and

Woods 1986). Zverlov gave a more recent review on ABE fermentation using hydrolyzed agricultural waste in the Soviet Union (Zverlov et al. 2006).

Butanol (and acetone, ethanol and isopropanol) are naturally produced by a variety of solvent-producing Clostridium species (Jones and Woods 1986; Lee et al.

2008). Clostridia are rod-shaped, spore-forming, gram-positive, and strict anaerobes

(Jones and Woods 1986). In a typical clostridial ABE fermentation, butanol is coproduced with acetone and ethanol with a butanol: solvent ratio of 0.6 and an A:B:E ratio of 3:6:1 (Green 2011; Jones and Woods 1986; Yang et al. 2013). Table 2.2 summarizes solventogenic Clostridium with their substrates utilization, pH, temperature and products. One distinct feature of solvent-producing Clostridium is the biphasic growth (Groot and Luyben 1986; Yang et al. 2013). The first phase is acidogenic phase, which mainly occurs during the exponential growth phase. During acidogenesis, acetate and butyrate are formed along with hydrogen and carbon dioxide, lowering the pH of

fermentation medium. The second phase is solventogenic phase, which usually occurs at the end of exponential growth phase. During the solventogenesis, previously formed acids are reassimilated and converted to solvents, including acetone, butanol and ethanol

(Gottschal and Morris 1981; Yang et al. 2013). Figure 2.1 shows the metabolic pathways in C. acetobutylicum (Lee et al. 2008). Dotted and solid arrows show the acidogenic and solventogenic pathways respectively.

The triggering of solventogenesis is a complex process and is often accompanied by morphological changes of the bacteria and the initiation of endospore formation (Yang et al. 2013). A huge change in gene expression was noticed (Alsaker et al. 2010; Jones et al. 2008). In the late 20th century, Jones reviewed and summarized several key factors involved in the metabolic transition from acidogenic to solventogenic phase including external pH, acid end products, internal pH, nutrient limitation, temperature and oxygen

(Jones and Woods 1986). Many reports mentioned that solvent production began only after the medium pH had decreased to certain level (Beesch 1953; Davies and Stephenson

1941; Reilly et al. 1920). Acids are the main products when maintained at high pH and solvent production predominates at low pH (Jones and Woods 1986). However, the optimal pH range for solvent production is quite wide, depending on the particular strain

(Jones and Woods 1986). Monot had reported that C. acetobutylicum ATCC 824 was able to produce good levels of solvents at the pH range of 4.3-5.5 (Monot et al. 1984). It has been suggested that high acid levels are toxic to the cells and the reassimilation of acids or the shift from acidogenesis to solventogenesis actually act as a detoxification process which allows the cells to avoid the inhibitory effects of acids. Gottschal and

Morris reported that the shift to solventogenic phase was rapidly induced when low levels of acetate and butyrate were introduced to the culture of C. acetobutylicum when pH maintained at 5.0 (Gottschal and Morris 1981). Wang also reported that the supplementation of 40 mM sodium butyrate to the medium of C. beijerinckii triggered solventogenesis during the mid-exponential growth phase, while in the control, solvent production was not initiated until late exponential phase (Wang et al. 2013b). Wang also mentioned that the addition of butyrate shortened the fermentation time, increased butanol titer, sugar-based yield, and butanol productivity (Wang et al. 2013b). Despite numerous studies on the biochemistry and molecular biology of ABE fermentation, the precise mechanism behind the metabolic shift from acidogenic to solventogenic phase in solvent-foaming Clostridium is still not totally clear.

Even with developed technologies, ABE fermentation still suffers from a number of challenges: high substrate cost, low butanol titer, yield and productivity, high recovery cost and high water usage (Green 2011; Jones and Woods 1986).

2.2.2 Strain development and metabolic engineering for improved ABE fermentation

Many Clostridium species can naturally produce butanol. The major solventogenic clostridia are C. acetobutylicum, C. beijerinckii, C. saccharobutylicum and

C. saccharoperbutylacetonicum (Berezina et al. 2012; Keis et al. 2001). These four strains are all mesophilic bacteria and have good butanol production levels and yields

(Lee et al. 2008); however, their carbon source utilization abilities differ, as well as 15

optimal pH and temperature (Berezina et al. 2012; Yang et al. 2013). Among all the solventogenic Clostridium species, C. acetobutylicum ATCC 824 is one of the most thoroughly studied and the first genome-sequenced strain (Yang et al. 2013). However, the genome of different solvent-forming clostridia may vary from one another (Yang et al.

2013).

Butanol is the only solvent that is produced to a level which becomes toxic to the cells and imposes a strong inhibitory effect on them (Jones and Woods 1986). High concentrations of butanol (>10 g/L) can increase cell membrane fluidity by disrupting the phospholipid component, which further destabilize the membrane and disrupts the membrane-associated functions (Bowles and Ellefson 1985; Gottwald and Gottschalk

1985; Vollherbstschneck et al. 1984). It is also reported that the addition of butanol can inhibit the maintenance of a cell’s internal pH and sugar uptake (Bowles and Ellefson

1985; Gottwald and Gottschalk 1985; Ounine et al. 1985). Cell growth ceases when butanol concentration reaches 12-16 g/L (Jones and Woods 1986). A batch fermentation usually gives a final butanol titer of ~12 g/L (Lee et al. 2008). Butanol inhibition greatly limits the concentration of substrate that can be utilized for fermentation and results in low butanol concentration and productivity (Lee et al. 2008).

Efforts are made to obtain mutants with improved butanol tolerance and yield by traditional mutagenesis and screening (Jones and Woods 1986). Dong reported two mutants C. acetobutylicum EA2018 with an improved butanol ratio of 70% (vs. 60%) among solvents and Rh9 with a butanol tolerance of 19 g/L (Dong 2012). With the improved butanol tolerance, however, these strains did not give a better butanol

production. Recently, a mutant strain C. acetobutylicum JB200 with high butanol production derived from C. acetobutylicum ATCC 55025 was obtained by spontaneous mutation in a fibrous-bed bioreactor (FBB) which can produce up to ~25 g/L butanol

(Zhao et al. 2009).

Equipped with modern recombinant DNA technology, many metabolically engineered strains have been obtained with desired genes. Many mutant strains of C. acetobutylicum ATCC 824 have been engineered to produce more butanol/solvents

(Huang et al. 2010; Papoutsakis 2008). For example, Jiang knocked out the acetoacetate decarboxylase gene (adc) and the mutant had an increased butanol/acetone ratio (Jiang et al. 2009). Besides native solventogenic clostridia, metabolic engineering also made butanol production in nonnative hosts possible. Escherichia coli is a well studied microbe and has been engineered to produce butanol. Atsumi constructed a recombinant E. coli for butanol production but the titer was low (< 1 g/L) (Atsumi et al. 2008). Dellomonaco also engineered E. coli to produce butanol with a much higer titer of ~14 g/L

(Dellomonaco et al. 2011). Shen also reported that ~30 g/L butanol was produced with high yield in engineered E. coli driven by external driving forces (Shen et al. 2011).

Another successfully engineered strained was obtained from C. tyrobutyricum by Yu (Yu et al. 2011). Originally C. tyrobutyricum ATCC 25755 is a butyric acid producing bacterium and Yu achieved butanol production in this heterologous host by overexpressing aldehyde/alcohol dehydrogenase 2 (adhE2), which converts butyryl-CoA to butanol (Yu et al. 2011). Besides, higher butanol titer was observed by knocking out the acetate kinase (ack) gene in the host bacterium (Yu et al. 2011). This engineered C.

tyrobutyricum (Δack-adhE2) can produce 10-16 g/L butanol depending on the carbon source used in the fermentation (Yu et al. 2011).

2.2.3 Butanol production from sugars and lignocellulosic biomass

Substrate cost has been an important economic factor of fermentative butanol production, which made up ~60% of the overall cost (Jones and Woods 1986; Ross 1961).

It is also reported that the cost of raw materials is about 57-116% of the selling price of the solvents during ABE fermentation (Jones and Woods 1986). Clearly, substrate cost is an important parameter in realizing economic fermentative butanol production. Recently, a review article was published about butanol production by clostridia from renewable biomass, which includes sugars and starch, lignocellulosic biomass, glycerol, algal biomass, Syngas (Jang et al. 2012). Here mainly discuss sugars, starch-based substrate, and lignocellulosic biomass.

Traditionally, butanol (along with other solvents) is produced from costly sugar

(molasses) and starch-based substrates (Jones and Woods 1986). Biofuels produced from these sugars and starch-based substrates are usually referred to as first generation biofuels, which only require a simple process to produce the desired biofuel (Nigam and Singh

2011). Parekh used glucose and corn steep liquor (CSL) as the feedstock and was able to produce 17.8 g/L butanol and a total ABE titer of 23.6 g/L using C. beijerinckii BA101

(Parekh et al. 1999). More recently, Lu produced 20.3 g/L butanol and 33.9 g/L ABE from cassava bagasse using a hyper-butanol-producing strain C. acetobutylicum JB200

(Lu et al. 2012). However, the high cost of these conventional food-based substrates has 18

limited the economic and large-scale production of butanol (Garcia et al. 2011; Jones and

Woods 1986; Kumar and Gayen 2011; Nigam and Singh 2011; Yang et al. 2013).

Lignocellulose is the most abundant renewable resource worldwide and is considered as substrate for second generation biofuel (Nigam and Singh 2011).

Lignocellulose consists of cellulose, hemicellulose and lignin. Although some

Clostridium species are capable of utilizing cellulose directly, for example Higashide obtained 0.66 g/L isobutanol from crystalline cellulose using C. cellulolyticum, the solvent production is too low to be practical (Gehin et al. 1996; Higashide et al. 2011).

Therefore, pretreatment and enzyme hydrolysis of lignocellulosic biomass is usually needed before fermentation. There are few reports about simultaneous saccharification, fermentation and recovery (SSFR) of butanol (Qureshi et al. 2014). Qureshi described the

SSFR process with corn stover using C. beijerinckii P260, from which an ABE productivity of 0.34 g/L∙h and yield of 0.39 g/g was obtained (Qureshi et al. 2014).

Table 2.3 summarizes ABE fermentation by solventogenic clostridia from renewable biomass including starch-based substrates and lignocellulosic biomass. A variety of lignocellulosic biomass has been used to produce butanol and other solvents, such as corn fiber, wheat straw, distiller’s dried grains and solubles, wheat bran, barley straw, corn stover, switchgrass, rice straw, corn straw, wood pulp, willow stem and bark

(Amiri et al. 2014; Ezeji and Blaschek 2008; Han et al. 2013; Lin et al. 2011; Liu et al.

2010; Lu et al. 2013; Qureshi et al. 2008a; Qureshi et al. 2007; Qureshi et al. 2010a;

Qureshi et al. 2008b; Qureshi et al. 2010b; Wang et al. 2013a; Wang et al. 2013c). Most of them were able to produce 20-30 g/L of ABE in total. Besides solventogenic clostridia,

other engineered species are also used for this purposed. For instance, Suhardi used recombinant E.coli to produce butanol from energy cane (Suhardi et al. 2013).

In addition, detoxification of lignocellulosic hydrolysate is usually needed before fermentation, because many inhibitors may be produced during the pretreatment and enzyme hydrolysis of lignocellulosic biomass, such as furan derivatives, phenolic compounds and weak acids (Ezeji et al. 2007b; Mussatto and Roberto 2004; Olsson and

HahnHagerdal 1996).

2.2.4 External driving forces for butanol production

Aside from metabolic engineering, there are other possible ways to direct the carbon and electron flows towards butanol synthesis during ABE fermentation. Many studies have been carried out to provide external driving forces for butanol production

(Fontaine et al. 2002; Lutke-Eversloh and Bahl 2011; Shen et al. 2011). Datta and Meyer examined the effect of carbon monoxide (CO) on butanol production by C. acetobutylicum (Datta and Zeikus 1985; Meyer et al. 1986). Carbon monoxide is an inhibitor of hydrogenase, which can reduce molecular hydrogen formation (Datta and

Zeikus 1985). Results show that the presence of carbon monoxide successfully reduced hydrogen production by 50%-100% and increased butyrate uptake and solvent productivity and yield, because of altered electron flow (Datta and Zeikus 1985; Meyer et al. 1986). Viologen dyes (methyl and benzyl viologen) and neutral red, acting as artificial electron carrier, are also used to modify the carbon and electron flow in C. acetobutylicum (Girbal et al. 1995; Peguin et al. 1994; Peguin and Soucaille 1995; Peguin 20

and Soucaille 1996; Rao and Mutharasan 1986; Rao and Mutharasan 1987). Altered electron flow directs carbon flow from acid forming to alcohol production along with reduced molecular hydrogen evolution (Rao and Mutharasan 1987). This saved reducing equivalent, previously released as free hydrogen, is directed to NADH formation, which results in enhanced alcohol production (Kim and Kim 1988; Rao and Mutharasan 1987).

Besides, iron limitations in the medium also affect carbon and electron flow during ABE fermentation (Junelles et al. 1988; Peguin and Soucaille 1995). Junelles reported that iron limitation increased butanol yield by 20-30% and also decreased hydrogenase specific activity (Junelles et al. 1988). However, Hipolito suggested that redox dyes, including methyl and benzyl viologen, neutral red and methylene blue had no effect on solvent production by C. saccharoperbutylacetonicum N1-4 (ATCC 13564) (Hipolito et al. 2008).

2.3 Advanced butanol recovery techniques

Butanol recovery is the most energy intensive step in the biobutanol fermentation process and largely determines the process efficiency (Ezeji et al. 2004b; Oudshoorn et al.

2009b). In ABE fermentation, the final butanol concentration is usually 1-2% in the fermentation broth (Jones and Woods 1986; Oudshoorn et al. 2009b). Separating butanol by conventional distillation is very energy intensive. Besides, butanol has a high boiling point (118 ºC) which makes its recovery by distillation even more difficult (Oudshoorn et al. 2009b). Figure 2.2 shows the effect of feed butanol concentration on specific energy requirements for butanol recovery by distillation (Matsumura et al. 1988). In a butanol- water binary system, the energy required to recover butanol from a 0.5 wt% solution to 21

99.9% pure butanol was estimated at 79.5 MJ/kg butanol (Matsumura et al. 1988), which is much higher than the energy content of butanol (36 MJ/kg). The energy consumption can be reduced drastically to ~36 MJ/kg and 6 MJ/kg when the butanol concentration is increased to ~1 wt% (or 10 g/L) and 10 wt% (100 g/L), respectively (Matsumura et al.

1988). Further increasing butanol concentration to above 40 wt % can reduce the energy consumption by distillation to less than 3 MJ/kg (Matsumura et al. 1988).

In order to solve this dilemma, alternative separation technologies are developed, which are more energy-efficient and suitable to recover butanol in low concentration

(Kraemer et al. 2011; Matsumura et al. 1988; Qureshi et al. 2005; Vane 2008; Xue et al.

2012). Generally, there are two different types of coupled processes: fermentation with product recovery integrated inside the fermentor, and fermentation and product recovery in a closed loop which is outside of fermentor (Groot et al. 1992). In the literature, the popular butanol recovery techniques include pervaporation, liquid-liquid extraction, adsorption, perstraction, and gas stripping. The basic principles, merits and limitations of those in-situ recovery techniques are summarized and compared in Table2.4 (Dhamole et al. 2012; Durre 1998; Ennis et al. 1986; Gapes et al. 1996; Groot et al. 1992; Qureshi and

Maddox 2005).

Vane divided primary alcohol removal modes into two categories: ‘end-of-pipe’, which recovers alcohol after fermentation has ceased and the fermentation broth is sent to the next processing unit, and ‘slip-stream’, which removes alcohol from the bioreactor while fermentation is ongoing and the alcohol depleted-stream is returned to or never leaves the bioreactor (Vane 2008). The end-of-pipe approach is usually employed in

ethanol recovery where alcohol concentration is high while the slip-stream approach is used under circumstances where alcohol concentration is relatively low, such as butanol fermentation (Vane 2008). The second approach is also referred to as in situ product recovery (ISPR), which offers many advantages (Nielsen and Prather 2009). Besides energy saving, ISRP is also helpful in releasing production inhibition due to in situ removal and therefore increasing substrate utilization, solvent production, prolonging the fermentation and reducing waste water (Abdehagh et al. 2014; Groot and Luyben 1986).

2.3.1 Adsorption

Adsorption is a widely studied separation technique for fermentation product recovery. It is based on the adherence of certain substances to the surface of the adsorbent, which is usually packed in a column (Vane 2008). The desired product from dilute solution, butanol in this case, is first adsorbed by adsorbents during the loading cycle and then desorbed to get a concentrated solution during the regeneration cycle, as depicted in

Figure 2.3 (Vane 2008). Adsorbent regeneration is needed to obtain concentrated product and adsorbent reuse. For the recovery of butanol, it is also achieved by sequential heating

(Qureshi et al. 2005).

The particular adsorbent fraction (Xr) is defined as (Nielsen and Prather 2009):

h h h Xr = (1) h h

The specific loading of adsorbent (L) is defined as:

L = (2)

Higher specific loading means this adsorbent can take up more solvent (butanol) from the solution, which is desired (Vane 2008).

Many adsorbents have been used to recover butanol from the ABE fermentation broth, including activated carbon (Abdehagh et al. 2013; Groot and Luyben 1986), polymeric resin (Ennis et al. 1987; Groot and Luyben 1986; Lin et al. 2012; Liu et al.

2014; Nielsen and Prather 2009; Nielsen et al. 1988; Qureshi et al. 2005; Wiehn et al.

2014), polyvinylpyridine (Yang et al. 1994), metal-organic framework (Saint Remi et al.

2011; Zhang et al. 2013) and zeolite/silicalite (Ennis et al. 1987; Maddox 1982;

Milestone and Bibby 1981; Oudshoorn et al. 2009a; Saravanan et al. 2010; Sharma and

Chung 2011). Table 2.5 lists commonly used adsorbents for butanol recovery with their performance. Milestone reported that a highly concentrated butanol solution (~98%) was obtained by sequential heating from a 0.5% solution using silicalite as the adsorbent

(Milestone and Bibby 1981). However, this result has not been duplicated during following research. ZSM-5 type zeolite is widely used as the adsorbent to recover butanol

(Oudshoorn et al. 2009a; Saravanan et al. 2010). Oudshoorn investigated three ZSM-5 type zeolites with different Si/Al ratio and found that high-silica zeolite CBV28014 had higher affinity for butanol and low-silica zeolite CBV901 had the highest butanol adsorption capacity (Oudshoorn et al. 2009a). Activated carbon has also been used to recover butanol, which usually has relatively high butanol adsorption capacity (~200-300 mg/g) (Abdehagh et al. 2013; Groot and Luyben 1986). Abdehagh reported that the presence of ethanol, glucose and xylose did not affect butanol adsorption by activated carbon, but the presence of acetone and acids did (Abdehagh et al. 2013). Recently, many

researchers explored butanol recovery by polymeric resins. Nielsen explored many polymeric resins for in situ butanol recovery and concluded that resins derived from poly(styrene-co-divinylbenzene) had the greatest butanol affinity, but the adsorption capacity was limited by their specific surface area (Nielsen and Prather 2009). Table 2.6 summarizes some fermentation processes integrated with adsorption by polymeric resins.

When integrated with adsorption, total butanol/ solvent production was significantly enhanced.

Qureshi reviewed different adsorbent materials as mentioned above, silicalite, polymeric resins, activated carbon and polyvinylpyridine and concluded that silicalite was the most attractive adsorbent, as it can concentrate a 5 g/L butanol solution to 790-

810 g/L (Qureshi et al. 2005). Qureshi also estimated energy consumption of butanol recovery by the adsorption-desorption process to be 1948 kcal/kg, in comparison with

5789 kcal/kg by distillation, 5220 kcal/kg by gas stripping and 3295 kcal/kg butanol by pervaporation (Qureshi et al. 2005). However, this energy consumption analysis may not be consistent with other reports.

The issue associated with adsorption, is the adsorption of other compounds including cells, substrates, nutrients and other metabolites (Nielsen et al. 1988). This not only lowers the adsorption capacity, but also negatively affects cell growth, substrate utilization. Cell fouling also hampers adsorbent regeneration for continued use (Nielsen et al. 1988). Cell removal (e.g. by membrane filtration) prior to adsorption is suggested to avoid fouling (Nielsen et al. 1988).

2.3.2 Gas stripping

Gas stripping is a simple, but effective technique. Figure 2.3 B shows the typical schematic diagram of gas stripping (Vane 2008). Nitrogen or fermentation gases are used as stripping gas to ensure the oxygen-free environment (Ezeji et al. 2004a) and are pumped into the fermentation broth (on the left side) to form bubbles. Those bubbles can carry volatile organic solvents out of the broth, like butanol, acetone, and ethanol. Then the organic vapors are condensed and collected.

Mass transfer mainly takes place at the feed vessel and the condenser. If all the organic gas vapors can be condensed and recovered in the condenser, then the efficiency of gas stripping depends on the mass transfer occurring in the feed vessel. Two-resistance film theory can be applied to the feed vessel. Consider the interface between gas bubbles and the bulk liquid phase, and the flux of some species across that interface. Assume steady state transfer and thermodynamic equilibrium at the interface, then the molar flux across the interface is continuous, and for any species A we may write

(3)

As shown in Figure 2.4, and represent gas and liquid side fluxes of specie

A, and represent gas and liquid side mass transfer coefficients. and represent gas pressure of A in the bulk gas and at the gas interface, while and represent the concentration of A in the bulk liquid and at the liquid interface.

At the interface

(4)

is given by an equilibrium relationship. In this case, Eq. (4) is the equilibrium partial pressure of species A above a solution of molar concentration . The driving force for mass transfer, on the gas side, is . (On the liquid side, it is

). Since interface compositions are hard to measure, fluxes expressed in terms of bulk composition differences are needed. Define overall mass transfer coefficients as

(5)

Partial pressure is defined as the partial pressure in equilibrium with the bulk liquid composition , KG and KL are the overall mass transfer coefficients. If a linear equilibrium relationship exists for Eq. (4), say = (Henry’s law), then

and

We’ll get

and (6)

These are the relationships between the overall (K) mass transfer coefficients and the individual (k) coefficients. For a highly soluble gas (e.g. NH3 in H2O), m is small and

and this indicates that there is no significant liquid phase resistance. In contrast,

for a sparingly soluble gas (e.g. CO2 in H2O), m is large and ; this means the

liquid phase resistance controls the flux. For butanol, the solubility in water is 73 g/L at room temperature. So it is partially soluble in water, and both gas and liquid side will contribute to the resistance during mass transfer.

The selectivity is defined as α = [y/(1-y)]/[x/(1-x)], where x and y are weight fractions of acetone, butanol, ethanol or together in the feed (fermentation broth or model

solution) and condensate, respectively (Ezeji et al. 2003). Partial pressure increases with elevating feed temperature, which will accelerate the mass transfer from liquid phase to vapor phase (Vane 2008). But, this is true for both water and solvents. Therefore, temperatures for gas stripping and condensation need to be optimized for higher selectivity of solvents over water (Vane 2008).

Many studies have shown successful butanol recovery by gas stripping from both model solution and fermentation broth (de Vrije et al. 2013; Mollah and Stuckey 1993;

Qureshi and Blaschek 2001; Xue et al. 2013). Table 2.7 lists solvent selectivities of processes integrated with gas stripping for butanol recovery and Table 2.8 summarizes solvent production in an integrated process from different substrates.

Qureshi showed that compared to sugar utilization of 30 g/L in a control batch reactor, 199 g/L sugar could be consumed and 70 g/L solvent was produced when integrated with gas stripping (Qureshi and Blaschek 2001). In a fed-batch reactor, 350 g/L sugar could be utilized (Qureshi and Blaschek 2001). This gas stripping integrated

ABE fermentation achieved a selectivity of 4-30.5 (Qureshi and Blaschek 2001).Also, gas stripping does not remove nutrients, cell or other nonvolatile metabolites; it reduces butanol toxicity and allows the use of concentrated sugar solution (Qureshi and Blaschek

2001). Ezeji studied the gas stripping integrated fermentation, using liquefied corn starch

(LCS) as the substrate. When the fed-batch reactor fed with saccharified liquefied corn starch (SLCS) was coupled with gas stripping, 81.3 g/L ABE was produced compared to

18.6 g/L (control) and 225.8 g/L SLCS sugar (487% of control) was consumed (Ezeji et al. 2007c). If there was no butanol removal, C beijerinckii BA101 could not use more than 46 g/L glucose (Ezeji et al. 2007c).

The work done by Mollah was the first attempt to integrate gas stripping for in situ removal of butanol with the continuous fermentation (Mollah and Stuckey 1993). It studied the effect of sparging gas flow rate and dilution rate on the continuous culture of alginate-immobilized C acetobutylicum (Mollah and Stuckey 1993). Solvent productivity was found to increase with the increasing sparging gas flow rate to a certain point, and then decrease as the gas flow rate increased further (Mollah and Stuckey 1993). It was also found that a dilution rate of 0.07h-1 could maximize the volumetric solvent production (Mollah and Stuckey 1993). Recently, Xue reported a two-stage gas stripping system which seems promising (Xue et al. 2014; Xue et al. 2013). After the second stage gas stripping, highly concentrated solvent solution was obained containing 500-600 g/L butanol and 600-700 g/L ABE (Xue et al. 2014; Xue et al. 2013).

Currently, gas stripping has not realized commercialization for solvent recovery.

The condensate needs at least one more purification step to get pure solvent. Vane suggested that innovative mass and energy integration schemes are needed in order to make this process economically feasible and attractive (Vane 2008).

2.3.3 Pervaporation

Recovering products from biomass fermentation processes by pervaporation offers many advantages compared to traditional method of distillation, including 29

increased energy efficiency, reduced capital cost for pervaporation systems, optimized integration of pervaporation with fermentor and etc. (Vane 2005)

Pervaporation is an efficient membrane process for liquid separation. It is a membrane-based separation technique that imposes a selective membrane between the feed side (liquid phase) and the permeate side (gas phase). Liquid feed containing volatile species flows on one side of the membrane, while the other side of the membrane is remained under vacuum. Components of the liquid stream, depending on the chemical properties, permeate and evaporate into vapor phase (hence the word ‘pervaporate’)

(Vane 2005). The resulting vapor, referred to as ‘the permeate’, is then condensed and collected in the cooling trap (Thongsukmak and Sirkar 2007). Due to different species in the feed mixture having different affinities for the membrane and different diffusion rates through the membrane, even a component at low concentration in the feed can be highly concentrated in the permeate (Vane 2005). Figure 2.3C shows the schematic diagram of the pervaporation process (Vane 2008).

There are two important parameters to characterize the performance of a membrane, flux (J) and separation factor (α). Flux J (kg/m2∙s) is inversely proportional to the overall mass transfer resistance and is defined as J=Q/At, where Q is the mass of collected permeate over a time interval t, A is the effective membrane area for mass transfer (Fouad and Feng 2009). Separation factor (α) indicates the ability of a membrane to enrich certain component and is actually the same as selectivity in gas stripping:

, where is the separation factor of species 1 relative to species 2, yi and

xi refer to the mass fraction of component i in the permeate and feed, respectively.

The separation performance is dominated by the properties of the membrane, whether it is hydrophobic or hydrophilic (Vane 2005). Feed species, temperature, composition, and permeate side pressure also have effects (Vane 2005). Various membranes are used in the pervaporation to recover butanol from the broth, but relatively few studies were carried out integrating pervaporation and fermentation directly. Table

2.9 summarizes the different membranes used in pervaporation and Table 2.10 lists integrated fermentation-pervaporation processes for ABE production. Among those membranes, polydimethylsiloxane (PDMS), also referred to as ‘silicone rubber’, shows good performance and promising potential application for butanol recovery (Li et al.

2010). It has highly hydrophobic properties, good thermal and chemical stability. Also, the fabrication is easy and economical (Li et al. 2010). Vane pointed out that PDMS is the current benchmark hydrophobic pervaporation membrane material, and can be used to fabricate hollow fiber, tubular, unsupported sheet, or supported sheet membranes (Vane

2005). Li reported a tri-layer PDMS/polyethylene (PE)/metal support membrane which gave a butanol separation factor of 32 and total flux of 132 g/m2∙h (Li et al. 2010).

In addition, different membrane filler materials are investigated for the improvement of butanol separation. Jonquieres investigated silicalite-filled GFT PDMS membrane for the separation of binary butanol/ water system and ternary butanol/ acetone/ water system (Jonquieres and Fane 1997). Wang and Wongchitphimon investigated the effect of polyethylene glycol (PEG) as an additive on the fabrication of polyvinylidene fluoride-co-hexafluropropylene (PVDF-HFP) asymmetric microporous hollow fiber membranes (Wang et al. 2011). Huang studied the thin film silicalite- filled

silicone composite membranes (Huang and Meagher 2001). Huang found that with the increase of silicalite content in the active layer, the selectivity for butanol flux increased, while the total flux decreased (Huang and Meagher 2001). Besides, Li studied the solution-diffusion model, specifically the mass transfer equation based on Fick’s first law, for the application of pervaporation from undefined fermentation broth (Li et al. 2011).

One problem with recovering butanol using pervaporation is membrane fouling due to the complex nature of fermentation broth (Qureshi and Blaschek 1999). Fouling is defined as a reduction in the rate of permeation (g/m2∙h) with time of membrane operation (Qureshi and Blaschek 1999). Fadeev reported that poly [1-(trimethylsilyl)-1- propyne] (PTMSP) membrane was fouled when used in pervaporation system due to polymer compaction under vacuum conditions, relaxation in alcohol/ water mixtures, and membrane contamination (Fadeev et al. 2000). Possible solutions to this situation include pre-removal of cells through ultrafiltration or centrifugation and/or developing anti- fouling membranes. Li developed a surface modified polyvinylidene fluoride (PVDF) membrane which is stable and anti-protein-fouling (Li et al. 2012a).

2.3.4 Other separation techniques

Other than adsorption, gas stripping and pervaporation, liquid-liquid extraction

(LLE) and perstraction are also used in butanol recovery. In liquid-liquid extraction, an extractant liquid is introduced to the fermentation broth/model solution. The contact between these two liquids can be direct or indirect, often using a membrane to separate them. The latter is also referred to as perstraction (Vane 2008). Perstraction is a 32

combination of liquid-liquid extraction and pervaporation. Table 2.11 summarizes the performance of fermentation processes integrated with liquid-liquid extraction or perstraction. Roffler reported that when fed-batch fermentation integrated with liquid- liquid extraction, 50.5-96.5 g/L of ABE was produced with a yield of 0.33-0.36 g/g and a productivity of 1.4-2.3 g/L∙h (Roffler et al. 1987). Recently, Bankar successfully integrated liquid-liquid extraction with continuous fermentation and achieved a high ABE productivity of 2.5 g/L∙h (25.3 g/L of ABE and 0.35 g/g of yield) (Bankar et al. 2012).

Qureshi integrated perstraction with batch fermentation and obtained 136.6 g/L of ABE, a yield of 0.44 and a productivity f 0.21 g/g from concentrated lactose/whey permeate

(Qureshi and Maddox 2005).

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2.5 Tables and figures

Fuel properties Gasoline Butanol Ethanol Density (g/cm3) 0.71-0.79 0.81 0.79 Energy density (MJ/L) 32 29.2 19.6 Heat of vaporization (MJ/kg) 0.36 0.43 0.92 Research octane number 91-99 96 129 Air to fuel ratio 11.2 14.6 9

Table 2.1 Properties of butanol and some other fuels

References: (Durre 2007; Lee et al. 2008)

Temp. Species / strain Substrates pH Products References (oC) C. acetobutylicum Glucose, xylose, arabinose, (Ezeji and 260 cellobiose, mannose, galactose Blaschek 2008) (Madihah et al. Starch 2001) P262 Lactose 5.5 – 6.5 35 Acetone, butanol, (Qureshi and ethanol, acetate, Glucose, mannose, galactose, Maddox 2005) butyrate fructose, arabinose, xylose, sucrose, lactose, maltose, cellobiose, starch (Servinsky et al. ATCC 824 2010) Cassava 3.5-6.0 37 (Li et al. 2012b) (Khamaiseh et al. NCIMB 13357 Date fruit 7.0 35 2014)

49 Acetate, ethanol, (Bruant et al. Syngas (H2, CO, CO2) 6.0 N/A C. carboxidivorans butyrate, butanol 2010) P7 Acetate, ethanol, (Rajagopalan et CO, CO 5.3 37 2 butanol al. 2002) (Thang et al. C. Glucose, maltose, starch 6.2 Acetone, butanol, 2010) saccharoperbutylacetonicum 30 ethanol, acetate, Not (Hipolito et al. N1-4 ATCC 13564 Glucose, molasses, starch butyrate controlled 2008) Acetone, butanol, C. saccharobutylicum Glucose, xylose, arabinose, (Ezeji and 5.5 – 6.5 35 ethanol, acetate, 262 cellobiose, mannose, galactose Blaschek 2008) butyrate Acetone, butanol, C. butylicum Glucose, xylose, arabinose, (Ezeji and 5.5 – 6.5 35 ethanol, acetate, NRRL 592 cellobiose, mannose, galactose Blaschek 2008) butyrate Continued

Table 2.2 Butanol production from solventogenic Clostridia (substrates, pH, temperature and products)

Table 2.2 continued

Temp. Species / strain Substrates pH Products References (oC) C. beijerinckii 5.5 – 6.5 (Ezeji and BA101 Glucose, xylose, arabinose, Blaschek 2008) 35 cellobiose, mannose, galactose Not (Ezeji et al. controlled 2007a) Room (Formanek et al. Glucose, maltodextrin Acetone, butanol, temp. 1997) NCIMB 8052 ethanol, acetate, Room (Formanek et al. Glucose, maltodextrin N/A butyrate temp. 1997) Glucose, fructose, galactose, 37 (Mitchell 1996) P260 glucitiol Glucose, xylose, arabinose, (Qureshi et al. 6.5 35 50 galactose, mannose 2014)

Glucose, , xylan, starch, pectin, C. aurantibutyricum (Somrutai et al. arabinose, xylose, galactose, 5.5 – 6.8 37 Acetone, butanol, ATCC 17777 1996) mannose isopropanol, acetate,

butyrate (George et al. NCIB 10659 Glucose 6.8 35 1983) 5.0 – 7.0 37 (Ahn et al. 2011) C. pasteurianum Butanol, ethanol, 1,3- 4.5 – 7.5 35 (Biebl 2001) DSM 525 (same as ATCC Glycerol propanediol, acetate, (Taconi et al. 6013) 7.0 35 butyrate, lactate 2009)

Pretreatment and ABE titer ABE yield Productivity Feedstock Strain References hydrolysis (g/L) (g/g) (g/L.h) Glucose + corn steep C. beijerinckii NCIMB 8052 19.2 N/A N/A (Parekh and None water (CSW) C. beijerinckii BA101 23.6 N/A N/A Blaschek 1999) Cassava starch 21.0 0.41 0.44 Corn starch None C. saccharoperbutylacetonicum 20.7 0.48 0.31 (Thang et al. Sago starch N1-4 ATCC 13564 19.6 0.43 0.27 2010) Cassava chips Enzyme 19.4 0.38 0.44 Cassava bagasse + None C. acetobutylicum JB200 33.9 0.39 0.62 (Lu et al. 2012) glucose Soy molasses + (Qureshi et al. None C. beijerinckii BA101 30.1 N/A N/A glucose 2001a) Degermed corn + (Campos et al. corn steep liquor None C. beijerinckii BA101 19.3 N/A 0.23 2002) 51 (CSL)

C. acetobutylicum NCIMB (Khamaiseh et Date fruit None N/A 0.35-0.61 0.025-0.24 13557 al. 2014) Dilute acid + (Qureshi et al. Corn fiber C. beijerinckii BA101 9.3 0.39 0.10 enzyme 2008a) (Qureshi et al. Dilute acid C. beijerinckii BA101 25.0 0.42 0.60 2007) Alkaline peroxide (Qureshi et al. Wheat straw C. beijerinckii P260 22.2 0.41 0.55 + enzyme 2008b) 7.05 0.155 0.141 (Wang et al. Enzyme C. acetobutylicum ATCC 824 (butanol) (butanol) (butanol) 2013c) Continued

Table 2.3 ABE fermentation by solventogenic clostridia from renewable biomass (AFEX, ammonia fiber explosion; N/A, not

available)

Table 2.3 continued

Pretreatment and ABE titer ABE yield Productivity Feedstock Strain References hydrolysis (g/L) (g/g) (g/L.h) C. acetobutylicum 260 Dilute acid C. acetobutylicum ATCC 824 (Ezeji and Distiller’s dried Liquid hot water C. saccharobutylicum 262 4.9-12.9 0.30-0.35 N/A Blaschek 2008) grains and solubles AFEX + enzyme C. butylicum 592 (DDGS) C. beijerinckii BA101 Electrolyzed water (Wang et al. C. beijerinckii BA101 3.5-5.5 N/A N/A + enzyme 2013a) (Liu et al. Wheat bran Dilute acid C. beijerinckii ATCC 55025 11.8 0.32 0.16 2010) (Qureshi et al. Barley straw Dilute acid C. beijerinckii P260 26.6 0.43 0.39 2010a) 52 Corn stover Dilute acid C. beijerinckii P260 26.3 0.44 0.31 (Qureshi et al. Switchgrass Dilute acid C. beijerinckii P260 14.6 0.39 0.17 2010b) Acid/alkaline + (Moradi et al. ~3.0 N/A N/A enzyme 2013) C. acetobutylicum NRRL B-591 Dilute acid + (Amiri et al. Rice straw 7.1 10.5 N/A enzyme 2014) (Gottumukkala Acid + enzyme C. sporogenes BE01 5.3 N/A N/A et al. 2013) Willow stem 9.4 N/A N/A (Han et al. Acid hydrolysis C. beijerinckii NCIMB 8052 Willow bark 8.9 N/A N/A 2013) (Lin et al. Corn straw Acid + enzyme C. acetobutylicum CICC 8008 6.2 (butanol) N/A N/A 2011) Cook + wash + Wood pulp acid + C. beijerinckii CC101 7.9 0.33 0.11 (Lu et al. 2013) detoxification

Butanol recovery Principle Advantages Limitations technique High adsorbent cost, low efficiency, low Adherence of solvents to silicalite Easy to operate, low energy selectivity (will absorb any component), Adsorption resin, clay, activated carbon, or other requirement low adsorbent capacity (loading: ~0.1 adsorptive materials g/g) Require a high temperature or vacuum for Volatile solvents being stripped out by Easy to operate, no harm to the Gas stripping sufficient volatility, low selectivity gases and then condensed culture, no fouling (separation factor: 6~20) Liquid-liquid Using the solubility differences of High selectivity, efficient Forming emulsion, toxic to the culture extraction solvents Membrane-based extraction, High selectivity, low toxic to the Poor stability, membrane fouling, high Perstraction separating the fermentation broth from culture compared to liquid-liquid cost the extractive solvents extraction Pervaporation Using membrane to selectively let the High selectivity (separation 53 Membrane fouling, high cost vaporous solvents pass through factor: 5~100)

Table 2.4 Comparison of advanced butanol separation techniques

References: (Dhamole et al. 2012; Durre 1998; Ennis et al. 1986; Gapes et al. 1996; Groot et al. 1992; Qureshi and Maddox 2005)

Butanol Feed Adsorbent adsorption Adsorbent material C loading References BuOH capacity (g/L) (g/L) (mg/g) (Groot and Activated carbon Norit W52 15.0 252 10 Luyben 1986) (Abdehagh et Activated carbon F-400, F-600 N/A 200-300 N/A al. 2013) (Ennis et al. XAD-16 9.2 75 85 1987) XAD-2 16.5 78 10 (Groot and XAD-4 14.4 100 10 Luyben 1986; Qureshi et al. XAD-8 15.5 66 10 2005) 4.0 – Amberlite XAD-4 27 – 83 100 – 200 20.0 4.0 – Amberlite XAD-7 22 – 69 100 – 200 20.0 (Nielsen et al. 4.0 – 1988) Bonopore 23 – 74 100 – 200 20.0 4.0 – Bonopore, nitrated 13 – 55 100 – 200 resin 20.0 Poly(styrene-co- divinylbenzene) (including Dowex Optipore L-493, SD-2, 5.0 22.3 – 56.3 100 (Nielsen and M43, Amberlite IR-120, IRA- Prather 2009) 900 and etc) Poly(methacrylate) 5.0 34.7 100 Poly(butrylene phthalate) 5.0 7.4 100 (Liu et al. 6.5 84-100 40-80 KA-I (cross-linked polystyrene 2014) framework) (Lin et al. 0.2-25 140-305 20 2012) (Wiehn et al. Dowex Optipore L-493 N/A ~300 40-160 2014) (Yang et al. Polyvinylpyridine 14.9 68 100 1994) Continued

Table 2.5 Performances of different adsorbents for butanol recovery by adsorption (N/A, not available)

Table 2.5 continued

Feed Butanol adsorption Adsorbent Adsorbent material C References BuOH capacity (mg/g) loading (g/L) (g/L) (Milestone and 21.5 97 40 Bibby 1981) 11.7 – Silicalite 64 – 85 168 (Maddox 1982) 16.8 (Ennis et al. 8.3 63.5 85 1987) Zeolite ZSM-5 (Oudshoorn et al. (CBV901, 811 and 4.8 – 9.0 98 – 117 7 – 25 2009a) 28014) Zeolite ZSM-5 (Saravanan et al. 10 ~120 64 (CBV901 and 28014) 2010) (Sharma and Zeolite (MEL3-6) 2 222 N/A Chung 2011) Metal-organic (Saint Remi et al. Framework (MOF) 1-70 ~300 N/A 2011) ZIF-8

Adsorbent (resin) Solvent production Fermentation model/ Equivalent Solvent Solvent Culture species Capacitya References adsorption Type total solvent productivi yield (mg/g) conc. (g/L) ty (g/L∙h) (g/g) Batch/ in situ C. beijerinckii (Groot and XAD-8 30 12.6 0.13 N/A adsorption LMD Luyben 1986) Two-stage continuous C. acetobutylicum 0.30- (Ennis et al. fermentation/ in-line XAD-16 64 11.0 1.2 P262 0.36 1987) adsorption Repeated batch/ batch C. acetobutylicum Bonopore, (Nielsen et al. 43 N/A N/A N/A adsorption ATCC 824 poly(styrene-co-DVB) 1988) Dowex Optipore SD- Batch/ in situ C. acetobutylicum 0.27- (Nielsen and 2, poly(styrene-co- 264 37 0.51 adsorption ATCC 824 0.40 Prather 2009) DVB) derivative Repeated fed-batch/ Reillex 425, (Yang and

56 C. acetobutylicum 61 47.2 1.69 0.32 fixed bed adsorption polyvinylpyridine Tsao 1995) Fed-batch/ fixed-bed 84 96.5 1.51 0.33 adsorption KA-I, cross-linked C. acetobutylicum (Liu et al. Fed-batch/ fixed bed polystyrene B3 2014) adsorption (MV framework 93 130.7 0.97 0.36 addition) Fed-batch/ expanded C. acetobutylicum Dowex Optipore L- (Wiehn et al. 300 40.7 0.72 0.28 bed adsorption ATCC 824 493 2014)

Table 2.6 Performance of different resins used for in situ butanol recovery coupled with fermentation (a Calculated based on

butanol; DVB, divinylbenzene; MV, methyl viologen; N/A, not available)

Stripping Condensation Stripping gas and gas Feed conditions Selectivity References Temp. (oC) Temp. (oC) recycle rate* (Qureshi and Integrated with fed-batch 34 -60 N2, 2.7 L/min ABE 4-30.5 Blaschek bioreactor 2001) Separate stripper, continuous (Qureshi et al. 65 - 67 3 - 4 N2, 2.5 L/min ABE 30.5 fermentation 1992)

Integrated with batch H2 and CO2, (Maddox et al. 34 -0.8 ABE 9.5 - 13 bioreactor 1.5 – 3.3 L/(L∙min) 1995) Butanol 10.3 - 13.8 57 Model solution 35 -2 N2, 4.6 L/min Acetone 4.1 - 6.4 Ethanol 4.9 - 7.9 (Ezeji et al. Butanol 6.7 - 13 2003) Integrated with batch 33 - 35 -2 H2 and CO2, 3 L/min Acetone 4.7 - 10.5 bioreactor Ethanol 4.7 - 9.3 Integrated with fed-batch (Ezeji et al. 33 - 35 -2 H2 and CO2, 6 L/min Butanol 10.3 - 22.1 bioreactor 2004a)

Table 2.7 Solvent selectivities of processes integrated with gas stripping for butanol recovery (* L/min: liter gas per minute;

L/(L∙min): liter gas per liter broth per minute)

Fermentation ABE ABE yield ABE Productivity Substrate Strain References mode (g/L) (g/g) (g/L∙h) C. acetobutylicum NCIB Glucose Continuous ~10 0.36 0.58 (Mollah and Stuckey 1993) 8052 Batch 70.0 0.35 0.32 (Maddox et al. 1995) Whey permeate C. acetobutylicum P262 Continuous 69.1 0.38 0.26 (Qureshi et al. 1992) Glucose C. beijerinckii BA101 Batch 79.5 0.47 0.60 (Ezeji et al. 2003) Glucose C. beijerinckii BA101 Fed-batch 232 0.47 1.16 (Ezeji et al. 2004a) Wheat straw C. beijerinckii P260 Batch 47.6 0.37 0.36 (Qureshi et al. 2007) Soluble corn starch Batch 23.9 0.43 0.31 Saccharified corn C. beijerinckii BA101 Batch 26.5 0.41 0.40 (Ezeji et al. 2007c) starch Fed-batch 81.3 0.36 0.59 C. acetobutylicum Cassava bagasse Fed-batch 108.5 037 0.47 (Lu et al. 2012) JB200

C. acetobutylicum Batch 31.8 0.40 0.66 (Xue et al. 2013) Glucose JB200 Fed-batch 73.3 0.40 0.36 (Xue et al. 2014) 70% WPH* 12.9 0.39 0.17 C. beijerinckii CC101 Batch (Lu et al. 2013) Detoxified WPH* 17.7 0.44 0.25 C. acetobutylicum ATCC Glucose Fed-batch N/A N/A 0.22-0.73 (Setlhaku et al. 2013) 824

Table 2.8 Integrated fermentation-gas stripping process for ABE production by Clostridia from various substrates (* WPH, wood

pulp hydrolysate)

Feed Membrane Active layer Feed 1-butanol Total flux BuOH flux Separation temperature References material thickness (µm) concentration (wt%) (g/m2·h) (g/m2·h) factor (ºC) PDMS/hollow (Dong et al. 10 1 40 1282 - 43 fiber 2014) (Liu et al. PDMS/ceramic 10 1 40 457 - 26 2011) (Niemisto et PDMS/PANa 4 3.5 42 - 800 22-29 al. 2013) (Li et al. Tri-layer PDMS 65-200 2 37 40-132 20-50 32-50 2010) (Hickey et al. PDMS 25 0-7 50 200-1000 0-700 15-35 1992) (Hickey et al. PMS - 0-7 50 130-380 0-200 10-15

59 1992)

Silicone 50 53-350 - 42-49 (Huang and Silicalite-filled 1 30-70 Meagher 19 63-607 - 86-111 silicone 2001) (Fadeev et al. PTMSP - 0.3-6 25-70 60-2097 16-347 41-78 2001) (Liu et al. PEBA 30-100 5 23 65-179 19-42 6-8 2005) ZIF-71 filled (Liu et al. 10-20 1 37 520 - 18.8 PEBAb 2013) ZSM-5 filled (Tan et al. - 2.5 30-45 - 90-240 22-30 PEBA 2013) Continued Table 2.9 Butanol separation performance of different membranes by pervaporation (PDMS: polydimethylsiloxane; PAN: polyacrylonitrile; PMS: poly(methoxy siloxane); PTMSP: poly(1-trimethylsilyl-1-propyne); PEBA: poly(ether block amide); ZIF: zeolitic imidazolate framework; EPDM: ethylene propylene diene rubber; SBR: styrene butadiene rubber; TOA: trioctylamine; PVDF: poly(vinylidene difluoride); PUR: polyurethane)

Table 2.9 continued

Feed Membrane Active layer Feed 1-butanol Total flux BuOH flux Separation temperature References material thickness (µm) concentration (wt%) (g/m2·h) (g/m2·h) factor (ºC) EPDM - 1-10 30 - 0-20 5 (Jitesh et al. SBR - 1-10 30 - 0-25 10-20 2000) (Thongsukm TOA/liquid - 1.5 54 - 11 275 ak and Sirkar membrane 2007) (Srinivasan et PVDF 120 7.5 50 4126 - 6.4 al. 2007) (Boddeker et PUR 50 1 50 88 10 9 al. 1990) 41-141 1.5 47 120-278 45-97 39-45

60 PDMS 100 0.5-3.0 47 120-213 21-118 41-45 This work 100 1.5 27-56 71-219 27-83 38-44 PDMS/zeolite 100 1.5 27-56 48-171 22-100 60-85

Fermentation ABE ABE yield Productivity Strain Membrane References mode (g/L) (g/g) (g/L.h)

C. acetobutylicum (Qureshi et al. Continuous Polyethylene hollow fiber 42.0 0.34 0.14 P262 1992) (Qureshi and Fed-batch C. beijerinckii BA101 Silicone membrane 165 0.43 0.98 Blaschek 2000) C. acetobutylicum Silicalite-silicone composite (Qureshi et al. Fed-batch 155 0.35 0.18 ATCC 824 membrane 2001b) Continuous C. acetobutylicum PDMS composite (Van Hecke et al. N/A 0.36 0.37 ATCC 824 membrane 2012)

C. acetobutylicum PDMS composite (Van Hecke et al. Continuous 41.8 0.35 1.13 ATCC 824 membrane 2013)

Table 2.10 Integrated fermentation-pervaporation process for ABE production

Recovery Fermentation ABE ABE yield Productivity Substrate Strain References technique mode (g/L) (g/g) (g/L.h)

C. acetobutylicum 50.5– (Roffler et al. Glucose Fed-batch 0.33–0.36 1.4–2.3 ATCC 824 96.5 1987) Liquid-liquid C. acetobutylicum (Qureshi et al. Whey permeate Continuous 23.8 0.35 0.14 extraction P262 1992) C. acetobutylicum B (Bankar et al. Glucose Continuous 25.3 0.35 2.5 5313 2012) Whey permeate (Qureshi and Batch 136.6 0.44 0.21 + lactose 62 Perstraction C. acetobutylicum Maddox 2005) P262 (Qureshi et al. Whey permeate Continuous 57.8 0.37 0.24 1992)

Table 2.11 Integrated fermentation-recovery process for ABE production (liquid-liquid extraction and perstraction)

Figure 2.1 Metabolic pathways in Clostridium acetobutylicum during both acidogenesis

(dotted arrows) and solventogenesis (solid arrows)

Reference: (Lee et al. 2008)

Figure 2.2 Effect of feed butanol concentration on specific energy requirement for butanol recovery by distillation ((XB)F: butanol mass fraction in the feed solution)

Reference: (Matsumura et al. 1988)

A B

C Figure 2.3 Schematic diagrams of alternative butanol recovery techniques. A. Adsorption, B. Gas stripping, C. Pervaporation

Reference: (Vane 2008)

Figure 2.4 Mass transfer at a gas/liquid interface

Chapter 3: Butanol Production in Fed-batch Fermentation with In Situ Product

Recovery by Adsorption

Abstract

Four commercial materials were identified as potential adsorbents for butanol separation. These four adsorbents, including activated carbon Norit ROW 0.8, zeolite

CBV901, polymeric resin Dowex Optipore L-493 and SD-2, showed high specific loading and adsorbent-aqueous partitioning coefficients for butanol. Adsorption isotherms and their regressions with Langmiur model were further studied for these adsorbents, which provided theoretical basis for prediction of the amount of butanol adsorbed on these adsorbents. In batch fermentation with in situ adsorption without pH control, activated carbon showed the best performance with 21.9 g/L total butanol production, and 71.3 g/L glucose consumption. Total butanol production with activated carbon increased by 87.2%, 51.0%, 44.1% and 90.4%, respectively, compared to the control (without adsorption), L-493, SD-2 and CBV901. The integration of adsorption by activated carbon, with both free and immobilized cell fermentation, was demonstrated to be successful. The control free cell fermentation produced 18.3 g/L butanol in 54 h with a butanol productivity of 0.34 g/L·h, while free cell fermentation with adsorption 67

produced >31.6 g/L butanol in 106 h with a butanol productivity of >0.30 g/L·h, offering a >70% increase in butanol titer. The control immobilized cell fermentation produced

16.4 g/L butanol in 47 h with a butanol productivity of 0.35 g/L·h, while immobilized cell fermentation with adsorption produced ~54.6 g/L butanol in 122 h with a butanol productivity of ~0.45 g/L·h, an increase of ~30% and ~200% in butanol productivity and butanol titer, respectively, compared to the control experiments. Furthermore, ~150 g/L of butanol in the condensate could be recovered from desorption of adsorbents, which was easily concentrated to ~640 g/L after a simple and naturally occurring phase separation. Therefore, based on the estimation on energy consumption of other separation technology (typically >10 kJ/g), our in situ product recovery (ISPR) process with activated carbon only required 4.8 kJ/g butanol, with greater energy return, showing its potential economical value for product recovery and integration with butanol fermentation to simultaneously remove inhibitory product.

3.1 Introduction

Due to concerns about the eventual depletion of crude oil and escalating prices of petroleum-derived products, n-butanol has attracted revived attention as a renewable transportation fuel alternative (Durre 1998; Durre 2007; Lee et al. 2008). However, due to severe product inhibition caused by end product butanol, conventional ABE fermentation is limited by low product titer, yield, and productivity, resulting in intensive energy consumption during recovery by distillation (Oudshoorn et al. 2009a). A massive effort has been made on improving butanol tolerance and production of butanol- 68

producing Clostridia through modern biotechnology, such as mutagenesis and metabolic engineering, whereas 2% w/v of butanol production was still a bottleneck in the development of ABE fermentation (Chen and Blaschek 1999; Kumar and Gayen 2011;

Papoutsakis 2008).

An integrated bioprocess, in which the potentially inhibitory product could be continuously removed from fermentation broth, has important advantages in improving conversion and productivity compared to the conventional process (Schugerl 2000).

Several online integrated butanol recovery methods, including adsorption (Nielsen and

Prather 2009; Qureshi et al. 2005), liquid-liquid extraction (Dhamole et al. 2012; Evans and Wang 1988), pervaporation (Matsumura et al. 1988; Qureshi and Blaschek 1999;

Qureshi et al. 1999) and gas stripping (Qureshi and Blaschek 2001; Xue et al. 2013) have been investigated for inhibitory product removal and process improvement. Among them, in situ product recovery (ISPR) by adsorption was considered to be effective in recovering butanol in situ and reducing the inhibition effect (Groot et al. 1992; Qureshi et al. 1992), even though it has been criticized for low adsorption capacity (Durre 1998). It was demonstrated that adsorption required less energy, and possessed the properties of rapid adsorption and ease of desorption and regeneration (Oudshoorn et al. 2009b).

The most commonly used materials for adsorption were generally from three types of adsorbent: activated carbon or bone charcoal, synthetic silicalite (silicalite-1), and polymeric resins (typically ion-exchange resins) (Nielsen and Prather 2009;

Oudshoorn et al. 2009a; Qureshi et al. 2005). Numerous studies have focused on evaluating the butanol adsorption capacity of various adsorbents from model solution and

fermentation broth (Qureshi et al. 2005). However, it is difficult to provide guidance for adsorbent selection.

Nielsen and Prather screened a variety of commercial resins and found that resin

Dowex Optipore SD-2 had the best performance, with an achievable butanol titer of 2.22% w/v and high butanol recovery rate, in spite of its expensive price (Nielsen and Prather

2009). About the desorption process of other materials: an extremely high butanol titer

(98% w/v) in the condensate was successfully obtained by sequential heating after adsorption of butanol in a 0.5% solution using silicalite, but biocompatibility of this silicalite was not tested (Milestone and Bibby 1981). In addition, when employing activated carbon Norit W52 (powder) for adsorption, it was found that the fermentation with Clostridium beijerinckii LMD 27.6 was drawn towards butyric and acetic acid production, which threw doubt on the application of activated carbon as an adsorbent integrated with butanol fermentation (Groot and Luyben 1986). Until now, some attempts have been made to investigate in situ product recovery (ISPR) by adsorption in butanol fermentation, but there are no detailed descriptions about either fermentation performance or butanol recovery by desorption using polymeric resin or silicalite, especially lacking those about activated carbon (Nielsen et al. 2010; Nielsen et al. 1988;

Yang et al. 1994).

The goal of this study was to investigate and demonstrate the feasibility of in situ adsorption for high-titer butanol production in fed-batch fermentation w/o immobilized cell fermentation of C. acetobutylicum JB200. A variety of commercial adsorbents, including activated carbon, resin and zeolite, were screened for their ability to take up n-

butanol from model solutions. Promising candidates were further studied by incorporating the adsorption isotherm, regression and in situ adsorption integrated with batch and fed-batch fermentation. The best adsorbent was then used in immobilized cell fermentation. The present study provides the first demonstration of the ability of activated carbon as an effective adsorbent for in situ butanol recovery from fed-batch fermentation. Activated carbon based adsorption and desorption processes for high-titer butanol production were investigated and are discussed in this paper.

3.2 Materials and methods

3.2.1 Screening adsorbents for butanol adsorption

Zeolite CBV901 was purchased from Zeolyst International. Active carbon Norit

ROW 0.8 was supplied by Sigma-Aldrich. Polymeric resin Dowex Optipore L-493 and

Dowex Optipore SD-2 were manufactured by Dow. Screening experiments of those adsorbents were performed in 50 mL capped tubes containing 25 mL solution. 1g of the desired adsorbents was added to ~10 g/L butanol solution to initiate the experiments.

Adsorbent fraction (Xr), specific loading (L) and partitioning coefficient (Kr) were determined to evaluate their ability to take up butanol from model solution, expressed by the following equations:

(7)

(8)

(9)

where VAq represents the volume of aqueous solution, md is the mass of adsorbent added, Xr is the fraction of adsorbent per volume solution, CAq is the butanol concentration in model solution, and and represent time at initial and equilibrium conditions, respectively.

3.2.2 Determination of adsorption isotherm

The desired adsorbents, ranging between 0.25 and 6 g, were added to 15 mL model solution with an initial butanol concentration of ~39.6±1.8 g/L. Mixtures equilibrated for 24 h at 37 or 60 oC with agitation at 150 rpm. The butanol adsorption capacity of the adsorbents was calculated from the butanol concentration difference between the initial and final state, which was at equilibrium with adsorbents.

3.2.3 Simulation and predictions

The temperature-dependent Langmuir isotherm was chosen for this study to represent adsorption isotherms. The Langmuir isotherm is the most common isotherm model, and is derived for monolayer adsorption on homogeneous surfaces. Aqueous- adsorbent 1-butanol equilibrium isotherms and predictions were made via least-squares regression by Minitab software. The equilibrium data was fitted to a simple Langmuir isotherm:

q K C q  m 1KC (10)

Where q (g/g) is the amount of 1-butanol adsorbed per unit weight of adsorbent

(g/g); C (g/L) is the equilibrium concentration of 1-butanol in the solution; K is the

Langmiur coefficients; qm (g/g) represents maximum adsorption capacity.

3.2.4 Culture and medium

Clostridium beijerinckii BA101 was used in serum bottle batch fermentation with adsorption, due to its excellent performance without PH control. Clostridium acetobutylicum strain JB200 derived from ATCC 55025 was used in this study. The seed culture for fermentation study was prepared in Clostridial growth medium (CGM) containing 30 g/L of glucose, 2 g/L of yeast extract, 1 g/L of Tryptone, minerals and vitamins in a phosphate buffer as described in (Lu et al. 2012), and incubated at 37 °C for

~16 h until active growth was observed. The medium was sterilized by autoclaving at 121 oC and 15 psig for 30 minutes. All solutions were purged with nitrogen for 1 h through a

3.2.5 Batch fermentation with in situ adsorption

In order to test the biocompatibility of selected adsorbents (activated carbon, resin

SD-2, resin L-493 and zeolite CBV901) in serum bottle experiment, batch fermentation was studied using P2 medium containing glucose (70 g/L), yeast extract (1 g/L),

phosphate buffer (0.5 g/L of KH2PO4 and 0.5 g/L of K2HPO4), ammonium acetate (2.2 g/L), vitamins (1 mg/L of para-amino-benzoic acid, 1 mg/L of thiamin and 0.01 mg/L of biotin), and mineral salts (0.2 g/L of MgSO4∙7H2O, 0.01 g/L of MnSO4∙H2O, 0.01 g/L of

FeSO4∙7H2O, 0.01 g/L of NaCl), prepared according to previously described procedures

(Qureshi and Blaschek 1999). The amount of adsorbents and medium volume were 4 g and 80 ml respectively, with a weight ratio of 5% in serum bottles. Liquid samples were drawn from the serum bottle periodically for analysis of glucose, free cell density and fermentation products.

3.2.6 Adsorption of broth components on selected adsorbent

The best adsorbent, from previous screening in batch fermentation with in situ adsorption, was then evaluated for its adsorption preference in complex model solution simulating the actual close-to-end fermentation broth (with low substrate concentration and high product concentration). In a solution originally containing ~20 g/L of glucose,

~20 g/L of acetone, ~4 g/L of ethanol, ~40 g/L of butanol, ~10 g/L of acetic acid, and ~4 g/L of butyric acid, different amounts of selected adsorbent were added. Then, the mixture of selected adsorbent and model solution was put in a shaking bed at 100 rpm, and 37 ºC for 24 hours. After equilibrium, the concentration of remaining solution was measured and the specific loading of every component was calculated.

3.2.7 Fed-batch fermentation with in situ adsorption with activated carbon

Figure 3.1 shows the integrated ABE fermentation and adsorption system consisting of a stirred-tank reactor (1.5 L working volume) and an external glass column

(i.d. 50 mm, length: 400 mm, 250 ml working volume) packed with 75 g of activated carbon (Norit ROW 0.8, Sigma-Aldrich, St. Louis, MO). The bioreactor with P2 medium

(same as mentioned before) and the column with activated carbon were autoclaved separately for 45 min, and aseptically connected after sterilization. Before inoculation with 100 ml of overnight culture in serum bottles, the whole system was sparged with nitrogen to ensure an oxygen-free environment. During fed-batch fermentation, a concentrated glucose solution (~360 g/L) was pulse-fed when glucose in the fermentation broth was nearly depleted. Adsorption was initiated by circulating the fermentation broth between the fermentor and adsorption column at ~60 ml/min when the butanol concentration in the fermentation broth had reached ~10 g/L (at ~30 h). Similar fed-batch fermentation was also carried out with cells immobilized in a fibrous bed inside the stirred-tank bioreactor. The internal fibrous bed was made of a piece of cotton towel wound together with a stainless steel mesh affixed to the inner wall of the bioreactor.

Batch fermentation without adsorption was also carried out with an initial glucose concentration of ~85 g/L in the P2 medium at 37 oC and pH 5.0 as the control.

3.2.8 Desorption and product recovery

Desorption curves of butanol, water and ABE mixture were made for activated carbon by thermogravimetry, with a heating rate of 10 oC/min. Butanol, water and ABE mixture with a ratio of 6:3:1 were adsorbed by activated carbon at 24h for adsorption saturation. After saturation, water/solvents were removed from the ‘wet’ activated carbon as much as possible with filter paper. Then, 30.37±0.1 g of initially ‘crude dried’ activated carbon was weighed for the analysis of desorption curves. The heating temperature increased from 25 oC to 250 oC until the “crude dried” activated carbon was dried to a constant weight.

For the sequential heating experiment of activated carbon, 10g of activated carbon was equilibrated in 300 ml model solution with initial concentration of ~15 g/L butanol at

37 oC while stirred at 25 rpm for 24 h in 500 ml flask. Recovery of butanol from equilibrated activated carbon was then performed by first removing as much liquid as possible by filter paper and aspiration through a 50 ml syringe. The crude dried adsorbents were then desorbed at 40 oC to remove water, followed by butanol recovery at

200 oC.

After butanol fermentation with in situ adsorption ceased, the adsorbents saturated with solvent and water were taken for desorption and product recovery. The closed (with circulation) desorption system was composed of an oven with temperature control, a cold trap and a peristaltic pump (figure not shown).

3.2.9 Analytical methods

Cell biomass in the fermentation broth was estimated by measuring optical density at 600 nm with a spectrophotometer (UV-16-1, Shimadzu, Columbia, MD).

Glucose and products in the fermentation broth were assayed after cell removal through centrifugation at 13,200 rpm for 5 min. The glucose concentration was determined with

YSI 2700 Select Biochemistry Analyzer (Yellow Springs, Ohio). Butanol, acetone, ethanol, acetic acid and butyric acid were determined with a gas chromatograph (GC-

2014 Shimadzu, Columbia, MD) equipped with a flame ionization detector (FID) and a fused silica column (Stabilwax- ness and 0.25 mm ID,

Restek, Bellefonte, PA) following previously described method (Yu et al., 2011).

3.3 Results and discussion

3.3.1 Screening adsorbents for butanol adsorption

Adsorption is a promising process for in situ recovery of butanol from fermentation broth, due to its simplicity and biocompatibility. Butanol is usually adsorbed by adsorbent materials in a packed column from dilute solution, and then desorbed by heating the adsorbent to obtain a concentrated butanol solution (adsorbent regeneration) (Vane 2008). Specific loading of the adsorbents is the key parameter in selecting desired adsorbent materials, which has been reviewed in Table 3.1. The common adsorbent materials for butanol adsorption included three types: silicate, polymeric resin and zeolite. It was obvious that the adsorption capacities of bone charcoal 77

and Norit Row 0.8 for butanol were high, at 0.206 and 0.252 g butanol/g carbon, respectively (Groot and Luyben 1986). To be mentioned, Dowex Optipore L-493 and

SD-2 were able to reduce the aqueous butanol concentration by 85% and 83% from ~2% w/v initial butanol concentration, while achieving specific loadings of 0.175 and 0.152 g butanol/g resin, respectively (Nielsen and Prather 2009). Other resins and zeolites had a relatively low butanol affinity, with a butanol capacity of ≤ 0.10 g/g adsorbent. Among reported zeolites, CBV901 offered the highest specific loading of 0.10 g butanol/g zeolite.

Since reported data was gathered under different conditions, in this study, four adsorbents from different categories with the highest specific loading of butanol were selected to evaluate their ability to take up butanol under same circumstance. They were activated carbon Norit Row 0.8 (0.252 g/g), zeolite CBV901 (0.10 g/g), resin Dowex

Optipore SD-2 (0.152 g/g) and L-493 (0.175 g/g). These selected adsorbents should have high butanol affinity or potential values for in situ butanol recovery, as described in the literature. The adsorbents with a high specific loading were further studied for their adsorption isotherms at various temperatures, and evaluated for their biocompatibility in butanol fermentation with Clostridium.

3.3.2 Adsorption isotherm and prediction

The relative butanol affinity of the four selected adsorbents was assessed above.

The Adsorption isotherms could further characterize and accurately predict the performance of dynamic fermentation. Furthermore, temperature during the adsorption process affected adsorption capacity and was also taken into account. Equilibrium 78

isotherms of these four selected adsorbents were investigated at 37 oC and 60 oC, plotted in Figure 3.2A, B.

At these temperatures, the isotherms of these four adsorbents fitted satisfactorily with the Langmuir isotherm model and their Langmuir parameters are shown in Table 3.2.

In general, the specific loadings of these adsorbents increased with the increase of butanol concentration in solution, whereas they decreased with the increase of temperature from 37 oC to 60 oC. The adsorption capacity was dependent on the variation of temperature and butanol concentration in solution. The maximum adsorption capacity

(qm) also decreased with the increase of temperature. However, qm of Dowex L-493 did not show much variance as temperatures changed from 37 oC to 60 oC. From the

Langmiur parameters shown in Table 3.2, it was indicated that the greatest qm was 0.451 g butanol/g Norit ROW 0.8 and followed with 0.426 g butanol/g Dowex Optipore SD-2 and 0.414 g/g of Dowex Optipore L-493 at 37 oC.

3.3.3 Batch fermentation with in situ adsorption in serum bottles

Although adsorption equilibrium and isotherm studies screened out several candidates with high affinity for butanol, adsorption capacity is inconsequential if the adsorbents themselves are toxic, or inhibit cell metabolism. In order to evaluate the biocompatibility of adsorbents, batch fermentation with/without adsorption was conducted with Clostridium beijerinckii BA101, employing activated carbon Norit Row

0.8, zeolite CBV901, polymeric resin Dowex Optipore L-493 and SD-2. Table 3.3 showed the performance of butanol fermentation with in-situ butanol adsorption by 79

various adsorbents. For the control experiment without adsorption, with the medium initially containing ~70 g/L of glucose, about 11.7 g/L of butanol was produced when the fermentation ceased, with about 28.5 g/L of residual glucose in the fermentation broth.

Final pH decreased to 4.85 due to acetic and butyric acids production during the acidogenesis phase in spite of partial reassimilation of acids by cells for butanol production (Jones and Woods 1986).

CBV901 is the commercial H-Y type microporous zeolite, with a more hydrophobic property than other silicalites, such as CBV780 and higher Si/Al ratio ZSM5 zeolites (Halasz et al. 2005). When applied to in situ butanol adsorption, batch fermentation ceased with less glucose consumption and total butanol production compared to those of the control experiment, which indicated its toxicity to cells and lesser biocompatibility in butanol fermentation. Furthermore, compared to other adsorbents, CBV901 had a light specific gravity and suspended together with free cells in the fermentation broth which made it difficult to recycle from fermentation broth by an energy-efficient process like sedimentation.

Dowex Optipore L-493 and SD-2 were highly cross-linked macroporous polymer beads with high surface area and improved adsorption capacity for organic compounds.

Using L-493 and SD-2 as the adsorbents for butanol adsorption, 14.5 g/L and 15.2 g/L of total butanol production were obtained, with 24.5 g/L and 20.4 g/L of final glucose concentration at the end of batch fermentation, respectively. Higher butanol production and glucose consumption revealed their better biocompatibility in butanol fermentation compared to CBV901.

Norit ROW 0.8 is an extruded activated carbon with a unique pore size distribution and can be easily thermally reactivated. With in situ adsorption by Norit

ROW 0.8, almost all the glucose was utilized for acid and butanol production, with 21.9 g/L of total butanol production and 1.2 g/L of residual glucose in fermentation broth.

Undoubtedly, activated carbon allowed C. beijerinckii BA101 to grow in an uninterrupted manner, achieving higher butanol production and substrate utilization compared to the control and batch fermentation with other adsorbents.

3.3.4 Adsorption of broth components on selected adsorbent

Since fermentation integrated with adsorption by activated carbon Norit ROW 0.8 provided the highest butanol production and substrate utilization among all studied adsorbents, the remainder of our study focused on the application of activated carbon

Norit DOW 0.8, to further evaluate its biocompatibility and feasibility in butanol fermentation with in situ adsorption in bioreactor for longer duration. On the other hand, activated carbon Norit DOW 0.8 was the most economical adsorbent with a commercially competitive price (activated carbon of 63$/kg vs SD-2 of 239$/kg vs L-493 of 216$/kg from commercial distributor Sigma-Aldrich in 2012), implying its potential value for industrial application (Nielsen and Prather 2009).

The model solution simulating close-to-end fermentation broth originally contained 18.9 g/L of glucose, 19.4 g/L of acetone, 4.3 g/L of ethanol, 38.7 g/L of butanol, 10.6 g/L of acetic acid, and 4.0 g/L of butyric acid (butanol and acetone concentration in model solution were high initially, but would be decreased after 81

equilibrium with adsorbent). The data at equilibrium and specific loading of every component are shown in Table 3.4, from which we can see that Norit Row 0.8 adsorbs large amount of butanol, small amount of acetone, and trace amount of other components.

Norit Row 0.8 adsorbed more butanol, even though the equilibrium butanol concentration was lower than acetone. Compared to acetone, butanol had a much larger affinity for activated carbon Norit Row 0.8.

3.3.5 Fed-batch fermentation with adsorption by activated carbon

With the medium initially containing ~85 g/L of glucose, the time course of free cell control fermentation (without adsorption) is shown in Figure 3.3A. About 18.3 g/L of butanol was produced in ~54 h in the batch fermentation after the glucose ran out, giving a butanol productivity of ~0.34 g/L·h and butanol yield of 0.22 g/g glucose.

It should be highlighted that C. acetobutylicum JB200 is an adaptive mutant strain derived from ATCC 55025, which is an asporogenic mutant strain of ATCC 4259 (Jain et al. 1993). The sporogenic strains, including ATCC 4259 and the type strain ATCC 824, usually can only produce up to 12-14 g/L of butanol due to sporulation and degeneration caused by the accumulated butanol (Ezeji et al. 2003; Maddox 1989). ATCC 55025 in the fibrous bed bioreactor (FBB) system was able to produce up to ~16 g/L of butanol, at which autolysis occurred and most cells died quickly (data not shown). In contrast, JB200 was able to produce butanol at a much higher concentration of 17~21 g/L, depending on whether employing FBB system. The hyper-butanol producing capability of JB200 would

allow its use in fed-batch fermentation with in situ adsorption to pursue a higher butanol titer in fermentation broth, as demonstrated in this study.

The time courses of free cell fed-batch fermentation with adsorption, the control immobilized cell batch fermentation (without adsorption), and immobilized cell fed-batch fermentation with adsorption are shown in Figure 3.3B, C, D, respectively. When butanol concentration in the broth had reached ~10 g/L, adsorption was initiated by circulating the broth between the fermentor and adsorption column. For the free cell fed-batch fermentation integrated with adsorption, final butanol concentration in the fermentation broth reached 18.7 g/L in ~106 h, as shown in Figure 3.3B. After desorption in a closed system, 112.5 ml butanol solution of 167.1 g/L was obtained, corresponding to a total amount of 18.8 g butanol.

For the immobilized cell fermentation, batch fermentation in P2 medium was conducted first, to immobilize cells onto the cotton fiber. After the fermentation ceased

(glucose concentration approached zero), the broth was drained and replaced with fresh medium. The batch fermentation was repeated several times until the O.D.600 was greater than 6. During the first three batches, butanol yield was around 0.21 g/g glucose and productivity was around 0.35 g/L·h, as shown in Figure 3.3C. In the subsequent batch, the broth was circulated between the fermentor and the adsorption column after the butanol concentration had reached ~10 g/L. During the fermentation, adsorption was switched to a new adsorption column filled with fresh (regenerated) activated carbon when the butanol concentration in the broth exceeded ~10 g/L. Concentrated glucose and other nutrition were added during the fermentation. A total of ~260 g/L glucose was

consumed in 122 h during the fed-batch fermentation with adsorption, and three columns of activated carbon were used for butanol adsorption.

Since activated carbon had a greater affinity to butanol than acetone, acetone gradually accumulated later in the fermentation and became the limiting factor, as can be seen in Figure 3.3D. It is thus necessary to avoid the accumulation of acetone in order to alleviate its toxic effect and extend the fermentation span. Table 3.5 summarizes and compares the performance of free and immobilized cell fermentation with and without adsorption.

Not all the butanol adsorbed in the immobilized cell fed-batch fermentation could be desorbed from the adsorbent. Based on the situation with the free cell fermentation, the yield from the control experiment was 0.22 g/g and that from free cell with adsorption was 0.20g/g which would turn into 0.22 based on 80% recovery rate. Therefore it is reasonable to assume the butanol yield of immobilized cell with adsorption to be similar to that of the control immobilized cell fermentation (0.21 g/g). Assuming that butanol yield remained unchanged (0.21g/g), total butanol production and productivity would be

~54.6 g /L and ~0.45 g/L·h, respectively, for immobilized cell fermentation coupled with adsorption by activated carbon. The butanol productivity and titer represented an increase of ~30% and ~200% compared to the control fermentation without adsorption. Among all the fermentations studied, the immobilized cell fermentation with adsorption gave the highest butanol productivity and titer of ~0.45 g/L·h and ~54.6 g/L.

The ability of butanol adsorption was strongly controlled by the specific surface area of the adsorbent (Nielsen and Prather 2009). In the present study, total butanol

prediction calculated from butanol titer in broth and isotherm in either fed-batch fermentation was higher than total butanol production from butanol in broth and butanol recovery by desorption. The tiny particles (proteins, cells and etc.) and other solvents such as acetone and ethanol produced in ABE fermentation correspondingly adsorbed on the activated carbon, which could compete for the specific surface area with butanol, and lower butanol adsorption capability.

3.3.6 Studies on desorption and butanol recovery

The desorption curves for water, butanol and ABE mixtures are shown in Figure

3.4. Due to the same amount of “crude dried” activated carbon prepared for thermogravimetrical desorption, it is revealed in the Figure 3.4 that more water could be adsorbed on activated carbon than butanol or ABE mixtures. Furthermore, all water could be readily desorbed at low temperatures of 120~130oC, but butanol could only be completely desorbed at higher temperatures (~200 oC), indicating that water adsorption is weaker than butanol. Therefore, it is possible to first remove most water by heating the sample at lower temperatures, and then recover high concentration butanol at higher temperatures.

Previously, desorption of alcohols and water adsorbed on silicalite from aqueous solution was studied (Milestone and Bibby 1981). Water molecules could penetrate the channel system of silicalite but were also readily lost at low temperatures (40~50 oC), revealing weak adsorption, as suggested in this study.

In our study, adsorption of butanol by activated carbon was equilibrated at 5.8 g/L of butanol from an initial butanol concentration of ~1.5% w/v. A mixture containing ~60 g/L of butanol was obtained when heated at 40 oC and then ~150 g/L of butanol was obtained when temperature increased to 200 oC.

3.3.7 Comparison to other studies

Adsorption for in situ butanol recovery from butanol fermentation has been studied as an effective method to alleviate butanol toxicity, increase substrate utilization, and increase butanol production. Table 3.1 summarizes and compares recent studies on the application of various adsorbents for butanol adsorption. In general, selection or evaluation of materials from model solution or fermentation broth was the main focus, but only a few previous reports presented the integrated process of butanol fermentation with in situ toxic products removal by adsorption. Using polyvinylpyridine (PVP) resin as the adsorbent, a batch operation mode in the adsorption-coupled system was developed with different weight ratios of the adsorbent to the fermentation broth ranging 5%~30%

(Yang et al. 1994). With the medium initially containing ~92 g/L glucose, total butanol production and productivity increased with the increasing amount of PVP resin added to the medium. A 17.5 g/L of total butanol production and ~0.42 g/L·h of butanol productivity was achieved in the adsorptive batch fermentation system with a 30 wt.% of adsorbent. The butanol desorption experiment was carried out in a preparative- scale chromatographic system, where no detailed description was shown referring to butanol recovery data from desorption process. In the present study, a 5 % w/w of adsorbent was 86

employed in the integrated fed-batch fermentation, with a total butanol production of 31.6 g/L, suggesting that activated carbon was more efficient in improving butanol production than PVP resin. It was demonstrated that the addition of resin Dowex Optipore SD-2 to batch culture facilitated high butanol titer of 2.22 % w/v, nearly double compared to inhibitory threshold of C. acetobutylicum ATCC 824. Recovery of butanol from resins via thermal treatment at 100oC oil bath had high efficiency and a recovery rate of 78~85%

(Nielsen and Prather 2009). However, neither the system design of desorption nor recovered butanol titer in the condensate was shown in these reports. A butanol concentration of 98% w/v or 980 g/L has been reported to be achieved by sequentially heating silicalite saturated in a 0.5% model solution. But butanol concentration could not exceed 810 g/L (specific gravity of pure butanol, ρ=0.81) (Milestone and Bibby 1981).

Hence, it was assumed that the actual butanol concentration was in the range of 790~810 g/L.

A similar butanol desorption approach was also designed in this study. From the desorption curves, water tends to be lost at low temperatures whereas butanol was completely desorbed at higher temperatures. However, there was no distinct range of temperatures to indicate that water and butanol could be desorbed separately. So based on desorption curves and the report described, ~60 g/L of butanol concentration in the condensate was obtained when heated at 40 oC and ~150 g/L of butanol was obtained when the temperature was increased to 200 oC in this study. Furthermore, it was noted that n-butanol has a low solubility of 7.7% w/w (20 oC) in water, and undergoes phase separation when concentration is higher than 8% w/w. Therefore the condensate

containing ~150 g/L of butanol from 200 oC heating treatment formed two phases and the upper organic phase had a butanol concentration of ~640 g/L, which could be easily further purified by distillation (Xue et al. 2012).

It was found that activated carbon/charcoal adsorption as a detoxification method could help remove more inhibitors and improve fermentation performance when pretreated lignocellulosic hydrolysates were used as substrates (Mussatto and Roberto

2004a; Mussatto and Roberto 2004b). When applying activated carbon (Norit W52) as an adsorbent for integrated butanol fermentation, it was found that acid production was enhanced, which resulted in the failure of butanol fermentation. The same phenomena also appeared on XAD2 and XAD4 (Groot and Luyben 1986). Adsorption data in the present study suggested that activated carbon had a high butanol affinity and better biocompatibility in butanol fermentation. Furthermore, adsorption strategy in fed-batch fermentation and desorption protocols are demonstrated as a model for further integration of this technology. To the best of our knowledge, this is the first study demonstrating the feasibility of stable high-titer butanol production in ABE fermentation via integrated adsorption with activated carbon as well as incorporating the design of adsorption and desorption strategy.

The greatest energy consumption for butanol recovery by distillation from conventional batch fermentation was primarily associated with water vaporization from fermentation broth containing 1~2% w/v butanol, accounting for approximately 98~99% of the total demand. However, since the specific heat capacity of activated carbon was

0.84 J/g·K, five-times lower than that of water (4.2 J/g·K), less energy was required to

heat the activated carbon phase to the temperature required for butanol separation. On the other hand, in the control batch fermentation with C. acetobutylicum JB200, butanol concentration could reach 17.2 g butanol/kg aqueous at its inhibitory threshold. In contrast, butanol adsorption on activated carbon could allow butanol concentration reach as high as 250.7 g butanol/kg activated carbon based on 100% recovery by desorption, a

15-fold increase. Therefore, butanol recovery costs could be dramatically reduced by collecting butanol in this activated carbon phase, where its local concentration was substantially increased. This meaningful result resembled that of Dowex Optipore SD-2 as the adsorbent for butanol adsorption, discussed in a previous report (Nielsen and

Prather 2009).

Considering the energy content of butanol, 36 KJ/g, minimization of energy requirement for recovery was essential in order to obtain the greatest net energy increase.

Table 3.6 compared the energy consumption in butanol recovery from fermentation broth by different separation methods. Conventional distillation is the most energy intensive, requiring more than ~79 kJ/g butanol produced from a butanol-water solution containing

0.5% w/v butanol (Matsumura et al. 1988),and dramatically decreasing to ~36 kJ/g butanol and 24 kJ/g butanol for a feed solution containing 1% and 1.5% butanol, respectively. As shown in Table 3.6, estimation of energy requirements for butanol recovery by gas stripping, pervaporation, extraction/perstraction and adsorption were different in these literatures depending on the process conditions (Groot et al. 1992;

Oudshoorn et al. 2009b; Qureshi et al. 2005). Every separation method had its energy- economical way for butanol recovery, if a desirable design and strategy were developed.

According to the data discussed above, our activated carbon-based ISPR process required

4.8 kJ/g butanol and offered a great energy return. Therefore, it was clear that integrated butanol fermentation with in situ butanol adsorption by activated carbon offers an energy- efficient and environmentally friendly process for the production of biobutanol that can be economically competitive to the petroleum-based butanol.

3.4 Conclusions

Adsorption is a promising process for butanol recovery and inhibitory product removal from fermentation broth, due to its simplicity and biocompatibility. From the commercially available candidate pool, a variety of materials have been screened and evaluated for their butanol adsorption capacity from model solution. Among these adsorbents, activated carbon (Norit ROW 0.8) showed the best specific loading and adsorbent-aqueous partitioning coefficient of butanol. In batch fermentation without pH control, 21.9 g/L of total butanol production could be achieved with in situ adsorption by activated carbon, increased by 87.2%, 51.0%, 44.1% and 90.4%, respectively, compared to those in the control, L-493, SD-2 and CBV901 experiments. In integrated fed-batch fermentation with in situ butanol adsorption by activated carbon, both free cell and immobilized cell fermentation can increase the total butanol titer. Especially immobilized cell fermentation, it increased the butanol productivity and titer by ~30% and ~200%, respectively, compared to the control fermentations. Furthermore, ~150 g/L of butanol solution could be recovered in the condensate by heating butanol-adsorbed activated carbon, which was easily concentrated to ~640 g/L after simple phase separation. The 90

specific energy cost of the ISPR process was estimated to be ~4.8kJ/g butanol with great energy efficiency, exhibiting its economical potential for applications in butanol fermentation to simultaneously remove inhibitory product and product recovery.

Acknowledgments

This work was supported in part by the Ohio Department of Development-Third

Frontier Advanced Energy Program (Tech 08-036), the National Science Foundation

STTR program (IIP-0810568, IIP-1026648), and Advanced Research Projects Agency-

Energy (DE-AR0000095). Financial support from the Fundamental Research Funds for the Central Universities (DUT11RC(3)77), China Postdoctoral Science Foundation

(20110491527), and China Scholarship Council (2009100607) are also acknowledged.

3.5 References

Chen CK, and Blaschek HP. 1999. Acetate enhances solvent production and prevents degeneration in Clostridium beijerinckii BA101. Appl Microbiol Biotechnol 52(2):170-173.

Dhamole PB, Wang ZL, Liu YQ, Wang B, and Feng H. 2012. Extractive fermentation with non-ionic surfactants to enhance butanol production. Biomass Bioenerg 40:112-119.

Durre P. 1998. New insights and novel developments in clostridial acetone/butanol/isopropanol fermentation. Appl Microbiol Biot 49(6):639-648.

Durre P. 2007. Biobutanol: an attractive biofuel. Biotechnol J 2(12):1525-1534.

Evans PJ, and Wang HY. 1988. Enhancement of Butanol Formation by Clostridium acetobutylicum in the Presence of Decanol-Oleyl Alcohol Mixed Extractants. Appl Environ Microbiol 54(7):1662-1667.

Ezeji TC, Qureshi N, and Blaschek HP. 2003. Production of acetone, butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping. World J Microb Biot 19:595-603.

Groot WJ, and Luyben KCAM. 1986. Insitu Product Recovery by Adsorption in the Butanol Isopropanol Batch Fermentation. Appl Microbiol Biot 25(1):29-31.

Groot WJ, Vanderlans RGJM, and Luyben KCAM. 1992. Technologies for Butanol Recovery Integrated with Fermentations. Process Biochem 27(2):61-75.

Halasz I, Agarwal M, Senderov E, Marcus B, and Cormier W. 2005. Molecular spectroscopic study of the fine structure of aluminum deficient, hydrophobic zeolites. Studies in Surface Science and Catalysis 158:647-654.

Jain M, Beacom D, and Datta R. 1993. Mutant strain of C. acetobutylicum and process for making butanol. Patent US5192673 A.

Jones DT, and Woods DR. 1986. Acetone-Butanol Fermentation Revisited. Microbiol Rev 50(4):484-524.

Kumar M, and Gayen K. 2011. Developments in biobutanol production: New insights. Appl Energ 88(6):1999-2012.

Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, and Jung KS. 2008. Fermentative butanol production by clostridia. Biotechnol Bioeng 101(2):209-228.

Maddox IS. 1982. Use of Silicalite for the Adsorption of Normal-Butanol from Fermentation Liquors. Biotechnol Lett 4(11):759-760.

Maddox IS. 1989. The acetone-butanol-ethanol fermentation: recent progress in technology. Biotechnol Genet Eng Rev 7:189-220.

Matsumura M, Kataoka H, Sueki M, and Araki K. 1988. Energy Saving Effect of Pervaporation Using Oleyl Alcohol Liquid Membrane in Butanol Purification. Bioprocess Eng 3(2):93-100.

Milestone NB, and Bibby DM. 1981. Concentration of Alcohols by Adsorption on Silicalite. J Chem Technol Biot 31(12):732-736.

Mussatto SI, and Roberto IC. 2004a. Alternatives for detoxification of diluted-acid lignocellulosic hydrolyzates for use in fermentative processes: a review. Bioresource Technol 93(1):1-10. 92

Mussatto SI, and Roberto IC. 2004b. Optimal experimental condition for hemicellulosic hydrolyzate treatment with activated charcoal for xylitol production. Biotechnol Prog 20(1):134-139.

Nielsen DR, Amarasiriwardena GS, and Prather KLJ. 2010. Predicting the adsorption of second generation biofuels by polymeric resins with applications for in situ product recovery (ISPR). Bioresource Technol 101(8):2762-2769.

Nielsen DR, and Prather KJ. 2009. In Situ Product Recovery of n-Butanol Using Polymeric Resins. Biotechnol Bioeng 102(3):811-821.

Nielsen L, Larsson M, Holst O, and Mattiasson B. 1988. Adsorbents for Extractive Bioconversion Applied to the Acetone-Butanol Fermentation. Appl Microbiol Biot 28(4-5):335-339.

Oudshoorn A, van der Wielen LAM, and Straathof AJJ. 2009a. Adsorption equilibria of bio-based butanol solutions using zeolite. Biochem Eng J 48(1):99-103.

Oudshoorn A, van der Wielen LAM, and Straathof AJJ. 2009b. Assessment of Options for Selective 1-Butanol Recovery from Aqueous Solution. Ind Eng Chem Res 48(15):7325-7336.

Papoutsakis ET. 2008. Engineering solventogenic clostridia. Curr Opin Biotech 19(5):420-429.

Qureshi N, and Blaschek HP. 1999. Butanol recovery from model solution/fermentation broth by pervaporation: evaluation of membrane performance. Biomass Bioenerg 17(2):175-184.

Qureshi N, and Blaschek HP. 2001. Recovery of butanol from fermentation broth by gas stripping. Renew Energ 22(4):557-564.

Qureshi N, Hughes S, Maddox IS, and Cotta MA. 2005. Energy-efficient recovery of butanol from model solutions and fermentation broth by adsorption. Bioproc Biosyst Eng 27(4):215-222.

Qureshi N, Meagher MM, and Hutkins RW. 1999. Recovery of butanol from model solutions and fermentation broth using a silicalite silicone membrane. J Membrane Sci 158(1-2):115-125.

Schugerl K. 2000. Integrated processing of biotechnology products. Biotechnol Adv 18(7):581-599.

Vane LM. 2008. Separation technologies for the recovery and dehydration of alcohols from fermentation broths. Biofuel Bioprod Bior 2(6):553-588.

Xue C, Zhao J, Liu F, Lu C, Yang ST, and Bai FW. 2013. Two-stage in situ gas stripping for enhanced butanol fermentation and energy-saving product recovery. Bioresour Technol 135:396-402.

Yang XP, Tsai GJ, and Tsao GT. 1994. Enhancement of in-Situ Adsorption on the Acetone-Butanol Fermentation by Clostridium acetobutylicum. Separ Technol 4(2):81-92.

3.6 Tables and figures

C in feed Specific loading, Type Adsorbents butanol References

(g/L) (g/g)

on (Groot and Luyben Norit Row 0.8 15.0 0.252

1986) Activate d carb d Norit Row 0.8 0.5~30 0.10~0.45 This study XAD-2 16.5 0.078 (Groot and Luyben XAD-4 14.4 0.100 1986) XAD-8 15.5 0.069 Polyvinylpyridine 14.9 0.068 (Yang et al. 1994) Dowex Optipore L- ~20 0.175 493 (Nielsen and Prather

Resin Dowex Optipore 2009) ~20 0.152 SD-2 Dowex Optipore 1.5~35 0.10~0.39 This study SD-2 Dowex Optipore L- 2.0~35 0.10~0.37 This study 493 (Oudshoorn et al. CBV811 4.8~9.0 0.98~0.117

2009a) Silicalite 11.7~16.8 0.064~0.085 (Maddox 1982) CBV28014 ~10 0.092 (Saravanan et al. Zeolite CBV901 ~10 0.10 2010) CBV901 1.0~35 0.10~0.23 This study

Table 3.1 Comparison of n-butanol adsorption capacity of various adsorbents (Butanol model solution was used except for the one by Maddox (1982))

37 oC 60 oC Adsorbents K qm (g/g) K qm (g/g)

Norit ROW 0.8 0.897 0.451 0.907 0.392

CBV901 0.857 0.216 1.104 0.185

Dowex L-493 0.183 0.414 0.144 0.420

Dowex SD-2 0.169 0.426 0.162 0.349

Table 3.2 Langmuir parameters from least-squares regression

Activated Control L-493 SD-2 CBV901 carbon

Initial glucose concn.(g/L) 71.5 72.5 70.5 69.0 71.0

Final glucose concn. (g/L) 28.5 1.2 24.5 20.4 30.1

Final butanol in the broth 11.7 4.1 4.8 5.2 4.5 (g/L)

Final pH 4.85 4.60 4.25 4.39 4.56

Total butanol production 11.7 21.9 14.5 15.2 11.5 (g/L) a

Yield (g butanol/ g 0.27 0.31 0.31 0.31 0.28 glucose)

Table 3.3 Comparison of n-butanol production in ABE fermentation with in situ butanol adsorption by various adsorbents (a: Total butanol production estimated from the final concentration in the fermentation broth and the adsorption isotherms)

97 Equilibrium concentration (g/L) Specific loading (g/g activated carbon) Selectivity of

Acetic Butyric Acetic Butyric butanol over Acetone Ethanol Butanol Acetone Ethanol Butanol acid acid acid acid acetone

18.2 5.0 29.6 10.1 2.8 0.038 0.000 0.290 0.016 0.038 7.6

4.8 20.6 10.1 1.9 0.029 0.000 0.272 0.008 0.032 9.4 17.5 11.9 3.9 5.2 9.8 0.8 0.048 0.003 0.216 0.005 0.021 4.5

9.1 3.6 3.1 10.0 0.8 0.051 0.003 0.177 0.003 0.016 3.5

4.4 2.7 1.2 11.0 1.1 0.053 0.006 0.132 0 0.010 2.5

Table 3.4 Specific loading of components in fermentation broth (All the model solutions initially contained 18.9 g/L glucose, 19.4

g/L acetone, 4.3 g/L ethanol, 38.7 g/L butanol, 10.6 g/L acetic acid, and 4.0 g/L butyric acid)

Immobilized cell Free cell fermentation fermentation

w/o w/ w/o w/ adsorption adsorption adsorption adsorption Total fermentation time (h) 54 106 48 122 Total amount of glucose 82 158 77 260 consumed (g/L) Final butanol concentration 18.3 18.7 16.4 8.9 in the broth (g/L) Amount of butanol adsorbed on activated - 18.8 - N/A carbon (g) Adsorbed butanol per unit - 12.9-16.1a - N/A volume (g/L) Butanol yield (g/g) 0.22 0.20-0.22a 0.21 0.21b Total butanol production 18.3 31.6-34.8 16.4 ~54.6 (g/L) Butanol productivity 0.34 0.30-0.33 0.35 ~0.45 (g/L·h)

Table 3.5 Fermentation performance with in-situ butanol adsorption by activated carbon

(a: The first value is calculated directly based on the actual amount of butanol in the broth and recovered from desorption, while the second value is based on a 80% recovery rate as not all the butanol was recovered during desorption; b: Assuming that butanol yield remained the same as the control fermentation)

Energy requirements (kJ/g) Separation Method Butanol (1) Butanol (2) ABE (3) Butanol (4)

Steam distillation >50 24 -- --

Gas stripping 14-31 22 21 --

Pervaporation 2-145 14 9 --

Extraction/perstraction 7.7 9 14 --

Adsorption 1.3-33 8 33 4.8

Table 3.6 Comparison of energy requirement for butanol recovery by different separation methods

References: (1) Oudshoorn et al. 2009; (2) Qureshi and Blaschek 2005; (3) Groot et al.

1992; (4) This study

100

Thermostat water

Activated carbon

Pump Temperature controlling unit PH controlling unit

Substrate tank Bioreactor

Figure 3.1 Bioreactor system with an external packed column for butanol adsorption

101

0.5

0.4

0.3

adsorbent

Loading ,

0.2

butanol/g

Specific -

g Activated carbon 0.1 CBV901 Dowex L-493 Dowex SD-2 0 0 10 20 30 40

ButanolAq, g/L A

0.5

0.4

adsorbent

- Loading ,

0.3

Special

butanol/g -

g 0.2

Activated carbon 0.1 CBV901 Dowex L-493 Dowex SD-2 0 0 10 20 30 40

ButanolAq, g/L B

Figure 3.2 Equilibrium isotherms of n-butanol with Norit ROW 0.8, CBV901, Dowex L-

493 and Dowex SD-2 A. at 37 °C; B. at 60 °C 102

Acetone (g/L) Butanol (g/L) Acetic acid (g/L) 90 Butyric acid (g/L) O.D. Glucose (g/L) 24

80 20 70

60 16

50 12 40

Glucose(g/L) 30 8 Products (g/L), O.D. (g/L), Products 20 4 10

0 0 0 10 20 30 40 50 60 A Time (h)

Acetone (g/L) Butanol (g/L) Acetic acid (g/L) 110 Butyric acid (g/L) O.D. Glucose (g/L) 24

100

90 20

80 16 70

60 12 50

40 Glucose(g/L) 8

30 O.D. Products(g/L),

20 4

0 0 0 20 40 60 80 100 120 B Time (h)

Continued Figure 3.3 Kinetics of ABE fermentation of C. acetobutylicum JB200 at 37 oC, pH 5 A. Batch free cell fermentation without adsorption (control); B. Fed-batch free cell fermentation with adsorption by activated carbon; C. Repeated batch immobilized cell fermentation without adsorption (control); D. Fed-batch immobilized cell fermentation with adsorption by activated carbon 103

Figure 3.3 continued Acetone (g/L) Butanol (g/L) Acetic acid (g/L) 110 Butyric acid (g/L) O.D. Glucose (g/L) 24

100

90 20

80 16 70

60 12 50

40 Glucose(g/L) 8

30 O.D. Products(g/L),

20 4

0 0 0 20 40 60 80 100 120 140 C Time (h)

Acetone (g/L) Butanol (g/L) Acetic acid (g/L) 110 Butyric acid (g/L) O.D. Glucose (g/L) 24

100 22

90 20 18 80 16 70 14 60 12 50 10 40 Glucose(g/L) 8

30 O.D. (g/L), Products 6

20 4

10 2

0 0 160 180 200 220 240 260 280 D Time (h)

104

100 Butanol

Water 80 ABE

Weight remaining (%) remaining Weight 20

0 25 50 75 100 125 150 175 200 225 250 Temperature, oC

Figure 3.4 Desorption of n-butanol, water, and ABE mixture from activated carbon determined thermogravimetrically

105

Chapter 4: Fed-batch Butanol Fermentation by Engineered Clostridium tyrobutyricum with External Driving Forces in a Fibrous-bed Bioreactor Integrated

with Gas Stripping

Abstract

As a second generation biofuel, butanol has been fermentatively produced by

ABE fermentation from lignocellulosic biomass. Traditional ABE fermentation usually suffers from low butanol yield and other limitations. Besides metabolic engineering, butanol production can be boosted by external driving forces which redirect the electron and carbon flow towards butanol synthesis. In this work, an engineered mutant strain

Clostridium tyrobutyricum overexpressing adhE2 and ack knock out CtΔack-adhE2 was used. When provided with external driving forces, butanol production with high yields

(>0.30 g/g) was achieved in bioreactor. Fed-batch butanol fermentation from different carbon sources in a fibrous-bed bioreactor with external driving forces integrated with gas stripping was studied, including soybean hull hydrolysate (SHH), sugarcane bagasse hydrolysate (SBH), and glucose-xylose mixture. Final butanol titer, yield and productivity of 12.1 g/L, 0.25 g/g and 0.12 g/L∙h, respectively, were obtained from soybean hull hydrolysate (SHH). A glucose and xylose mixture mimicking sugar 106

composition in SHH was also used to produce butanol, which resulted in a total butanol production of 24.7 g/L. This work demonstrated the feasibility of butanol fermentation from soybean hull and sugarcane bagasse hydrolysate integrated with butanol recovery by gas stripping.

4.1 Introduction

Butanol is an important second generation biofuel and can be produced by traditional acetone-butanol-ethanol (ABE) fermentation. However, there are several drawbacks of clostridial ABE fermentation: high substrate cost, low solvent concentration in fermentation broth, production of low-value by-products (acetone and ethanol), and costly product recovery (Gu et al. 2011; Jones and Woods 1986). Instead of traditional starchy feedstock and molasses, many studies have been focused on butanol production from lignocellulosic biomass, including wood pulp (Lu et al. 2013), corn fiber

(Qureshi et al. 2008a), corn stover (Qureshi et al. 2010b), corn straw (Lin et al. 2011), wheat straw (Qureshi et al. 2007; Qureshi et al. 2008b; Wang et al. 2013), wheat bran

(Liu et al. 2010), barley straw (Qureshi et al. 2010a), switchgrass (Qureshi et al. 2010b), rice straw (Amiri et al. 2014; Gottumukkala et al. 2013; Moradi et al. 2013), willow stem and bark (Han et al. 2013). Besides carbon sources, alternative nitrogen sources have also been explored. Corn steep liquor is a by-product of corn wet-milling and has been used to replace the original nitrogen sources in P2 medium (Qureshi et al. 2004).

Efforts have also been devoted to enhance the final butanol titer. A mutant strain

C. acetobutylicum JB200 with high butanol production derived from C. acetobutylicum 107

ATCC 55025 was obtained by spontaneous mutation in a fibrous-bed bioreactor (FBB) which can produce up to ~25 g/L butanol (Zhao et al. 2009). Shen engineered an

Escherichia coli mutant strain, which was able to produce ~30 g/L butanol with external driving forces (Shen et al. 2011). Meanwhile, researchers have tried to increase butanol yield by lowering byproduct formation. Dong reported a mutant strain C. acetobutylicum

EA2018 with an improved butanol ratio of 70% (vs. 60%) among solvents (Dong 2012).

Yu engineered a mutant strain CtΔack-adhE2 from C. tyrobutyricum, which mainly produced butanol as solvents and acids (Yu et al. 2012; Yu et al. 2011).

Other than mutagenesis and metabolic engineering, external driving forces can direct carbon and electron flows toward butanol synthesis during ABE fermentation as well (Fontaine et al. 2002; Lutke-Eversloh and Bahl 2011; Shen et al. 2011). The introduction of carbon monoxide (CO) can increase solvent productivity and yield in C. acetobutylicum due to an altered electron flow (Datta and Zeikus 1985; Meyer et al.

1986). Artificial electron carriers including viologen dyes (methyl and benzyl viologen) and neutral red are also used to modify the carbon and electron flows in C. acetobutylicum (Girbal et al. 1995; Peguin et al. 1994; Peguin and Soucaille 1995; Peguin and Soucaille 1996; Rao and Mutharasan 1986; Rao and Mutharasan 1987). Altered electron flow directs carbon flow from acid forming to alcohol production, along with reduced molecular hydrogen evolution (Rao and Mutharasan 1987). This saved reducing equivalent, previously released as free hydrogen, is directed to NADH formation, which results in enhanced alcohol production (Kim and Kim 1988; Rao and Mutharasan 1987).

108

In order to reduce butanol recovery cost, many alternative separation techniques have been developed such as pervaporation, perstraction, adsorption, gas stripping and liquid-liquid extraction (Vane 2008). Among them, gas stripping is a simple technique and easy to integrate with bioreactors.

In this study, lignocellulosic biomass was used for butanol production. Butanol fermentative production by mutant strain CtΔack-adhE2 from different carbon sources, which was supplemented with certain amount of methyl viologen, was first studied in serum bottles. Then, immobilized cell fermentation was carried out in a fibrous-bed bioreactor. Finally, fed-batch n-butanol fermentation from different carbon sources, including lignocellulosic biomass hydrolysates and glucose-xylose mixture with external driving forces in a fibrous-bed bioreactor integrated with gas stripping, was studied.

4.2 Materials and methods

4.2.1 Pretreatment and enzymatic hydrolysis of lignocellulosic biomass

Soybean hull (yellow pellet) and sugarcane bagasse (brown powder) were used in this study. Before enzymatic hydrolysis, 100 g of soybean hull or sugarcane bagasse was well-mixed with 900 mL 0.04 HCl or 0.02 H2SO4 solution (corresponding to a 10% (w/w) solid loading) in a 2 L flask and then autoclaved at 121 °C and 15 psi for 30 min. The pH of the sterile mixture was adjusted to ~5.5 by 7 N NaOH after cooling to room temperature. And then 6 g of cellulase (Novozymes Cellic CTec2 VCNI0018) was added to the mixture corresponding to the enzyme loading of 0.06 g/g biomass to hydrolyze the

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cellulose into glucose. The enzymatic hydrolysis was operated at 50 °C and 150 rpm for

72 h. The obtained hydrolysates were then centrifuged at 8000 rpm for 10 min to remove the solid wastes to get soybean hull hydrolysate (SHH) and sugarcane bagasse hydrolysate (SBH). The resulting SHH contained 27.5±0.7 g/L of glucose and 12.7±0.4 g/L of xylose and SBH contained 18.7±0.8 g/L of glucose and 11.9±0.8 g/L of xylose, respectively, as shown in Table 4.1. SHH contained higher total sugar concentration and xylose percentage was lower than SBH. Generally, microbes prefer glucose over xylose.

This lower xylose percentage in SHH may favor fermentation than SBH. These lignocellulosic biomass hydrolysates were concentrated to desired sugar concentrations by rotary evaporation under vacuum at 60 °C and 60 rpm.

4.2.2 Culture and medium

C. tyrobutyricum mutant strain CtΔack-adhE2 overexpressed adhE2 gene and knocked out ack gene was used in this study (Liu et al. 2006; Yu et al. 2011). The stock culture of this mutant strain was stored in a 15% glycerol-Reinforced Clostridial Medium

(RCM; Difco, Detroit, MI) in a -80 °C fridge supplemented with 30 µg/mL thiamphenicol. All the fermentation studies were carried out anaerobically at 37 °C.

Except for when comparing the different nitrogen and carbon sources in serum bottle fermentation, all the other fermentation was carried out in a CSL medium containing ~60 g/L sugars (glucose and/or xylose or hydrolysates), 40 g/L corn steep liquor (CSL; Dow

AgroScience, Indianapolis, IN), 3 g/L (NH4)2SO4, 1.5 g/L K2HPO4, 0.6 g/L MgSO4∙7H2O,

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0.03 g/L FeSO4∙7H2O, 0.5 g/L cysteine, 30 µg/mL thiamphenicol, certain amount of methyl viologen (MV) or benzyl viologen (BV). All medium components except thiamphenicol and viologen dyes were autoclaved under 121 °C and 15 psi for 30 min and purged with nitrogen through a sterile 0.2 µm membrane filter to ensure the anaerobic condition either before or after autoclave. Thiamphenicol and MV or BV were filter-sterilized separately through sterile 0.2 µm membrane filters and added to medium before inoculation. To prepare the seed culture for serum bottle and bioreactor fermentation studies, 0.2 mL of the glycerol stock stored at -80 °C with high cell density was inoculated into 60 mL of RCM medium supplemented with 30 µg/mL thiamphenicol in serum bottle, and incubated for 6-12 h until high active cell growth was observed.

4.2.3 Serum bottle fermentation

To order to study the preliminary effect of different nitrogen sources (mixture of tryptone and yeast extract vs. CSL) and carbon sources (glucose, xylose or SHH) on the fermentation performance, six in total and three groups of serum bottle fermentation were carried out. Except for differences in nitrogen and carbon sources, the medium had the same composition as previously mentioned supplemented as 250 µM MV as the artificial electron carrier. The three groups used glucose, xylose or SHH as carbon source, respectively. Within each group, the first one used 4 g of tryptone and 2 g of yeast extract as the nitrogen sources and the second one used 40 g/L CSL as the nitrogen sources.

These six serum bottle fermentation were label as 1-6 (group 1: 1-2; group 2: 3-4; group

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3: 5-6). 3 mL of actively growing seed culture was inoculated to 57 mL medium in serum bottle which corresponded to 5% inoculation. Samples were taken every 24 h for analysis of O.D., glucose, xylose, butanol, ethanol, butyric and acetic acids. Besides, pH was adjusted to ~6.5 once per day by adding 7 N NaOH solution.

4.2.4 Immobilized cell fermentation in a fibrous-bed bioreactor

Immobilized cell fermentation in a fibrous-bed bioreactor was carried out in a 5 L bioreactor connected with a fibrous-bed bioreactor (FBB). 60 mL of actively growing cells were inoculated into the fermentor containing 1140 mL sterile medium resulting in a total volume of 1.2 L, supplemented with 250 µM MV. After the O.D.600 had reached 6.0

(~36 h), medium circulation between the fermentor and FBB unit was initiated to allow cell immobilization onto the fibrous matrix. When cell growth ceased or sugars were about to be depleted, the old fermentation broth was drained and replaced with fresh medium. Throughout the process, pH was controlled at 6.0 by adding ammonia solution.

The reactor setup for FBB has been previously described in detail in chapter 3 and also by Jiang (Jiang et al. 2010). Two different medium compositions were studied, mixture of

60 g/L of glucose and 10 g/L xylose, 60 g/L of xylose.

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4.2.5 Fed-batch fermentation in a fibrous-bed bioreactor integrated with gas stripping

In the fermentation-recovery integrated process, the first batch contained 60 g/L of xylose as the carbon source supplemented with 0 or 5 µM BV. The subsequent batches studied three different carbon sources: SHH, SBH and mixture of 3:1 glucose and xylose

(60 g/L of glucose and 20 g/L xylose, a ratio similar to that in SHH). The experimental setup for this integrated process has been described previously (Lu et al. 2012; Xue et al.

2012).

4.2.6 Analytical methods

Ethanol, butanol, acetic and butyric acid concentrations were measured by gas chromatography (GC, SHIMADZU GC-2014, Columbia, MD) equipped with an auto- injector, flame ionization detector (FID) and a 30 m fused silica column (Stabilwax-DA,

0.25 µm film thickness and 0.25 mm ID, Restek, Bellefonte, PA). Glucose and xylose concentrations were measured by high performance liquid chromatography (HPLC, LC-

20AD, Shimadzu, Columbia, MD) following the method described by a previous group member (Yu et al. 2011). Cell density was measured at the optical density at 600 nm as

O.D.600 using a spectrophotometer (Shimadzu, Columbia, MD, UV-16-1).

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4.3 Results and discussion

4.3.1 Serum bottle fermentation from glucose, xylose and soybean hull hydrolysate

Butanol production by C. tyrobutyricum mutant strain CtΔack-adhE2 using different nitrogen and carbon sources was studied in serum bottles to preliminarily evaluate the effects of CSL as nitrogen source and xylose, SHH as carbon sources, supplemented with 250 µM MV. The detailed fermentation kinetics and results summary are shown in Figure 4.1 and Table 4.2. In the control experiment (glucose + tryptone and yeast extract), 7.5 g/L of butanol was produced in 96 h with a butanol yield and productivity of 0.33 g/g and 0.07 g/L∙h, respectively. When the nitrogen source was replaced by CSL, the same butanol titer and productivity were achieved but with higher butanol yield (0.41 vs. 0.33 g/g), lower acids production and higher solvents to acids ratio

(3.4 vs. 2.3). When xylose was used as the carbon source, almost no cell growth was observed, despite the nitrogen source type. In the fermentation from soybean hull hydrolysate (SHH), 5.7-6.0 g/L of butanol (~80% of control) was obtained with a butanol yield and productivity of .021-0.25 g/g and 0.59-0.63 g/L∙h, respectively. Significantly higher amounts of acids (7-8g/L vs. 3-4 g/L) were produced from SHH, probably due to the inhibitors present in the biomass hydrolysate.

4.3.2 Immobilized cell fermentation in a fibrous-bed bioreactor

Immobilized cell fermentation was carried out in a fibrous-bed bioreactor to investigate the effect of different carbon sources supplemented with 250 µM MV. 114

Fermentation kinetics of CtΔack-adhE2 from the mixture of glucose and xylose, solely xylose are shown in Figure 4.2 and 4.3 respectively and Table 4.3 summarizes the fermentation results. Much higher butanol titer (14-15 vs. 6-8 g/L) and productivity

(0.20-0.22 vs. 0.06-0.0 g/L∙h) were achieved from a mixture of glucose and xylose compared to serum bottle fermentation. In this medium containing 6:1 glucose and xylose, xylose was hardly used by the end of fermentation. As can be further seen, when xylose was used as the sole carbon source, cell growth was strongly inhibited by MV as shown in Figure 4.3, probably due to the coupled effect of unfavorable carbon source (xylose) and inhibition effect on cell growth of MV. Cell density O.D.600 only reached 2.3 over

142 h and large percentage of acid was produced resulting in low butanol yield (0.17 g/g) along with low butanol titer (3. g/L), productivity (0.03 g/L∙h) and solvents to acids ratio

(0.8).

4.3.3 Fed-batch immobilized cell fermentation in a fibrous-bed bioreactor integrated with gas stripping

Fed-batch immobilized cell fermentation was carried out in a fibrous-bed bioreactor integrated with butanol recovery by gas stripping. Different carbon sources, including soybean hull and sugarcane bagasse hydrolysate (SHH and SBH) and a mixture of glucose and xylose (3:1) mimicking the sugar composition in SHH, were investigated.

Xylose is one of the major building molecules of hemicellulose and present in lignocellulosic biomass hydrolysates. However, xylose is fermented by cells at a slower rate compared to glucose (Ounine et al. 1983; Ounine et al. 1985). In order to facilitate 115

cell growth and induce the culture to utilize xylose, xylose was used as the carbon source and no artificial electron carrier was added to the medium for the first batch, when immobilizing cells onto the fibrous matrix in fermentation of lignocellulosic biomass hydrolysates. Over 16 g/L of butyric acid and ~5 g/L butanol were produced in ~48 hours.

During the subsequent batches using hydrolysates, 250 µM MV was added to inhibit acid production. The fermentation kinetics of immobilized cell fermentation from SHH and

SBH integrated with gas stripping are in Figure 4.4 and 4.5, respectively. 12.1 g/L of butanol was obtained with a butanol yield and productivity of 0.25 g/g and 0.12 g/L∙h from SHH; 10.7 g/L of butanol was obtained with a butanol yield and productivity of

0.31 g/g and 0.0 1 g/L∙h from SBH. Comparing fermentation performance of SHH and

SBH, less acids were produced from SHH, with higher butanol titer and productivity.

This can be explained by the fact SBH is more toxic to cells as cell density only reached

~3 in SBH medium while O.D.600 reached over 14 in SHH medium, shown in Figure 4.4 and 4.5. Performance results are summarized in Table 4.3. Detoxification of these lignocellulosic biomass hydrolysates may be exploited to enhance the fermentation performance (Lu et al. 2013).

Further fed-batch immobilized cell fermentation integrated with gas stripping was performed using a mixture containing 3:1 glucose and sugar, mimicking the sugar composition in SHH. During the first batch utilizing glucose-xylose mixture, high butanol titer, yield and productivity of 17.0 g/L, 0.36 g/g and 0.21 g/L∙h, were observed respectively. During the second batch, more butanol was produced but at a lower rate resulting in a total butanol production of 24.7 g/L with an average butanol yield and

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productivity of 0.32 g/g and 0.10 g/L∙h. Further optimization could lead to a stable process, for example replenishment of nutrients.

4.4 Conclusions

Preliminary effects of different nitrogen (mixture of tryptone and yeast extract vs. corn steep liquor) and carbon sources (glucose, xylose, and lignocellulosic biomass) were studied in serum bottle fermentation by engineered mutant strain CtΔack-adhE2 with external driving forces. Then, fed-batch butanol fermentation production in the co- existence of glucose and xylose in a fibrous-bed bioreactor integrated with gas stripping was further studied. Final butanol titer, yield and productivity of 12.1 g/L, 0.25 g/g and

0.12 g/L∙h, respectively, were obtained from soybean hull hydrolysate (SHH). Due to a higher level of or more inhibitors present in sugarcane bagasse hydrolysate (SBH), butanol titer, yield and productivity of 10.7 g/L, 0.31 g/g and 0.0 1 g/L∙h, respectively, were achieved in medium containing sugarcane bagasse hydrolysate. Detoxification of lignocellulosic biomass hydrolysates before fermentation can further boost butanol production. A glucose and xylose mixture was also used to produce butanol, which resulted in a total butanol production of 24.7 g/L. Further optimization is needed for stable butanol production. To the best of our knowledge, this is the first attempt to produce butanol from soybean hull hydrolysate by fermentation integrated with gas stripping. This work demonstrated the feasibility of butanol fermentative production from soybean hull and sugarcane bagasse hydrolysates and mixture of glucose and xylose in a gas stripping integrated system. 117

Acknowledgements

This work was supported by the ARPA-E Electrobiofuel Program.

4.5 References

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Datta R, and Zeikus JG. 1985. Modulation of Acetone-Butanol-Ethanol Fermentation by Carbon-Monoxide and Organic-Acids. Appl Environ Microb 49(3):522-529.

Dong HJT, W. W. Dai, Z. J. . 2012. Biobutanol. Advances in biochemical engineering/biotechnology 128:85-100.

Han SH, Cho DH, Kim YH, and Shin SJ. 2013. Biobutanol production from 2-year-old willow biomass by acid hydrolysis and acetone-butanol-ethanol fermentation. Energy 61:13-17.

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Jiang L, Wang J, Liang S, Wang X, Cen P, and Xu Z. 2010. Production of butyric acid from glucose and xylose with immobilized cells of Clostridium tyrobutyricum in a fibrous-bed bioreactor. Appl Biochem Biotechnol 160(2):350-359.

Jones DT, and Woods DR. 1986. Acetone-Butanol Fermentation Revisited. Microbiol Rev 50(4):484-524.

Kim TS, and Kim BH. 1988. Electron Flow Shift in Clostridium acetobutylicum Fermentation by Electrochemically Introduced Reducing Equivalent. Biotechnol Lett 10(2):123-128.

Liu X, Zhu Y, and Yang ST. 2006. Construction and characterization of ack deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid and hydrogen production. Biotechnol Prog 22(5):1265-1275.

Liu ZY, Ying Y, Li FL, Ma CQ, and Xu P. 2010. Butanol production by Clostridium beijerinckii ATCC 55025 from wheat bran. J Ind Microbiol Biot 37(5):495-501.

Lu CC, Dong J, and Yang ST. 2013. Butanol production from wood pulping hydrolysate in an integrated fermentation-gas stripping process. Bioresource Technol 143:467-475.

Lutke-Eversloh T, and Bahl H. 2011. Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production. Curr Opin Biotech 22(5):634-647.

Moradi F, Amiri H, Soleimanian-Zad S, Ehsani MR, and Karimi K. 2013. Improvement of acetone, butanol and ethanol production from rice straw by acid and alkaline pretreatments. Fuel 112:8-13.

Ounine K, Petitdemange H, Raval G, and Gay R. 1983. Acetone-Butanol Production from Pentoses by Clostridium acetobutylicum. Biotechnol Lett 5(9):605-610.

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Ounine K, Petitdemange H, Raval G, and Gay R. 1985. Regulation and Butanol Inhibition of D-Xylose and D-Glucose Uptake in Clostridium acetobutylicum. Appl Environ Microb 49(4):874-878.

Peguin S, Goma G, Delorme P, and Soucaille P. 1994. Metabolic Flexibility of Clostridium acetobutylicum in Response to Methyl Viologen Addition. Appl Microbiol Biot 42(4):611-616.

Peguin S, and Soucaille P. 1995. Modulation of Carbon and Electron Flow in Clostridium acetobutylicum by Iron Limitation and Methyl Viologen Addition. Appl Environ Microb 61(1):403-405.

Qureshi N, Saha BC, and Cotta MA. 2007. Butanol production from wheat straw hydrolysate using Clostridium beijerinckii. Bioproc Biosyst Eng 30(6):419-427.

Qureshi N, Saha BC, Dien B, Hector RE, and Cotta MA. 2010a. Production of butanol (a biofuel) from agricultural residues: Part I - Use of barley straw hydrolysate. Biomass Bioenerg 34(4):559-565.

Rao G, and Mutharasan R. 1986. Alcohol Production by Clostridium acetobutylicum Induced by Methyl Viologen. Biotechnol Lett 8(12):893-896.

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Rao G, and Mutharasan R. 1987. Altered Electron Flow in Continuous Cultures of Clostridium acetobutylicum Induced by Viologen Dyes. Appl Environ Microb 53(6):1232-1235.

Shen CR, Lan EI, Dekishima Y, Baez A, Cho KM, and Liao JC. 2011. Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli. Appl Environ Microb 77(9):2905-2915.

Vane LM. 2008. Separation technologies for the recovery and dehydration of alcohols from fermentation broths. Biofuel Bioprod Bior 2(6):553-588.

Yu MR, Zhang YL, Tang IC, and Yang ST. 2011. Metabolic engineering of Clostridium tyrobutyricum for n-butanol production. Metab Eng 13(4):373-382.

Zhao JB, Yang ST, Jiang WH, and Yang S. 2009. BIOT 305-Evolution of solvent- producing Clostridium beijerinckii toward high butanol tolerance. Abstr Pap Am Chem S 238.

4.6 Tables and figures

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Lignocellulosic biomass Glucose (g/L) Xylose (g/L)

Soybean hull 27.5±0.7 12.7±0.4

Sugarcane bagasse 18.7±0.8 11.9±0.8

Table 4.1 Composition of soybean hull and sugarcane bagasse hydrolysate (before rotary evaporation)

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Initial sugar Final sugar Butanol Acids Solvents Butanol conc. (g/L, conc. (g/L, Butanol Solvents Acids Substrate yield yield to acids productivity glucose/xylose/ glucose/xylose/ (g/L) (g/L) (g/L) (g/g) (g/g) ratio (g/L∙h) total) total) 1: glucose + tryptone and 40.6/0/40.6 18.0/0/18.0 7.5 8.5 3.7 0.33 0.16 2.3 0.078 YE* 2: glucose + 44.7/0/44.7 26.4/0/26.4 7.5 8.8 2.6 0.41 0.14 3.4 0.078 CSL 3: xylose+ 0/49.1/49.1 0/48.2/48.2 0 0.1 0.5 0 0.56 0.2 0 tryptone and YE 4: xylose + CSL 0/49.5/49.5 0/39.5/39.5 0.4 0.6 1.0 0.04 0.1 0.6 0.004 5: SHH + 23.9/12.0/35.9 2.2/9.3/11.5 6 7.7 6.9 0.25 0.28 1.1 0.063

123 tryptone and YE 6: SHH + CSL 25.9/13.3/39.2 2.8/9.7/12.5 5.7 7.7 7.9 0.21 0.30 1.0 0.059

Table 4.2 Effect of different nitrogen and carbon sources on fermentation of mutant strain CtΔack-adhE2 in serum bottles

supplemented with 250 µM methyl viologen (MV) (* YE, yeast extract)

Butanol Acids Solvents Butanol Fermentation Fermentation Butanol Solvents Acids Substrate yield yield to acids productivity condition time (h) (g/L) (g/L) (g/L) (g/g) (g/g) ratio (g/L∙h) 1st Mixture 71 14.0 16.6 3.8 0.30 0.07 4.4 0.20 batch of glucose 250 µM 2nd and MV 55 14.8 19.6 8.6 0.28 0.17 2.3 0.22 batch xylose Xylose 142 3.8 4.1 4.9 0.17 0.22 0.8 0.027 250 µM SHH 103 12.1 14.7 10.7 0.25 0.14 1.4 0.12

124 MV + gas SBH 116 10.7 11.8 15.0 0.31 0.19 0.8 0.081

stripping 1st Mixture 70 17.0 17.0 7.8 0.36 0.13 2.2 0.21 25 µM BV batch of glucose + gas 2nd and 145 24.7 25.1 10.1 0.27 0.08 3.5 0.053 stripping batch xylose Overall 214 24.7 25.1 10.1 0.32 0.11 2.5 0.10

Table 4.3 Results of immobilized cell fermentation in a fibrous-bed bioreactor from different carbon sources

50 1 60 1

50 40 2 2 40 30 3 3 30 20 4 20 4

10 (g/L) Xylose

Glucose (g/L) Glucose 5 10 5 0 6 0 6 0 20 40 60 80 100 0 20 40 60 80 100 A Time (h) B Time (h)

4 1 2.5 1 2 2.0 2

600 3 1.5 3 2

O.D. 4 1.0 4 1 5 (g/L) Ethanol 0.5 5 0 6 0.0 6 0 20 40 60 80 100 0 20 40 60 80 100 C Time (h) D Time (h)

Continued

Figure 4.1 Effect of different nitrogen and carbon sources on fermentation kinetics of mutant strain CtΔack-adhE2 in serum bottles supplemented with 250 µM methyl viologen (MV) (A: glucose; B: xylose; C: O.D.600; D: ethanol; E: butanol; F: acetic acid;

G: butyric acid) (Figure legend: 1, glucose + yeast extract + tryptone; 2, glucose + corn steep liquor; 3, xylose + yeast extract + tryptone; 4, xylose + corn steep liquor; 5, soybean hull hydrolysate + yeast extract + tryptone; 6, soybean hull hydrolysate + corn steep liquor) 125

Figure 4.1 continued

8 1 2.5 1

6 2 2.0 2 3 1.5 3 4 4 1.0 5 2 Butanol (g/L) Butanol 5 0.5 6 0 6 (g/L)acid Acetic 0.0 4 0 20 40 60 80 100 0 20 40 60 80 100 E Time (h) F Time (h)

8 1

6 2 3 4 4 2 5 Butyric acid (g/L) acid Butyric 0 6 0 20 40 60 80 100 G Time (h)

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70 16

60 14 Glucose 12 50 Xylose 10 O.D. 40 8 Ethanol 30 6 Butanol 20 Acetic acid 4 (g/L) products O.D.,

Glucose/xylose (g/L) Glucose/xylose Butyric acid 10 2 0 0 0 30 60 90 120 150 Time (h)

Figure 4.2 Fermentation kinetics of immobilized cell fermentation in a fibrous-bed bioreactor from mixture of glucose and xylose supplemented with 250 µM methyl viologen (MV)

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60 5

50 Glucose 4 Xylose 40 3 O.D. 30 Ethanol 2 Butanol 20 Acetic acid 1 (g/L) products O.D., Glucose/ xylose (g/L) xylose Glucose/ 10 Butyric acid

0 0 0 50 100 150 Time (h)

Figure 4.3 Fermentation kinetics of immobilized cell fermentation in a fibrous-bed bioreactor from xylose supplemented with 250 µM methyl viologen (MV)

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70 Gas stripping start 18

60 16 Glucose

14 Xylose 50 12 O.D. 40 10 Ethanol 30 8 Butanol 6 Acetic acid 20

4 (g/L) products O.D., Butyric acid Glucose/ xylose (g/L)xylose Glucose/ 10 2 Total Butanol 0 0 0 30 60 90 120 150 180 Time (h)

Figure 4.4 Fed-batch immobilized cell fermentation in a fibrous-bed bioreactor integrated with gas stripping from soybean hull hydrolysate (SHH)

129

70 20 Gas stripping start 18

60 Glucose

16 Xylose 50 14 O.D. 12 40 Ethanol 10 Butanol 30 8 Acetic acid 20 6 O.D., products (g/L) products O.D., Butyric acid

Glucose/ xylose (g/L)xylose Glucose/ 4 10 2 Total butanol 0 0 0 30 60 90 120 150 180 Time (h)

Figure 4.5 Fed-batch immobilized cell fermentation in a fibrous-bed bioreactor integrated with gas stripping from sugarcane bagasse hydrolysate (SBH)

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70 25 Gas stripping start

60 Glucose

20 Xylose 50 O.D. 15 40 Ethanol Butanol 30 10 Acetic acid 20 5 (g/L) products O.D., Butyric acid Glucose/ xylose (g/L)xylose Glucose/ 10 Total butanol 0 0 0 50 100 150 200 250 300 350 Time (h)

Figure 4.6 Fed-batch immobilized cell fermentation in a fibrous-bed bioreactor integrated with gas stripping from mixture of glucose and xylose mimicking soybean hull hydrolysate (SHH)

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Chapter 5: High Performance PDMS (Mixed Matrix) Membrane for Butanol

Recovery from Aqueous Solution by Pervaporation

Abstract

Butanol is an important second generation biofuel. However, the lack of an efficient recovery method of butanol from dilute solution has always been a concern. In this work, high performance polydimethylsiloxane (PDMS) membranes and zeolite filled

PDMS mixed matrix membranes (MMMs) were developed to recovery butanol from model solutions. The effect of membrane filler zeolite, feed butanol concentration, operating (feed) temperature on pervaporation performance of PDMS membranes and

PDMS MMMs was studied. With the feed solution of 1.5 wt% butanol at 47°C, the

PDMS MMM filled with 40 wt% zeolite was found to have the highest butanol separation factor of 77 with a butanol and total flux of 62 and 118 g/m2·h, respectively.

and 44.2 kJ/mol, respectively. Compared with literature, this work demonstrated higher butanol separation performance by pervaporation and showed huge energy saving compared to traditional distillation.

Keywords: pervaporation; butanol; separation; PDMS; zeolite; mixed matrix membrane

(MMM)

5.1 Introduction

N-butanol (hereafter ‘butanol’) is a four carbon primary alcohol and has many applications. It is used as a solvent, an intermediate in the production of other chemicals, etc. Recently, butanol has attracted increasing research interest as a second generation biofuel (Xue et al. 2013). Compared to ethanol, which is also considered to be a promising fossil fuel alternative, butanol has higher energy density, lower water adsorption and easier application to the existing gasoline engine (Lee et al. 2008).

Butanol can be produced through fermentation processes, which is environmentally friendly and sustainable, other than petrochemical process. Traditionally, biobutanol is natively produced by the acetone-butanol-ethanol (ABE) fermentation (typically in the ratio of 3:6:1) by many Clostridium strains, such as C. acetobutylicum or C. beijerinckii

(Jones and Woods 1986; Lee et al. 2008). This fermentative butanol production usually suffers from low butanol yield and productivity. Much work have been dedicated to solve these issues. Heterologous species, such as Escherichia coli and C. tyrobutyricum, have been engineered to produce butanol without the formation of byproduct acetone or ethanol, which greatly increased butanol yield (Atsumi et al. 2008; Yu et al. 2011). Both 133

native and heterologous species can only produce up to ~2 wt% butanol, due to butanol toxicity (Xue et al. 2012; Yu et al. 2011). Due to low butanol titer in the fermentation broth, recovery by traditional distillation is very energy intensive and uneconomical.

Conventional distillation for recovering and purifying butanol from a dilute aqueous solution requires great energy input. In a butanol-water binary system, the energy required to recover butanol from a 0.5 wt% solution to 99.9% pure butanol was estimated at 79.5 MJ/kg butanol (Matsumura et al. 1988), which is much higher than the energy content of butanol (36 MJ/kg). The energy consumption can be reduced drastically to 36 MJ/kg and 6 MJ/kg when the butanol concentration is increased to ~1 wt% and 10 wt%, respectively (Matsumura et al. 1988). Further increasing butanol concentration to above 40 wt % can reduce energy consumption in distillation to less than

3 MJ/kg (Matsumura et al. 1988). Energy consumption in butanol purification by distillation is very sensitive to feed butanol concentration when feed concentration is below 10 wt% (Matsumura et al. 1988). Therefore, it is crucial to pre-concentrate butanol before recovery by distillation.

Alternative separation technologies, which are more energy-efficient and suitable to recover butanol from low concentration solutions, have been developed, such as gas stripping (Ezeji et al. 2003; Xue et al. 2013), adsorption (Lin et al. 2012; Qureshi et al.

2005), pervaporation (Dong et al. 2014; Li et al. 2010), extraction (Dhamole et al. 2012) and etc. Among these advanced butanol recovery techniques, pervaporation is outstanding for its high selectivity and low energy consumption (Groot et al. 1992;

Thongsukmak and Sirkar 2007). Pervaporation is a membrane based technique, wherein

134

the selective membrane is the key factor. Many membranes have been proposed to recover butanol from aqueous solutions, including polydimethyl siloxane (PDMS) (Dong et al. 2014; Li et al. 2013; Liu et al. 2011a; Liu et al. 2011b; Liu et al. 2011c; Niemisto et al. 2013), poly-1-trimethylsilyl-1-propyne (PTMSP) (Fadeev et al. 2001; Yakovlev et al.

2013), poly-4-methyl-2-pentyne (PMP) (Yakovlev et al. 2013), polyvinylidene fluoride

(PVDF) (Srinivasan et al. 2007), polytetrafluoroethylene (PTFE) (Li et al. 2010), poly(ether block amide) (PEBA 2533) (Liu et al. 2005), ethylene propylene diene rubber membrane (EPDM) (Jitesh et al. 2000), Styrene butadiene rubber (SBR) (Jitesh et al.

2000), polyurethane (PUR) (Boddeker et al. 1990) and trioctylamine (TOA) - based liquid membrane (Thongsukmak and Sirkar 2007). Among the reported membranes,

PDMS membranes are the most widely studied and have good butanol separation performance (Li et al. 2010). In order to improve the flux and separation factor, different filler materials were incorporated to the polymeric membrane matrix. Silicates (Huang and Meagher 2001) and zeolitic imidazolate frameworks (ZIF-71) (Liu et al. 2013) have been filled to PDMS membranes, and ZSM-5 zeolite has been filled to PEBA membrane

(Tan et al. 2013), which all increased the butanol separation factor to some extent.

In this work, PDMS membranes and zeolite filled PDMS MMMs were fabricated.

Comprehensive study on these membranes for butanol recovery from aqueous model solutions by pervaporation was carried out. The effects of membrane filler zeolite, feed butanol concentration, membrane thickness, and operating (feed) temperature on the performance of butanol separation were investigated. Activation energies of butanol and water permeation in PDMS membranes and PDMS MMMs were evaluated. Energy

135

consumption in this pervaporation system was estimated, in comparison with traditional method of distillation.

5.2 Experimental

5.2.1 Materials

Hexane (>99.9%), and butanol (>99.95%) were purchased from Fisher Scientific.

Sylgard® 184 silicone elastomer kit was supplied by Dow Corning Corporation. ZSM-5 type zeolite CBV 28014 with SiO2/Al2O3 mole ratio of 300 was obtained from Zeolyst international. Deionized water was used for the preparation of aqueous model solution.

5.2.2 Flat sheet membrane fabrication

The base of silicone elastomer kit was mixed with a curling agent in the ratio of

10:1using hexane as the solvent (together with membrane filler material when preparing zeolite filled PDMS MMMs). This mixture was stirred, sonicated, and centrifuged alternatively for ½ h and then cast evenly on a glass plate. Then, the plate was placed in a

70 ºC oven and dried overnight. After that, the flat sheet PDMS membranes/ PDMS

MMMs were carefully peeled off and then cut to fit the pervaporation module.

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5.2.3 Experimental setup

Pervaporation experiments were conducted using a pervaporation system as shown in Figure 5.1. Feed vessel containing binary butanol-water model solution was maintained at certain temperature by the heating unit. Feed solution (1 L) was circulated between the feed vessel and pervaporation unit at a flow rate of 90 ml/min. The permeate side was maintained as a vacuum using a vacuum pump. Mass transfer across the membrane was induced by the partial pressure/ chemical activity difference (Vane 2005).

Permeate was collected in the cold trap and analyzed by gas chromatograph (GC). The membrane in the pervaporation unit had an effective mass transfer area of 36 cm2.

The pervaporation performance of a membrane is usually characterized by two parameters: flux and separation factor (Feng and Fouad 2008), which are defined as follows

(11)

(12)

where is the weight of component i in the permeate, A is the effective membrane area, t is the permeation time, and are the mass fractions of component i in the permeate and feed, respectively.

5.2.4 Analytical methods

Butanol concentration was measured by GC (SHIMADZU GC-2014, Columbia,

MD) equipped with an auto-injector, flame ionization detector (FID) and a 30 m fused 137

silica column (Stabilwax-DA, 0.25 µm film thickness and 0.25 mm ID, Restek,

Bellefonte, PA) following the method described by a previous group member (Yu et al.

2011). Membrane thickness was measured by Mitutoyo (ID-C112EB) ABSOLUTE digimatic dial indicator (Mitutoyo Corp., Japan). Surface and cross section images of membranes were taken via scanning electron microscopy (SEM, Quanta 200).

5.3 Results and discussion

5.3.1 Membrane fabrication and characterization

PDMS membranes of different thickness (41, 85, 90, 115 and 141 µm) and PDMS mixed matrix membranes (MMMs) filled with different amount of ZSM-5 zeolite CBV

28014 (10, 20, 30 and 40 wt%) were fabricated. Figure 5.2 shows the SEM images of surface and cross section PDMS membrane and PDMS MMMs, from which we can see that both membranes were non-porous and defect-free and zeolite was uniformly dispersed in the polymeric PDMS matrix.

5.3.2 Effect of membrane filler on membrane separation

Membrane filler materials were incorporated into the PDMS membranes to improve the pervaporation performance. ZSM-5 type zeolite CBV28014 was selected due to its hydrophobic nature. These zeolite filled PDMS MMMs would have higher selectivity for butanol, as the hydrophobic zeolite would selectively let organic solvent

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pass though while inhibiting water. PDMS membrane and PDMS MMMs filled with different amount of zeolite (10, 20, 30, 40 wt%) with a thickness of ~100 µm were tested at 47 ºC and 1.5 wt% butanol feed solution. As shown in Figure 5.3, the zeolite incorporation greatly reduced water flux, from 100 to 56 g/m2·h, while not much affecting butanol flux, remained at ~60 g/m2·h, resulting in a reduced total flux. The major function that zeolite played in this mix matrix membrane was to block water. This lowered water flux is favorable for further purification by distillation which will be discussed in more detail in section 3.6. Therefore, the butanol separation factor was greatly enhanced, from 41 (control, no zeolite) to 77 (40 wt% zeolite), which is also shown in Figure 5.3. Notice that zeolite CBV28014 had a low density. When filling too much zeolite to PDMS membrane, zeolite took up the majority volume in the preparation mixture and resulted in a solid-like mixture of zeolite and monomer dimethylsiloxane

(DMS), which was impossible to make a membrane out of. The PDMS MMM with the highest possible amount of zeolite incorporation was 40 wt%.

5.3.3 Effect of feed concentration on membrane separation

Mass transfer in pervaporation usually can be described by the solution-diffusion model (Wijmans and Baker 1995). The flux of component across the membrane can be described as follows (Niemisto et al. 2013)

(13)

139

where is the partial flux of component , is the membrane permeability of component , is membrane thickness, and are the mole fraction of component at

feed and permeate side, respectively, is the activity coefficient of component , is the saturation pressure of species and is the total vapor pressure of the permeate side.

Due to the vacuum, is small enough to be neglected and Eq. (13) is reduced to

(14)

Therefore, for one membrane operated at a constant temperature and assuming does not change much, partial flux of component is approximately proportional to the feed concentration.

The effect of feed butanol concentration on PDMS membrane with a thickness of

~100 µm was studied at 47 ºC. Four different feed concentrations (5, 10, 15, 30 g/L) were tested. At low butanol concentrations (< 3 wt%), the assumption that remained almost the same was acceptable. As shown in Figure 5.4, butanol flux increased linearly with increasing feed butanol concentration; water flux decreased slightly and total flux increased linearly with increasing butanol concentration. In the tested butanol concentration range, there seems to be no obvious correlation between the butanol separation factor and feed butanol concentration; the butanol separation factor remained almost unchanged.

140

5.3.4 Effect of membrane thickness on membrane separation

As discussed in the previous section, partial flux of component can be expressed

as follows, . When operated at the same feed concentration and temperature, partial flux is inversely proportional to the membrane thickness. PDMS membranes of five different thicknesses (41, 85, 90, 115, 141 µm) were tested at 47 ºC and 1.5 wt% butanol feed solution, and results are shown in Figure 5.5. It is clear that the butanol separation factor remained approximately unchanged while butanol, water and total fluxes increased greatly with the decreasing membrane thickness, due to reduced mass transfer resistance. The butanol and total fluxes were 97 and 278 g/m2·h, respectively, at the lowest tested membrane thickness (41 µm). Based on this model, the

PDMS membrane with a thickness of 5 µm would have butanol and total fluxes of ~600 and ~2000 g/m2·h, respectively, comparable to those previously reported (Dong et al.

2014; Niemisto et al. 2013). Previously, we concluded that the incorporation of zeolite to

PDMS MMMs did not much affect butanol flux, but greatly reduced water flux.

Therefore, zeolite filled PDMS MMMs with a thickness of 5 µm would also have a butanol flux of ~600 g/m2·h. A thinner PDMS membrane can be casted on a support such as ceramic hollow fiber (Dong et al. 2014) and polyacrylonitrile (PAN) (Niemisto et al.

2013).

141

5.3.5 Effect of feed temperature on membrane separation

The effect of feed temperature on partial fluxes (butanol or water) and the butanol separation factor was investigated. The temperature dependence of partial fluxes usually follows the Arrhenius equation (Feng and Huang 1996):

(15) where is a constant, is the apparent activation energy of permeation, is the gas constant and is the feed temperature in Kelvin.

PDMS membrane and PDMS MMM (filled with 40 wt% zeolite) with the same thickness of ~100 µm were tested under four different temperatures (27, 37, 47, and 56

ºC) for their ability to recover butanol from aqueous solutions, as shown in Figure 5.6

(Note that the y axis is in log scale). Clearly, both butanol and water fluxes increased with increasing temperature in both membranes. The presence of zeolite in the PDMS membrane greatly reduced the water flux due to its strong hydrophobicity, as previously discussed in section 3.2 (the effect of membrane filler). As shown in Figure 5.6 and Table

5.1, the apparent activation energies of butanol permeation in PDMS membrane and zeolite-filled PDMS MMM were found to be 34.3 and 44.2 kJ/mol, respectively. The higher activation energy of the zeolite-filled PDMS MMM indicated that it was more sensitive to temperature than a pure PDMS membrane. The result of the activation energy increase for butanol permeation with zeolite incorporation was different from previously reported (Tan et al. 2013). Tan concluded that the incorporation of zeolite in PEBA membranes could decrease the activation energy of butanol permeation (61.1 in control vs. 48.2 kJ/mol in zeolite filled PEBA MMM) (Tan et al. 2013). This inconsistency may 142

be explained by the different interaction between the inorganic matrix (zeolite) and polymeric matrix (PDMS or PEBA).

The apparent activation energies of water permeation in PDMS and zeolite filled

PDMS MMM were 33.2 and 31.8 kJ/mol, respectively, which was almost the same. For both membranes, the activation energy of water permeation was smaller than that of butanol, indicating that butanol flux was more sensitive to temperature than water. This explains why the separation factor increased with temperature, as shown in Figure 5.7. It is also noted that the activation energy difference between butanol and water permeation in PDMS MMM was much larger than that of PDMS membrane (12.4 vs. 1.1 kJ/mol), which perfectly explains the more obvious increase in the butanol separation factor of zeolite filled PDMS MMM with increasing temperature, shown in Figure 5.7. In terms of butanol flux, zeolite filled PDMS MMMs are more suitable at a relatively high temperature (> 30 ºC), while pure PDMS membrane performance better at a relatively low temperature (< 30 ºC).

5.3.6 Energy consumption analysis in pervaporation

Energy consumption in pervaporation is mainly consisted of evaporation/condensation of permeate and energy consumption of vacuum pump. The normalized energy requirement for evaporating the permeate can be calculated as follows

(Vane 2005):

(16)

143

where is the energy requirement, normalized to per unit of butanol permeated,

is the heat of vaporization of species . This equation can be rewritten in terms of the butanol-water separation factor α as (Vane 2005):

(17)

where is the total molar concentration in the feed, and are the water and butanol molar concentration in the feed, respectively. Theoretically, the energy required to condense the permeate vapor is the same as the heat required for the

evaporation (ie ) (Vane 2005).

The energy consumption of the vacuum pump can be calculated from the following equation (Matsumura et al. 1988):

(18)

where is the energy required to exhaust one mole of gas, subscripts 1 and 2 refer to the inlet and outlet of the vacuum pump, T, P, and γ refer to temperature, pressure and adiabatic constant, respectively.

When purifying butanol by distillation from 1.5 wt% butanol concentration, the energy requirement was ~30 MJ/kg butanol. When pervaporation was integrated, from

Eq. (17) and (18), recovery energy consumption was reduced to ~6 MJ/kg butanol, assuming the butanol separation factor was 77, which was the performance of 40 wt% zeolite filled PDMS mixed matrix membrane at 47°C. This energy requirement was only

20% of that in the original distillation. This indicates pervaporation can greatly save

144

energy consumption during butanol recovery and have great potential for future application.

5.3.7 Membrane separation performance compared with literature

Table 5.2 summarizes the performance of pervaporative butanol recovery from aqueous solutions. The results presented in this study is comparable to the results Huang et al. (Huang and Meagher 2001) previously reported, in terms of both the butanol separation factor and flux, which can be further enhanced by elevating operating temperature. The incorporation of zeolite to PDMS mixed matrix membrane blocked a large portion of water permeation, while not affecting butanol flux. Therefore, the butanol separation factor was significantly enhanced. Butanol and total flux can be increased greatly by reducing membrane thickness. Butanol flux can be enhanced to hundreds g/m2·h when membrane thickness is reduced to several micron, comparable to recent results reported by Dong and Niemisto (Dong et al. 2014; Niemisto et al. 2013).

But, fermentation broth was not tested in this study. Nevertheless, the results exhibit huge energy savings and great potential application for future butanol recovery in fermentative butanol production.

5.4 Conclusions

PDMS membranes and ZSM-5 zeolite filled PDMS mixed matrix membranes were developed and tested for butanol recovery by pervaporation from model solutions. 145

The incorporation of zeolite to PDMS membrane blocked a large amount of water, therefore greatly increasing the butanol separation factor. At the highest zeolite filling of

40 wt%, the PDMS MMM gave a butanol separation factor of 77 and a butanol and total flux of 62 and 118 g/m2·h, respectively, with 1.5 wt% butanol feed concentration at 47°C.

This means permeate containing ~480 g/L of butanol can be obtained via pervaporation from 15 g/L butanol solutions. The butanol separation factor can be further enhanced by elevating operating temperature. Besides, butanol and total fluxes can be further boosted by reducing membrane thickness, increasing feed butanol concentration, or elevating operating temperature. A butanol flux of ~600 g/m2·h is expected with a membrane thickness of 5 µm. The apparent activation energies of butanol permeation in PDMS membrane and zeolite filled (40 wt%) PDMS MMM were evaluated to be 34.3 and 44.2 kJ/mol, respectively. Butanol recovery incorporating pervaporation saves a great portion of energy consumption compared to distillation alone. Compared with literature, this work demonstrated higher butanol separation performance by pervaporation and had great potential application for fermentative butanol recovery.

Acknowledgments

This work was supported by the ARPA-E Electrobiofuel Program.

5.5 References

Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, Chou KJ, Hanai T, and Liao JC. 2008. Metabolic engineering of Escherichia coli for 1-butanol production. Metab Eng 10(6):305-311. 146

Boddeker KW, Bengtson G, and Pingel H. 1990. Pervaporation of Isomeric Butanols. J Membrane Sci 54(1-2):1-12.

Dhamole PB, Wang ZL, Liu YQ, Wang B, and Feng H. 2012. Extractive fermentation with non-ionic surfactants to enhance butanol production. Biomass Bioenerg 40:112-119.

Dong ZY, Liu GP, Liu SN, Liu ZK, and Jin WQ. 2014. High performance ceramic hollow fiber supported PDMS composite pervaporation membrane for bio-butanol recovery. J Membrane Sci 450:38-47.

Ezeji TC, Qureshi N, and Blaschek HP. 2003. Production of acetone, butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping. World J Microb Biot 19:595-603.

Feng XS, and Fouad EA. 2008. Use of pervaporation to separate butanol from dilute aqueous solutions: Effects of operating conditions and concentration polarization. J Membrane Sci 323(2):428-435.

Feng XS, and Huang RYM. 1996. Estimation of activation energy for permeation in pervaporation processes. J Membrane Sci 118(1):127-131.

Groot WJ, Vanderlans RGJM, and Luyben KCAM. 1992. Technologies for Butanol Recovery Integrated with Fermentations. Process Biochem 27(2):61-75.

Jitesh K, Pangarkar VG, and Niranjan K. 2000. Pervaporative stripping of acetone, butanol and ethanol to improve ABE fermentation. Bioseparation 9(3):145-154.

Jones DT, and Woods DR. 1986. Acetone-Butanol Fermentation Revisited. Microbiol Rev 50(4):484-524.

Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, and Jung KS. 2008. Fermentative butanol production by clostridia. Biotechnol Bioeng 101(2):209-228.

Li SF, Qin F, Qin PY, Karim MN, and Tan TW. 2013. Preparation of PDMS membrane using water as solvent for pervaporation separation of butanol-water mixture. Green Chem 15(8):2180-2190. 147

Li SY, Srivastava R, and Parnas RS. 2010. Separation of 1-butanol by pervaporation using a novel tri-layer PDMS composite membrane. J Membrane Sci 363(1- 2):287-294.

Liu FF, Liu L, and Feng XS. 2005. Separation of acetone-butanol-ethanol (ABE) from dilute aqueous solutions by pervaporation. Sep Purif Technol 42(3):273-282.

Liu GP, Hou D, Wei W, Xiangli FJ, and Jin WQ. 2011a. Pervaporation Separation of Butanol-Water Mixtures Using Polydimethylsiloxane/Ceramic Composite Membrane. Chinese J Chem Eng 19(1):40-44.

Liu GP, Wei W, Wu H, Dong XL, Jiang M, and Jin WQ. 2011b. Pervaporation performance of PDMS/ceramic composite membrane in acetone butanol ethanol (ABE) fermentation-PV coupled process. J Membrane Sci 373(1-2):121-129.

Liu SN, Liu GP, Zhao XH, and Jin WQ. 2013. Hydrophobic-ZIF-71 filled PEBA mixed matrix membranes for recovery of biobutanol via pervaporation. J Membrane Sci 446:181-188.

Liu XL, Li YS, Liu Y, Zhu GQ, Liu J, and Yang WS. 2011c. Capillary supported ultrathin homogeneous silicalite-poly(dimethylsiloxane) nanocomposite membrane for bio-butanol recovery. J Membrane Sci 369(1-2):228-232.

Matsumura M, Kataoka H, Sueki M, and Araki K. 1988. Energy Saving Effect of Pervaporation Using Oleyl Alcohol Liquid Membrane in Butanol Purification. Bioprocess Eng 3(2):93-100.

Niemisto J, Kujawski W, and Keiski RL. 2013. Pervaporation performance of composite poly(dimethyl siloxane) membrane for butanol recovery from model solutions. J Membrane Sci 434:55-64.

Qureshi N, Hughes S, Maddox IS, and Cotta MA. 2005. Energy-efficient recovery of butanol from model solutions and fermentation broth by adsorption. Bioproc Biosyst Eng 27(4):215-222.

Srinivasan K, Palanivelu K, and Gopalakrishnan AN. 2007. Recovery of 1-butanol from a model pharmaceutical aqueous waste by pervaporation. Chem Eng Sci 62(11):2905-2914.

Tan HF, Wu YH, and Li TM. 2013. Pervaporation of n-butanol aqueous solution through ZSM-5-PEBA composite membranes. J Appl Polym Sci 129(1):105-112. 148

Thongsukmak A, and Sirkar KK. 2007. Pervaporation membranes highly selective for solvents present in fermentation broths. J Membrane Sci 302(1-2):45-58.

Vane LM. 2005. A review of pervaporation for product recovery from biomass fermentation processes. J Chem Technol Biot 80(6):603-629.

Wijmans JG, and Baker RW. 1995. The Solution-Diffusion Model - a Review. J Membrane Sci 107(1-2):1-21.

Xue C, Zhao J, Liu F, Lu C, Yang ST, and Bai FW. 2013. Two-stage in situ gas stripping for enhanced butanol fermentation and energy-saving product recovery. Bioresour Technol 135:396-402.

Yakovlev AV, Shalygin MG, Matson SM, Khotimskiy VS, and Teplyakov VV. 2013. Separation of diluted butanol-water solutions via vapor phase by organophilic membranes based on high permeable polyacetylenes. J Membrane Sci 434:99-105.

Yu MR, Zhang YL, Tang IC, and Yang ST. 2011. Metabolic engineering of Clostridium tyrobutyricum for n-butanol production. Metab Eng 13(4):373-382.

5.6 Tables and figures

149

Ea of butanol Ea of water ΔEa (Ea,butanol – Ea, Membrane R2 R2 (kJ/mol) (kJ/mol) water) (kJ/mol)

PDMS 34.3 0.994 33.2 0.998 1.1

Zeolite filled 44.2 0.997 31.8 0.996 12.4 PDMS MMM*

Table 5.1 Apparent activation energies (Ea) of butanol and water permeation in PDMS membrane and zeolite filled (40 wt%) PDMS MMM (* MMM: mixed matrix membrane)

150

Feed butanol Butanol Active layer Temperatu Total flux Separation Membrane material concentration flux Reference thickness (µm) re (ºC) (g/m2·h) factor (wt%) (g/m2·h) (Dong et al. PDMS/hollow fiber 10 1 40 1282 - 43 2014) (Liu et al. PDMS/ceramic 10 1 40 457 - 26 2011a) (Niemisto et PDMS/PAN 4 3.5 42 - 800 22-29 al. 2013) Tri-layer PDMS 65-200 2 37 40-132 20-50 32-50 (Li et al. 2010) Silicone 50 53-350 - 42-49 (Huang and Silicalite-filled 1 30-70 19 63-607 - 86-111 Meagher 2001) silicone

151 (Fadeev et al. PTMSP - 0.3-6 25-70 60-2097 16-347 41-78 2001) (Liu et al. PEBA 30-100 5 23 65-179 19-42 6-8 2005) (Liu et al. ZIF-71 filled PEBA 10-20 1 37 520 - 18.8 2013) (Tan et al. ZSM-5 filled PEBA - 2.5 30-45 - 90-240 22-30 2013) Continued

Table 5.2 Pervaporation performance of different membranes for butanol recovery from aqueous solutions (PDMS: polydimethylsiloxane; PAN: polyacrylonitrile; PTMSP: poly(1-trimethylsilyl-1-propyne); PEBA: poly(ether block amide); ZIF: zeolitic imidazolate framework; EPDM: ethylene propylene diene rubber; SBR: styrene butadiene rubber; TOA: trioctylamine; PVDF: poly(vinylidene difluoride); PUR: polyurethane)

Table 5.2 continued

Feed butanol Butanol Active layer Temperatu Total flux Separation Membrane material concentration flux Reference thickness (µm) re (ºC) (g/m2·h) factor (wt%) (g/m2·h) EPDM - 1-10 30 - 0-20 5 (Jitesh et al. SBR - 1-10 30 - 0-25 10-20 2000) (Thongsukmak TOA/liquid - 1.5 54 - 11 275 and Sirkar membrane 2007) (Srinivasan et PVDF 120 7.5 50 4126 - 6.4 al. 2007) (Boddeker et

152 PUR 50 1 50 88 10 9 al. 1990)

41-141 1.5 47 120-278 45-97 39-45 PDMS 100 0.5-3.0 47 120-213 21-118 41-45 This work 100 1.5 27-56 71-219 27-83 38-44 PDMS/zeolite 100 1.5 27-56 48-171 22-100 60-85

Figure 5.1 Pervaporation system used in this study

153

A B

C D

Figure 5.2 Surface and cross-sectional scanning electron microscope (SEM) images of

PDMS membrane and zeolite filled PDMS MMM (A and B: surface of cross-sectional image of PDMS membrane; C and D: surface and cross-sectional image of zeolite filled

PDMS MMM)

154

200 Separation factor Butanol flux Water flux

150 Total flux

·h), separation separation ·h),

2 factor 100

50 Fluxes (g/m Fluxes

0 PDMS 10% zeolite 20% zeolite 30% zeolite 40% zeolite

Figure 5.3 Effect of ZSM-5 zeolite CBV28014 in PDMS membrane on butanol separation by pervaporation (47 °C, 1.5 wt% butanol feed solution, ~100 µm in thickness)

155

250 Butanol flux 70

·h) Water flux 2 y = 3.999x + 100 Total flux 60 200 Separation factor 50

150 40

100 30 y = -0.0938x + 98.8 20 factor Separation 50 y = 4.088x + 1.30 10

Butanol, water and total flux (g/mflux total and water Butanol, 0 0 0 5 10 15 20 25 30 Feed 1-butanol concentration (g/L)

Figure 5.4 Effect of feed butanol concentration on pervaporation performance of PDMS membrane (47 °C, ~100 µm in thickness)

156

300

BuOH flux ·h) 2 Water flux 250 Total flux y = 9.4082x + 54.95 200

150 y = 6.3514x + 30.016

100 y = 3.0545x + 24.984

50 Butanol, water and total flux (g/mflux total and water Butanol, 0 0 5 10 15 20 25 30 1000/thickness (1/µm) A

120 BuOH flux Separation factor 100

·h), separation separation ·h),

2 60 factor 40

BuOH flux (g/m flux BuOH 0 141 115 90 75 41 B Thickness (µm)

Figure 5.5 Effect of membrane thickness on the performance of PDMS membranes: A, butanol, water and total flux; B, butanol flux and separation factor (47 ºC, 1.5 wt% butanol feed solution) 157

BuOH flux of PDMS MMM 134.1 BuOH flux of PDMS

Water flux of PDMS MMM ·h)

2 Water flux of PDMS

EH2O, PDMS=33.2 kJ/mol 49.7

EH2O, PDMS MMM=31.8 kJ/mol EBuOH, PDMS=34.3 kJ/mol

Butanol and water flux flux (g/mwater and Butanol EBuOH, PDMS MMM=44.2 kJ/mol 18.4 3.0 3.1 3.2 3.3 3.4 1000/T (K-1)

Figure 5.6 Arrhenius plots of butanol and water fluxed for PDMS membrane and 40 wt% zeolite filled PDMS MMM (y axis in log scale, 1.5 wt% butanol solution, ~100 µm in thickness)

158

Total flux of PDMS 300 Total flux of PDMS MMM 100 α of PDMS

250 α of PDMS MMM 80

·h) 200 2 60 150 40

100 Separation factor Separation Total flux (g/mflux Total 50 20

0 0 20 25 30 35 40 45 50 55 60 Temperature (°C)

Figure 5.7 Effect of temperature on total flux and separation factor of PDMS membrane and 40 wt% zeolite filled PDMS membranes (α represents separation factor, 1.5 wt% butanol solution, ~100 µm in thickness)

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Chapter 6: Conclusions and Recommendations

6.1 Conclusions

This study demonstrated the feasibility and advantages of butanol fermentative production integrated with online/in situ product recovery. Adsorption and gas stripping were integrated with butanol fermentation. Higher butanol concentration was achieved in those integrated process. High yield butanol production was also achieved in C. tyrobutyricum mutant strain CtΔack-adhE2 with external driving forces. For pervaporation, it was not integrated with the actual fermentation process due to membrane fouling. Performance of polydimethylsiloxane (PDMS) membrane was greatly enhanced by zeolite incorporation.

6.1.1 Butanol production integrated with adsorption

Many alternative recovery techniques have been developed to recovery butanol from dilute fermentation broth. Adsorption is a promising process for butanol recovery and inhibitory product removal from fermentation broth. A variety of adsorbent materials have been screened and evaluated for their butanol uptake capacity from the

160

commercially available candidate pool. Four adsorbents were selected for further study: activated carbon (Norit ROW 0.8), zeolite CBV901, polymeric resin SD-2 and L-493.

Among these, activated carbon (Norit ROW 0.8) showed the best specific loading and adsorbent-aqueous partitioning coefficient of butanol. In batch fermentation in serum bottles without pH control, 21.9 g/L of total butanol production was achieved with in situ adsorption by activated carbon, increased by 87.2% compared to the control experiment.

In integrated fed-batch fermentation with in situ butanol adsorption by activated carbon, total butanol titer in both free cell and immobilized cell fermentation was increased compared to control without adsorption. Especially in immobilized cell fermentation, butanol productivity and titer were enhanced by ~30% and ~200%, respectively, compared to control fermentation. Furthermore, ~150 g/L of butanol solution could be recovered in the condensate by heating butanol-adsorbed activated carbon, which was easily concentrated to ~640 g/L after simple phase separation. The specific energy cost of the in situ product recovery (ISPR) process was estimated to be ~4.8kJ/g butanol with great energy efficiency, exhibiting its economical potential for the application in butanol fermentation to simultaneously remove inhibitory product and product recovery.

6.1.2 Butanol production from lignocellulosic biomass integrated with gas stripping

As a second generation biofuel, butanol can be produced from lignocellulosic biomass. Preliminary studies on effects of different nitrogen (mixture of tryptone and yeast extract vs. corn steep liquor) and carbon sources (glucose, xylose, and lignocellulosic biomass) were performed in serum bottle fermentation by engineered C. 161

tyrobutyricum mutant strain CtΔack-adhE2 with external driving forces. Corn steep liquor (CSL) is a good nitrogen source replacement and ~6.0 g/L butanol was produced from soybean hull hydrolysate compared to 7.5 g/L butanol production in the control in serum bottles. Fed-batch butanol fermentation production in the co-existence of glucose and xylose in a fibrous-bed bioreactor integrated with gas stripping was further studied.

Final butanol titer, yield and productivity of 12.1 g/L, 0.25 g/g and 0.12 g/L∙h, respectively, were obtained from soybean hull hydrolysate (SHH). Due to a higher level of or more inhibitors present in sugarcane bagasse hydrolysate (SBH), butanol titer, yield and productivity of 10.7 g/L, 0.31 g/g and 0.0 1 g/L∙h, respectively, were achieved in medium containing sugarcane bagasse hydrolysate. Detoxification of lignocellulosic biomass hydrolysates before fermentation can further boost butanol production. A glucose and xylose mixture was also used to produce butanol, which resulted in a total butanol production of 24.7 g/L, an increase of ~67% in butanol titer compared to control.

To the best of our knowledge, this is the first attempt to produce butanol from soybean hull hydrolysate by fermentation integrated with gas stripping. This work demonstrated the feasibility of butanol fermentative production from soybean hull and sugarcane bagasse hydrolysates and a mixture of glucose and xylose in a gas stripping integrated system.

6.1.3 Butanol recovery by pervaporation using PDMS membranes

Pervaporation can recover butanol from aqueous solution with high selectivity.

PDMS membranes and ZSM-5 zeolite filled PDMS mixed matrix membranes (MMM) 162

were developed and tested for 1-butanol recovery by pervaporation from model solutions.

The incorporation of zeolite to PDMS membrane blocked a large amount of water, and therefore greatly increased the butanol separation factor. At the highest zeolite filling of

40 wt%, the PDMS MMM gave a butanol separation factor of 77 and a butanol and total flux of 62 and 118 g/m2·h, respectively, with 1.5 wt% 1-butanol feed concentration at

47°C. This means permeate containing an average of ~480 g/L of 1-butanol can be obtained by pervaporation from 15 g/L 1-butanol solutions. After natural phase separation, even higher concentration of butanol solution can be obtained. The butanol separation factor of PDMS MMM can be further enhanced by elevating operating temperature. Besides, butanol and total fluxes can be further boosted by reducing membrane thickness, increasing feed 1-butanol concentration, or elevating operating temperature. A butanol flux of ~600 g/m2·h is expected with a membrane thickness of 5

µm. The apparent activation energies of 1-butanol permeation in PDMS membrane and zeolite filled (40 wt%) PDMS MMM were evaluated to be 34.3 and 44.2 kJ/mol, respectively. 1-butanol recovery incorporating pervaporation saves a great portion of energy consumption compared to distillation. Compared with literature, this work demonstrated higher 1-butanol separation performance by pervaporation and had great potential application for fermentative 1-butanol recovery.

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6.2 Recommendations

6.2.1 Butanol production integrated with adsorption

Activated carbon was selected as the adsorbent to integrate with fermentation. In the integrated process, acetone gradually accumulated to 18 g/L and finally became toxic to cells due to a higher affinity for butanol compared to acetone in activated carbon.

Therefore, acetone free strains such as C. tyrobutyricum mutant strain CtΔack-adhE2 can be used for this purpose (Yu et al. 2012; Yu et al. 2011), which is also used in the study of chapter 4. Besides, since activated carbon also adsorbs other medium components including water, sugars and nutrients, butanol specific adsorbent materials are of interest.

Research can be done reaching out for high selective adsorbent materials targeting butanol only.

6.2.2 Butanol production from lignocellulosic biomass integrated with gas stripping

Lignocellulosic biomass hydrolysates were used in fermentation without any detoxification in this study. Different detoxification methods could be explored to remove some inhibitors present in the hydrolysates, and further facilitate the butanol fermentative production. Nutrients can be replenished in the fed-batch fermentation to ensure active cell growth and stable butanol production. Furthermore, other than acid pretreatment and enzymatic hydrolysis, different biomass pretreatment methods can also be investigated like alkaline pretreatment.

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6.2.3 Butanol recovery by pervaporation

Butanol recovery by pervaporation from model solution, but not fermentation broth, was studied. Future work can be done to recover butanol in a pervaporation- fermentation integrated process. Before the integration, anti-fouling membranes can be developed. Other than zeolite, many other filler materials, such as carbon nanotubes, can also be explored. In order to enhance butanol flux, thinner membranes can be fabricated for this purpose.

6.3 References

Yu MR, Zhang YL, Tang IC, and Yang ST. 2011. Metabolic engineering of Clostridium tyrobutyricum for n-butanol production. Metab Eng 13(4):373-382.

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