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

By

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

Copyright by

Fangfang Liu

2014

ii

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

iii

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.

iv

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

v

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.

vi

Dedication

Dedicated to my parents and sisters

vii

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.

viii

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.

ix

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

x

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

xi

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

xii

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

xiii

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

xiv

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

xv

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

xvi

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

xvii

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

xviii

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

xix

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

xx

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

xxi

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

1

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

2

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

3

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).

4

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

5

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.

6

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8

Moradi F, Amiri H, Soleimanian-Zad S, Ehsani MR, and Karimi K. 2013. Improvement

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19(5):420-429.

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liquor on bioreactor performance. Appl Biochem Biotech 113:713-721.

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9

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batch reactors. Biomass Bioenerg 32(12):1353-1358.

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1.3 Figures

10

Figure 1.1 Overview of research objective and tasks in this study

Research Objective

To develop integrated processes for n-butanol fermentation and

recovery by adsorption, gas stripping and pervaporation

Task 1

Butanol production

integrated with

adsorption using

activated carbon and

other adsorbents

(Chapter 3)

Task 2

Butanol production in

the co-existence of

glucose and xylose

with external driving

forces integrated with

gas stripping

(Chapter 4)

Task 3

Butanol recovery

from aqueous model

solution by

pervaporation using

polydimethylsiloxane

(PDMS) membranes

(Chapter 5)

11

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,

12

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

13

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

14

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

15

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

16

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

17

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.

18

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

19

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,

20

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

21

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

22

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

23

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):

Xr = h h h

h h (1)

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

L =

(2)

24

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

25

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).

26

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)

27

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.

28

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

29

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

30

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.

31

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

32

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

33

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

48

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)

Species / strain Substrates pH Temp.

(oC) Products References

C. acetobutylicum 260

P262

ATCC 824

Glucose, xylose, arabinose,

cellobiose, mannose, galactose

5.5 – 6.5 35 Acetone, butanol,

ethanol, acetate,

butyrate

(Ezeji and

Blaschek 2008)

Starch (Madihah et al.

2001)

Lactose (Qureshi and

Maddox 2005) Glucose, mannose, galactose,

fructose, arabinose, xylose, sucrose,

lactose, maltose, cellobiose, starch (Servinsky et al.

2010) Cassava 3.5-6.0 37 (Li et al. 2012b)

NCIMB 13357 Date fruit 7.0 35 (Khamaiseh et al.

2014)

C. carboxidivorans P7

Syngas (H2, CO, CO2) 6.0 N/A Acetate, ethanol,

butyrate, butanol (Bruant et al.

2010)

CO, CO2 5.3 37 Acetate, ethanol,

butanol (Rajagopalan et

al. 2002)

C.

saccharoperbutylacetonicum N1-4 ATCC 13564

Glucose, maltose, starch 6.2 30

Acetone, butanol,

ethanol, acetate,

butyrate

(Thang et al.

2010)

Glucose, molasses, starch Not

controlled (Hipolito et al.

2008)

C. saccharobutylicum 262

Glucose, xylose, arabinose,

cellobiose, mannose, galactose 5.5 – 6.5 35

Acetone, butanol,

ethanol, acetate,

butyrate

(Ezeji and

Blaschek 2008)

C. butylicum NRRL 592

Glucose, xylose, arabinose,

cellobiose, mannose, galactose 5.5 – 6.5 35

Acetone, butanol,

ethanol, acetate,

butyrate

(Ezeji and

Blaschek 2008)

Continued

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

49

Table 2.2 continued

Species / strain Substrates pH Temp.

(oC) Products References

C. beijerinckii BA101

NCIMB 8052

P260

Glucose, xylose, arabinose,

cellobiose, mannose, galactose

5.5 – 6.5

35

Acetone, butanol,

ethanol, acetate,

butyrate

(Ezeji and

Blaschek 2008) (Ezeji et al.

2007a)

Not

controlled

Glucose, maltodextrin

N/A

Room

temp. (Formanek et al.

1997)

Glucose, maltodextrin Room

temp. (Formanek et al.

1997) Glucose, fructose, galactose,

glucitiol 37 (Mitchell 1996)

Glucose, xylose, arabinose,

galactose, mannose 6.5 35

(Qureshi et al.

2014)

C. aurantibutyricum ATCC 17777 NCIB 10659

Glucose, , xylan, starch, pectin,

arabinose, xylose, galactose,

mannose 5.5 – 6.8 37 Acetone, butanol,

isopropanol, acetate,

butyrate

(Somrutai et al.

1996)

Glucose 6.8 35 (George et al.

1983)

C. pasteurianum DSM 525 (same as ATCC

6013) Glycerol

5.0 – 7.0 37 Butanol, ethanol, 1,3-

propanediol, acetate,

butyrate, lactate

(Ahn et al. 2011) 4.5 – 7.5 35 (Biebl 2001)

7.0 35 (Taconi et al.

2009)

50

Feedstock Pretreatment and

hydrolysis Strain

ABE titer

(g/L) ABE yield

(g/g) Productivity

(g/L.h) References

Glucose + corn steep

water (CSW) None

C. beijerinckii NCIMB 8052 19.2 N/A N/A (Parekh and

Blaschek 1999) C. beijerinckii BA101 23.6 N/A N/A Cassava starch

None C. saccharoperbutylacetonicum

N1-4 ATCC 13564

21.0 0.41 0.44 (Thang et al.

2010) Corn starch 20.7 0.48 0.31 Sago starch 19.6 0.43 0.27 Cassava chips Enzyme 19.4 0.38 0.44 Cassava bagasse +

glucose None C. acetobutylicum JB200 33.9 0.39 0.62 (Lu et al. 2012)

Soy molasses +

glucose None C. beijerinckii BA101 30.1 N/A N/A

(Qureshi et al.

2001a) Degermed corn +

corn steep liquor

(CSL) None C. beijerinckii BA101 19.3 N/A 0.23

(Campos et al.

2002)

Date fruit None C. acetobutylicum NCIMB

13557 N/A 0.35-0.61 0.025-0.24

(Khamaiseh et

al. 2014)

Corn fiber Dilute acid +

enzyme C. beijerinckii BA101 9.3 0.39 0.10

(Qureshi et al.

2008a)

Wheat straw

Dilute acid C. beijerinckii BA101 25.0 0.42 0.60 (Qureshi et al.

2007) Alkaline peroxide

+ enzyme C. beijerinckii P260 22.2 0.41 0.55

(Qureshi et al.

2008b)

Enzyme C. acetobutylicum ATCC 824 7.05

(butanol) 0.155

(butanol) 0.141

(butanol) (Wang et al.

2013c)

Continued

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

available)

51

Table 2.3 continued

Feedstock Pretreatment and

hydrolysis Strain

ABE titer

(g/L) ABE yield

(g/g) Productivity

(g/L.h) References

Distiller’s dried

grains and solubles

(DDGS)

Dilute acid Liquid hot water AFEX + enzyme

C. acetobutylicum 260 C. acetobutylicum ATCC 824 C. saccharobutylicum 262 C. butylicum 592 C. beijerinckii BA101

4.9-12.9 0.30-0.35 N/A (Ezeji and

Blaschek 2008)

Electrolyzed water

+ enzyme C. beijerinckii BA101 3.5-5.5 N/A N/A

(Wang et al.

2013a)

Wheat bran Dilute acid C. beijerinckii ATCC 55025 11.8 0.32 0.16 (Liu et al.

2010)

Barley straw Dilute acid C. beijerinckii P260 26.6 0.43 0.39 (Qureshi et al.

2010a) Corn stover Dilute acid C. beijerinckii P260 26.3 0.44 0.31 (Qureshi et al.

2010b) Switchgrass Dilute acid C. beijerinckii P260 14.6 0.39 0.17

Rice straw

Acid/alkaline +

enzyme C. acetobutylicum NRRL B-591

~3.0 N/A N/A (Moradi et al.

2013) Dilute acid +

enzyme 7.1 10.5 N/A

(Amiri et al.

2014)

Acid + enzyme C. sporogenes BE01 5.3 N/A N/A (Gottumukkala

et al. 2013) Willow stem

Acid hydrolysis C. beijerinckii NCIMB 8052 9.4 N/A N/A (Han et al.

2013) Willow bark 8.9 N/A N/A

Corn straw Acid + enzyme C. acetobutylicum CICC 8008 6.2 (butanol) N/A N/A (Lin et al.

2011)

Wood pulp Cook + wash +

acid +

detoxification C. beijerinckii CC101 7.9 0.33 0.11 (Lu et al. 2013)

52

Butanol recovery

technique Principle Advantages Limitations

Adsorption

Adherence of solvents to silicalite

resin, clay, activated carbon, or other

adsorptive materials

Easy to operate, low energy

requirement

High adsorbent cost, low efficiency, low

selectivity (will absorb any component),

low adsorbent capacity (loading: ~0.1

g/g)

Gas stripping Volatile solvents being stripped out by

gases and then condensed

Easy to operate, no harm to the

culture, no fouling

Require a high temperature or vacuum for

sufficient volatility, low selectivity

(separation factor: 6~20)

Liquid-liquid

extraction

Using the solubility differences of

solvents High selectivity, efficient Forming emulsion, toxic to the culture

Perstraction

Membrane-based extraction,

separating the fermentation broth from

the extractive solvents

High selectivity, low toxic to the

culture compared to liquid-liquid

extraction

Poor stability, membrane fouling, high

cost

Pervaporation Using membrane to selectively let the

vaporous solvents pass through

High selectivity (separation

factor: 5~100) Membrane fouling, high cost

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)

53

54

Adsorbent material

Feed

CBuOH

(g/L)

Butanol

adsorption

capacity

(mg/g)

Adsorbent

loading

(g/L)

References

Activated carbon Norit W52 15.0 252 10 (Groot and

Luyben 1986)

Activated carbon F-400, F-600 N/A 200-300 N/A (Abdehagh et

al. 2013)

XAD-16

resin

9.2 75 85 (Ennis et al.

1987)

XAD-2 16.5 78 10 (Groot and

Luyben 1986;

Qureshi et al.

2005)

XAD-4 14.4 100 10

XAD-8 15.5 66 10

Amberlite XAD-4 4.0 –

20.0 27 – 83 100 – 200

(Nielsen et al.

1988)

Amberlite XAD-7 4.0 –

20.0 22 – 69 100 – 200

Bonopore 4.0 –

20.0 23 – 74 100 – 200

Bonopore, nitrated 4.0 –

20.0 13 – 55 100 – 200

Poly(styrene-co-

divinylbenzene) (including

Dowex Optipore L-493, SD-2,

M43, Amberlite IR-120, IRA-

900 and etc)

5.0 22.3 – 56.3 100 (Nielsen and

Prather 2009)

Poly(methacrylate) 5.0 34.7 100

Poly(butrylene phthalate) 5.0 7.4 100

KA-I (cross-linked polystyrene

framework)

6.5 84-100 40-80 (Liu et al.

2014)

0.2-25 140-305 20 (Lin et al.

2012)

Dowex Optipore L-493 N/A ~300 40-160 (Wiehn et al.

2014)

Polyvinylpyridine 14.9 68 100 (Yang et al.

1994)

Continued

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

not available)

55

Table 2.5 continued

Adsorbent material

Feed

CBuOH

(g/L)

Butanol adsorption

capacity (mg/g)

Adsorbent

loading (g/L) References

Silicalite

21.5 97 40 (Milestone and

Bibby 1981)

11.7 –

16.8 64 – 85 168 (Maddox 1982)

8.3 63.5 85 (Ennis et al.

1987)

Zeolite ZSM-5

(CBV901, 811 and

28014)

4.8 – 9.0 98 – 117 7 – 25 (Oudshoorn et al.

2009a)

Zeolite ZSM-5

(CBV901 and 28014) 10 ~120 64

(Saravanan et al.

2010)

Zeolite (MEL3-6) 2 222 N/A (Sharma and

Chung 2011)

Metal-organic

Framework (MOF)

ZIF-8

1-70 ~300 N/A (Saint Remi et al.

2011)

Fermentation model/

adsorption Culture species

Adsorbent (resin) Solvent production

References Type

Capacitya

(mg/g)

Equivalent

total solvent

conc. (g/L)

Solvent

productivi

ty (g/L∙h)

Solvent

yield

(g/g)

Batch/ in situ

adsorption

C. beijerinckii

LMD XAD-8 30 12.6 0.13 N/A

(Groot and

Luyben 1986)

Two-stage continuous

fermentation/ in-line

adsorption

C. acetobutylicum

P262 XAD-16 64 11.0 1.2

0.30-

0.36

(Ennis et al.

1987)

Repeated batch/ batch

adsorption

C. acetobutylicum

ATCC 824

Bonopore,

poly(styrene-co-DVB) 43 N/A N/A N/A

(Nielsen et al.

1988)

Batch/ in situ

adsorption

C. acetobutylicum

ATCC 824

Dowex Optipore SD-

2, poly(styrene-co-

DVB) derivative

264 37 0.51 0.27-

0.40

(Nielsen and

Prather 2009)

Repeated fed-batch/

fixed bed adsorption C. acetobutylicum

Reillex 425,

polyvinylpyridine 61 47.2 1.69 0.32

(Yang and

Tsao 1995)

Fed-batch/ fixed-bed

adsorption C. acetobutylicum

B3

KA-I, cross-linked

polystyrene

framework

84 96.5 1.51 0.33

(Liu et al.

2014) Fed-batch/ fixed bed

adsorption (MV

addition)

93 130.7 0.97 0.36

Fed-batch/ expanded

bed adsorption

C. acetobutylicum

ATCC 824

Dowex Optipore L-

493 300 40.7 0.72 0.28

(Wiehn et al.

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)

56

Feed conditions Stripping

Temp. (oC)

Condensation

Temp. (oC)

Stripping gas and gas

recycle rate* Selectivity References

Integrated with fed-batch

bioreactor 34 -60 N2, 2.7 L/min ABE 4-30.5

(Qureshi and

Blaschek

2001)

Separate stripper, continuous

fermentation 65 - 67 3 - 4 N2, 2.5 L/min ABE 30.5

(Qureshi et al.

1992)

Integrated with batch

bioreactor 34 -0.8

H2 and CO2,

1.5 – 3.3 L/(L∙min) ABE 9.5 - 13

(Maddox et al.

1995)

Model solution 35 -2 N2, 4.6 L/min

Butanol 10.3 - 13.8

Acetone 4.1 - 6.4

Ethanol 4.9 - 7.9 (Ezeji et al.

2003) Integrated with batch

bioreactor 33 - 35 -2 H2 and CO2, 3 L/min

Butanol 6.7 - 13

Acetone 4.7 - 10.5

Ethanol 4.7 - 9.3

Integrated with fed-batch

bioreactor 33 - 35 -2 H2 and CO2, 6 L/min Butanol 10.3 - 22.1

(Ezeji et al.

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)

57

Substrate Strain Fermentation

mode ABE (g/L)

ABE yield (g/g)

ABE Productivity

(g/L∙h) References

Glucose C. acetobutylicum NCIB

8052 Continuous ~10 0.36 0.58 (Mollah and Stuckey 1993)

Whey permeate C. acetobutylicum P262 Batch 70.0 0.35 0.32 (Maddox et al. 1995) 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

C. beijerinckii BA101 Batch 23.9 0.43 0.31

(Ezeji et al. 2007c) Saccharified corn

starch Batch 26.5 0.41 0.40 Fed-batch 81.3 0.36 0.59

Cassava bagasse C. acetobutylicum

JB200 Fed-batch 108.5 037 0.47 (Lu et al. 2012)

Glucose

C. acetobutylicum

JB200

Batch 31.8 0.40 0.66 (Xue et al. 2013)

Fed-batch 73.3 0.40 0.36 (Xue et al. 2014)

70% WPH* C. beijerinckii CC101 Batch

12.9 0.39 0.17 (Lu et al. 2013)

Detoxified WPH* 17.7 0.44 0.25

Glucose C. acetobutylicum ATCC

824 Fed-batch N/A N/A 0.22-0.73 (Setlhaku et al. 2013)

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

pulp hydrolysate)

58

Membrane

material

Active layer

thickness (µm)

Feed 1-butanol

concentration (wt%)

Feed

temperature

(ºC)

Total flux

(g/m2·h)

BuOH flux

(g/m2·h)

Separation

factor References

PDMS/hollow

fiber 10 1 40 1282 - 43

(Dong et al.

2014)

PDMS/ceramic 10 1 40 457 - 26 (Liu et al.

2011)

PDMS/PANa 4 3.5 42 - 800 22-29 (Niemisto et

al. 2013)

Tri-layer PDMS 65-200 2 37 40-132 20-50 32-50 (Li et al.

2010)

PDMS 25 0-7 50 200-1000 0-700 15-35 (Hickey et al.

1992)

PMS - 0-7 50 130-380 0-200 10-15 (Hickey et al.

1992)

Silicone 50

1 30-70

53-350 - 42-49 (Huang and

Meagher

2001) Silicalite-filled

silicone 19 63-607 - 86-111

PTMSP - 0.3-6 25-70 60-2097 16-347 41-78 (Fadeev et al.

2001)

PEBA 30-100 5 23 65-179 19-42 6-8 (Liu et al.

2005)

ZIF-71 filled

PEBAb 10-20 1 37 520 - 18.8

(Liu et al.

2013)

ZSM-5 filled

PEBA - 2.5 30-45 - 90-240 22-30

(Tan et al.

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)

59

Table 2.9 continued

Membrane

material

Active layer

thickness (µm)

Feed 1-butanol

concentration (wt%)

Feed

temperature

(ºC)

Total flux

(g/m2·h)

BuOH flux

(g/m2·h)

Separation

factor References

EPDM - 1-10 30 - 0-20 5 (Jitesh et al.

2000) SBR - 1-10 30 - 0-25 10-20

TOA/liquid

membrane - 1.5 54 - 11 275

(Thongsukm

ak and Sirkar

2007)

PVDF 120 7.5 50 4126 - 6.4 (Srinivasan et

al. 2007)

PUR 50 1 50 88 10 9 (Boddeker et

al. 1990)

PDMS

41-141 1.5 47 120-278 45-97 39-45

This work 100 0.5-3.0 47 120-213 21-118 41-45

100 1.5 27-56 71-219 27-83 38-44

PDMS/zeolite 100 1.5 27-56 48-171 22-100 60-85

60

Fermentation

mode Strain Membrane

ABE

(g/L)

ABE yield

(g/g)

Productivity

(g/L.h) References

Continuous C. acetobutylicum

P262 Polyethylene hollow fiber 42.0 0.34 0.14

(Qureshi et al.

1992)

Fed-batch C. beijerinckii BA101 Silicone membrane 165 0.43 0.98 (Qureshi and

Blaschek 2000)

Fed-batch C. acetobutylicum

ATCC 824

Silicalite-silicone composite

membrane 155 0.35 0.18

(Qureshi et al.

2001b)

Continuous C. acetobutylicum

ATCC 824

PDMS composite

membrane N/A 0.36 0.37

(Van Hecke et al.

2012)

Continuous C. acetobutylicum

ATCC 824

PDMS composite

membrane 41.8 0.35 1.13

(Van Hecke et al.

2013)

Table 2.10 Integrated fermentation-pervaporation process for ABE production

61

Recovery

technique Substrate Strain

Fermentation

mode

ABE

(g/L)

ABE yield

(g/g)

Productivity

(g/L.h) References

Liquid-liquid

extraction

Glucose C. acetobutylicum

ATCC 824 Fed-batch

50.5–

96.5 0.33–0.36 1.4–2.3

(Roffler et al.

1987)

Whey permeate C. acetobutylicum

P262 Continuous 23.8 0.35 0.14

(Qureshi et al.

1992)

Glucose C. acetobutylicum B

5313 Continuous 25.3 0.35 2.5

(Bankar et al.

2012)

Perstraction

Whey permeate

+ lactose C. acetobutylicum

P262

Batch 136.6 0.44 0.21 (Qureshi and

Maddox 2005)

Whey permeate Continuous 57.8 0.37 0.24 (Qureshi et al.

1992)

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

62

63

Figure 2.1 Metabolic pathways in Clostridium acetobutylicum during both acidogenesis

(dotted arrows) and solventogenesis (solid arrows)

Reference: (Lee et al. 2008)

64

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)

65

66

Figure 2.4 Mass transfer at a gas/liquid interface

67

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

68

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-

69

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

70

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-

71

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)

72

(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:

73

1

mq K Cq

K C

(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),

74

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.

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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.

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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).

77

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

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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

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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 o

C 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

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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.

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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

82

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

83

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

84

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

85

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 o

C),

revealing weak adsorption, as suggested in this study.

86

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

87

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

88

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

89

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.

90

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

91

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.

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production by clostridia. Biotechnol Bioeng 101(2):209-228.

Lu CC, Zhao JB, Yang ST, and Wei D. 2012. Fed-batch fermentation for n-butanol

production from cassava bagasse hydrolysate in a fibrous bed bioreactor with

continuous gas stripping. Bioresource Technol 104:380-387.

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.

93

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.

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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

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Papoutsakis ET. 2008. Engineering solventogenic clostridia. Curr Opin Biotech

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4(2):81-92.

3.6 Tables and figures

95

Type Adsorbents Cbutanol in feed

(g/L)

Specific loading,

(g/g) References

Act

ivat

e

d c

arb

on

Norit Row 0.8 15.0 0.252 (Groot and Luyben

1986)

Norit Row 0.8 0.5~30 0.10~0.45 This study

Res

in

XAD-2 16.5 0.078 (Groot and Luyben

1986) XAD-4 14.4 0.100

XAD-8 15.5 0.069

Polyvinylpyridine 14.9 0.068 (Yang et al. 1994)

Dowex Optipore L-

493 ~20 0.175

(Nielsen and Prather

2009) Dowex Optipore

SD-2 ~20 0.152

Dowex Optipore

SD-2 1.5~35 0.10~0.39 This study

Dowex Optipore L-

493 2.0~35 0.10~0.37 This study

Zeo

lite

CBV811 4.8~9.0 0.98~0.117 (Oudshoorn et al.

2009a)

Silicalite 11.7~16.8 0.064~0.085 (Maddox 1982)

CBV28014 ~10 0.092 (Saravanan et al.

2010) CBV901 ~10 0.10

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))

96

Adsorbents

37 oC 60

oC

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

97

Control

Activated

carbon

L-493 SD-2 CBV901

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

(g/L)

11.7 4.1 4.8 5.2 4.5

Final pH 4.85 4.60 4.25 4.39 4.56

Total butanol production

(g/L) a

11.7 21.9 14.5 15.2 11.5

Yield (g butanol/ g

glucose)

0.27 0.31 0.31 0.31 0.28

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)

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

butanol over

acetone Acetone Ethanol Butanol

Acetic

acid

Butyric

acid

Acetone Ethanol Butanol

Acetic

acid

Butyric

acid

18.2 5.0 29.6 10.1 2.8 0.038 0.000 0.290 0.016 0.038 7.6

17.5 4.8 20.6 10.1 1.9 0.029 0.000 0.272 0.008 0.032 9.4

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)

98

99

Free cell fermentation Immobilized cell

fermentation

w/o

adsorption

w/

adsorption

w/o

adsorption

w/

adsorption

Total fermentation time (h) 54 106 48 122

Total amount of glucose

consumed (g/L) 82 158 77 260

Final butanol concentration

in the broth (g/L) 18.3 18.7 16.4 8.9

Amount of butanol

adsorbed on activated

carbon (g)

- 18.8 - N/A

Adsorbed butanol per unit

volume (g/L) - 12.9-16.1

a - N/A

Butanol yield (g/g) 0.22 0.20-0.22a 0.21 0.21

b

Total butanol production

(g/L) 18.3 31.6-34.8 16.4 ~54.6

Butanol productivity

(g/L·h) 0.34 0.30-0.33 0.35 ~0.45

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)

100

Separation Method

Energy requirements (kJ/g)

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

101

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

Thermostat water

Activated carbon

Pump

Substrate tank Bioreactor

Temperature controlling unit

PH controlling unit

102

A

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

0

0.1

0.2

0.3

0.4

0.5

0 10 20 30 40

Activated carbon

CBV901

Dowex L-493

Dowex SD-2

Sp

ecif

ic L

oad

ing , L

g-b

uta

no

l/g

-adso

rben

t

ButanolAq, g/L

0

0.1

0.2

0.3

0.4

0.5

0 10 20 30 40

Activated carbon CBV901 Dowex L-493 Dowex SD-2

Sp

ecia

l L

oad

ing , L

g-b

uta

no

l/g

-adso

rben

t

ButanolAq, g/L

103

A

0 10 20 30 40 50 60

0

10

20

30

40

50

60

70

80

90 Glucose (g/L)

Glu

co

se

(g

/L)

Time (h)

0

4

8

12

16

20

24

Acetone (g/L) Butanol (g/L) Acetic acid (g/L)

Butyric acid (g/L) O.D.

Pro

du

cts

(g

/L),

O.D

.

B

0 20 40 60 80 100 120

0

10

20

30

40

50

60

70

80

90

100

110 Glucose (g/L)

Glu

co

se

(g

/L)

Time (h)

0

4

8

12

16

20

24

Acetone (g/L) Butanol (g/L) Acetic acid (g/L)

Butyric acid (g/L) O.D.

Pro

du

cts

(g

/L),

O.D

.

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

104

Figure 3.3 continued

C

0 20 40 60 80 100 120 140

0

10

20

30

40

50

60

70

80

90

100

110 Glucose (g/L)

Glu

co

se

(g

/L)

Time (h)

0

4

8

12

16

20

24

Acetone (g/L) Butanol (g/L) Acetic acid (g/L)

Butyric acid (g/L) O.D.

Pro

du

cts

(g

/L),

O.D

.

D

160 180 200 220 240 260 280

0

10

20

30

40

50

60

70

80

90

100

110 Glucose (g/L)

Glu

co

se

(g

/L)

Time (h)

0

2

4

6

8

10

12

14

16

18

20

22

24

Acetone (g/L) Butanol (g/L) Acetic acid (g/L)

Butyric acid (g/L) O.D.

Pro

du

cts

(g

/L),

O.D

.

105

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

determined thermogravimetrically

0

20

40

60

80

100

25 50 75 100 125 150 175 200 225 250

Butanol

Water

ABE W

eight

rem

ainin

g (

%)

Temperature, oC

106

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

107

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

108

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).

109

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

110

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,

111

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

112

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.

113

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).

114

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.

115

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

116

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

117

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.

118

Acknowledgements

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

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4.6 Tables and figures

122

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)

Substrate

Initial sugar

conc. (g/L,

glucose/xylose/

total)

Final sugar

conc. (g/L,

glucose/xylose/

total)

Butanol

(g/L)

Solvents

(g/L)

Acids

(g/L)

Butanol

yield

(g/g)

Acids

yield

(g/g)

Solvents

to acids

ratio

Butanol

productivity

(g/L∙h)

1: glucose +

tryptone and

YE*

40.6/0/40.6 18.0/0/18.0 7.5 8.5 3.7 0.33 0.16 2.3 0.078

2: glucose +

CSL 44.7/0/44.7 26.4/0/26.4 7.5 8.8 2.6 0.41 0.14 3.4 0.078

3: xylose+

tryptone and YE 0/49.1/49.1 0/48.2/48.2 0 0.1 0.5 0 0.56 0.2 0

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 +

tryptone and YE 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

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)

123

Fermentation

condition Substrate

Fermentation

time (h)

Butanol

(g/L)

Solvents

(g/L)

Acids

(g/L)

Butanol

yield

(g/g)

Acids

yield

(g/g)

Solvents

to acids

ratio

Butanol

productivity

(g/L∙h)

250 µM

MV

1st

batch

Mixture

of glucose

and

xylose

71 14.0 16.6 3.8 0.30 0.07 4.4 0.20

2nd

batch 55 14.8 19.6 8.6 0.28 0.17 2.3 0.22

Xylose 142 3.8 4.1 4.9 0.17 0.22 0.8 0.027

250 µM

MV + gas

stripping

SHH 103 12.1 14.7 10.7 0.25 0.14 1.4 0.12

SBH 116 10.7 11.8 15.0 0.31 0.19 0.8 0.081

25 µM BV

+ gas

stripping

1st

batch Mixture

of glucose

and

xylose

70 17.0 17.0 7.8 0.36 0.13 2.2 0.21

2nd

batch 145 24.7 25.1 10.1 0.27 0.08 3.5 0.053

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

124

125

A B

C D

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)

0

10

20

30

40

50

0 20 40 60 80 100

Glu

cose

(g

/L)

Time (h)

1

2

3

4

5

6 0

10

20

30

40

50

60

0 20 40 60 80 100

Xyl

ose

(g

/L)

Time (h)

1

2

3

4

5

6

0

1

2

3

4

0 20 40 60 80 100

O.D

. 60

0

Time (h)

1

2

3

4

5

6 0.0

0.5

1.0

1.5

2.0

2.5

0 20 40 60 80 100

Eth

ano

l (g

/L)

Time (h)

1

2

3

4

5

6

126

Figure 4.1 continued

E F

G

0

2

4

6

8

0 20 40 60 80 100

Bu

tan

ol (

g/L

)

Time (h)

1

2

3

4

5

6 0.0

0.5

1.0

1.5

2.0

2.5

0 20 40 60 80 100

Ace

tic

acid

(g

/L)

Time (h)

1

2

3

5

6

4

0

2

4

6

8

0 20 40 60 80 100

Bu

tyri

c ac

id (

g/L

)

Time (h)

1

2

3

4

5

6

127

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)

0

2

4

6

8

10

12

14

16

0

10

20

30

40

50

60

70

0 30 60 90 120 150

O.D

., p

rod

uct

s (g

/L)

Glu

cose

/xyl

ose

(g

/L)

Time (h)

Glucose

Xylose

O.D.

Ethanol

Butanol

Acetic acid

Butyric acid

128

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

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

0

1

2

3

4

5

0

10

20

30

40

50

60

0 50 100 150

O.D

., p

rod

uct

s (g

/L)

Glu

cose

/ xy

lose

(g

/L)

Time (h)

Glucose

Xylose

O.D.

Ethanol

Butanol

Acetic acid

Butyric acid

129

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

with gas stripping from soybean hull hydrolysate (SHH)

0

2

4

6

8

10

12

14

16

18

0

10

20

30

40

50

60

70

0 30 60 90 120 150 180

O.D

., p

rod

uct

s (g

/L)

Glu

cose

/ xy

lose

(g

/L)

Time (h)

Glucose

Xylose

O.D.

Ethanol

Butanol

Acetic acid

Butyric acid

Total Butanol

Gas stripping start

130

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

with gas stripping from sugarcane bagasse hydrolysate (SBH)

0

2

4

6

8

10

12

14

16

18

20

0

10

20

30

40

50

60

70

0 30 60 90 120 150 180

O.D

., p

rod

uct

s (g

/L)

Glu

cose

/ xy

lose

(g

/L)

Time (h)

Glucose

Xylose

O.D.

Ethanol

Butanol

Acetic acid

Butyric acid

Total butanol

Gas stripping start

131

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)

0

5

10

15

20

25

0

10

20

30

40

50

60

70

0 50 100 150 200 250 300 350

O.D

., p

rod

uct

s (g

/L)

Glu

cose

/ xy

lose

(g

/L)

Time (h)

Glucose

Xylose

O.D.

Ethanol

Butanol

Acetic acid

Butyric acid

Total butanol

Gas stripping start

132

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.

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 to be 34.3

133

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

134

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

135

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

136

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.

137

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

138

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

139

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)

140

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.

141

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).

142

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

143

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)

144

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

145

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.

146

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

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Liu SN, Liu GP, Zhao XH, and Jin WQ. 2013. Hydrophobic-ZIF-71 filled PEBA mixed

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model pharmaceutical aqueous waste by pervaporation. Chem Eng Sci

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Vane LM. 2005. A review of pervaporation for product recovery from biomass

fermentation processes. J Chem Technol Biot 80(6):603-629.

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

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production by Clostridium acetobutylicum JB200 in fed-batch fermentation with

intermittent gas stripping. Biotechnol Bioeng 109(11):2746-2756.

Yakovlev AV, Shalygin MG, Matson SM, Khotimskiy VS, and Teplyakov VV. 2013.

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5.6 Tables and figures

150

Membrane

Ea of butanol

(kJ/mol)

R2

Ea of water

(kJ/mol)

R2

ΔEa (Ea,butanol – Ea,

water) (kJ/mol)

PDMS 34.3 0.994 33.2 0.998 1.1

Zeolite filled

PDMS MMM*

44.2 0.997 31.8 0.996 12.4

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)

Membrane material Active layer

thickness (µm)

Feed butanol

concentration

(wt%)

Temperatu

re (ºC)

Total flux

(g/m2·h)

Butanol

flux

(g/m2·h)

Separation

factor Reference

PDMS/hollow fiber 10 1 40 1282 - 43 (Dong et al.

2014)

PDMS/ceramic 10 1 40 457 - 26 (Liu et al.

2011a)

PDMS/PAN 4 3.5 42 - 800 22-29 (Niemisto et

al. 2013)

Tri-layer PDMS 65-200 2 37 40-132 20-50 32-50 (Li et al. 2010)

Silicone 50

1 30-70

53-350 - 42-49 (Huang and

Meagher 2001) Silicalite-filled

silicone 19 63-607 - 86-111

PTMSP - 0.3-6 25-70 60-2097 16-347 41-78 (Fadeev et al.

2001)

PEBA 30-100 5 23 65-179 19-42 6-8 (Liu et al.

2005)

ZIF-71 filled PEBA 10-20 1 37 520 - 18.8 (Liu et al.

2013)

ZSM-5 filled PEBA - 2.5 30-45 - 90-240 22-30 (Tan et al.

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)

151

Table 5.2 continued

Membrane material Active layer

thickness (µm)

Feed butanol

concentration

(wt%)

Temperatu

re (ºC)

Total flux

(g/m2·h)

Butanol

flux

(g/m2·h)

Separation

factor Reference

EPDM - 1-10 30 - 0-20 5 (Jitesh et al.

2000) SBR - 1-10 30 - 0-25 10-20

TOA/liquid

membrane - 1.5 54 - 11 275

(Thongsukmak

and Sirkar

2007)

PVDF 120 7.5 50 4126 - 6.4 (Srinivasan et

al. 2007)

PUR 50 1 50 88 10 9 (Boddeker et

al. 1990)

PDMS

41-141 1.5 47 120-278 45-97 39-45

This work 100 0.5-3.0 47 120-213 21-118 41-45

100 1.5 27-56 71-219 27-83 38-44

PDMS/zeolite 100 1.5 27-56 48-171 22-100 60-85

152

153

Figure 5.1 Pervaporation system used in this study

154

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)

155

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)

0

50

100

150

200

PDMS 10% zeolite 20% zeolite 30% zeolite 40% zeolite

Flu

xes

(g/m

2·h

), s

epar

atio

n

fact

or

Separation factor Butanol flux Water flux Total flux

156

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

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

y = 4.088x + 1.30

y = -0.0938x + 98.8

y = 3.999x + 100

0

10

20

30

40

50

60

70

0

50

100

150

200

250

0 5 10 15 20 25 30

Sep

arat

ion

fac

tor

Bu

tan

ol,

wat

er

and

to

tal

flu

x (g

/m2·h

)

Feed 1-butanol concentration (g/L)

Butanol flux Water flux Total flux

Separation factor

157

A

B

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)

y = 3.0545x + 24.984

y = 6.3514x + 30.016

y = 9.4082x + 54.95

0

50

100

150

200

250

300

0 5 10 15 20 25 30

Bu

tan

ol,

wat

er

and

to

tal f

lux

(g/m

2·h

)

1000/thickness (1/µm)

BuOH flux

Water flux

Total flux

0

20

40

60

80

100

120

141 115 90 75 41

Bu

OH

flu

x (g

/m2·h

), s

ep

arat

ion

fa

cto

r

Thickness (µm)

BuOH flux

Separation factor

158

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)

18.4

49.7

134.1

3.0 3.1 3.2 3.3 3.4

Bu

tan

ol a

nd

wat

er

flu

x (g

/m2·h

)

1000/T (K-1)

BuOH flux of PDMS MMM BuOH flux of PDMS Water flux of PDMS MMM Water flux of PDMS

EH2O, PDMS=33.2 kJ/mol

EBuOH, PDMS=34.3 kJ/mol

EBuOH, PDMS MMM=44.2 kJ/mol

EH2O, PDMS MMM=31.8 kJ/mol

159

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)

0

20

40

60

80

100

0

50

100

150

200

250

300

20 25 30 35 40 45 50 55 60

Sep

arat

ion

fac

tor

Tota

l flu

x (g

/m2·h

)

Temperature (°C)

Total flux of PDMS Total flux of PDMS MMM α of PDMS α of PDMS MMM

160

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

161

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.

162

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)

163

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.

164

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.

165

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.

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Yu MR, Zhang YL, Tang IC, and Yang ST. 2011. Metabolic engineering of Clostridium

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