Butanol Production from Lignocellulosic Biomass and Agriculture Residues by

Acetone-Butanol-Ethanol Fermentation

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in

the Graduation School of The Ohio State University

By

Jie Dong, M.S.

Graduate Program in Chemical and Biomolecular Engineering

The Ohio State University

2014

Dissertation Committee:

Professor Shang-Tian Yang, Advisor

Professor David Wood

Professor Aravind Asthagiri

Copyright by

Jie Dong

2014
 Abstract

Butanol is an important intermediate and solvent in the chemical industry.

Moreover, compared to ethanol, butanol has higher energy density and lower vapor

pressure, so butanol is also considered a preferred fuel additive or even a potential

replacement for gasoline. As the oil price rises, the cost of producing butanol from

petrochemical processes increases dramatically. Therefore, more and more research is

being done on ways to produce butanol from fermentation of renewable resources. It is

necessary to reduce the cost of fermentation-derived butanol in order to make it

competitive with petrochemically produced butanol. One of the most influential economy

factors in producing butanol by fermentation is the cost of substrate. In this research, in

order to reduce the cost of substrate, different types of lignocellulosic biomass, such as

sugarcane bagasse, soybean hull, cotton stalk and corn fibers were utilized as substrates

in ABE (Acetone-Butanol-Ethanol) fermentation. However, lignocellulosic biomass

cannot be used by the solventogenic clostridia directly and needs to be hydrolyzed first.

The hydrolysis process usually produces some inhibitory compounds that could severely

inhibit bacteria growth and butanol production. Therefore, the inhibitors in the

hydrolysate must be reduced or removed by certain detoxification processes before

fermentation. To make the detoxification process more efficient, a better understanding

of these inhibitors is needed. In this research, the effects of 9 inhibitors derived from the

degradation of carbohydrate and lignin on the fermentation kinetics of several

ii
solventogenic Clostridia strains were investigated at various concentration levels. The

inhibitors' effects on butanol and butyraldehyde dehydrogenase activities were also

investigated. Among the 9 inhibitors studied, four lignin derived inhibitors

(syringaldehyde, ferulic acid, vanillin, and p-coumaric acid) were found to strongly

inhibit cell growth and butanol and butyraldehyde dehydrogenases. These four phenolic

inhibitors were further tested in C. tyrobutyricum mutants. They were very toxic to one

mutant overexpressing ctfAB and adhE2 genes, but they had no effects on the mutant

overexpressing only adhE2 gene. Therefore, the phenolic compounds are inhibitors to

CoA-transferase expressed by ctfAB. This was further confirmed by their toxicity to C.

beijerinckii and C. acetobutylicum. This also explained the toxicity of corn steep liquor as

nitrogen source in the butanol fermentation.

Then four lignocellulosic biomass, cotton stalk, corn fiber, soybean hull and

sugarcane bagasse, were pretreated with acid and then hydrolyzed by enzymes. The

obtained hydrolysates were further detoxified to remove the inhibitors in them. The

detoxified hydrolysates were then used as carbon source in ABE fermentation of

Clostridium. All hydrolysates gave very high butanol production. The acid and alkali

pretreatments followed with removing the supernatant and washing before enzymatic

hydrolysis were studied, and their effects on biomass compositions and subsequent

fermentation were also studied and compared.

iii
 Other than lignocellulosic biomass, another possibility for substrates is some

other agriculture residues generated during the bioprocessing. In this study, four of these

agriculture residues were investigated: cassava bagasse, Jerusalem artichoke, soy

molasses and soybean meal. Starch based cassava bagasse only needs enzyme

(glucoamylase) hydrolysis. Its hydrolysate contained no toxic inhibitors and performed

very well in ABE fermentation. Inulin based Jerusalem artichoke was hydrolyzed only by

dilute acid. But its hydrolysate contained high concentration of ferulic acid which is an

inhibitor to ABE fermentation. Soy molasses contain mainly oligosaccharides and can be

hydrolyzed by α-galactosidase. But in the ABE fermentation using soy molasses

hydrolysate, the butanol production was partly inhibited. Soybean meal was also proved

to be a good nitrogen source after the acid hydrolysis. The detailed capital and operating

cost of large scale butanol plants were also analyzed.

iv
 Dedication

Dedicated to my wife Yao Nie,

my son Felix

and my parents

v
 Acknowledgements

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

Yang, for his academic and financial support during my four years Ph.D. study. His great

personalities, his patience, his insights have greatly influenced me, both on academic

research and personal life. I'm really grateful that he gave me the opportunity to enter the

field of biotechnology and bioengineering. It's a great honor to do research under his

guidance and encouragement. As an excellent scientist and admirable person, he sets a

brilliant example that someday I wish I can be. I gained really a lot from him.

I would also thank Dr. Aravind Asthagiri and Dr. David Wood for taking time to be

on my committee, as well as their valuable advice to my research.

Then I would like to thank Dr. Congcong Lu. She taught me everything about

microbiology at the beginning, all the basic knowledge and techniques. She taught me how to

use every piece of equipment in the lab and every detailed experiment skills. I'm really

grateful. I am thankful to Dr. Jingbo Zhao for teaching me about the fibrous bed bioreactor

and giving me great suggestions on my research. My thanks also go to all the previous and

current group members, especially Dr. Chuang Xue, Dr. Mingrui Yu, Dr. Zhongqiang Wang,

Dr. Yinming Du, Dr. Chih-Chin Chen, Fangfang Liu, Le Yu, Mengmeng Xu, for their helps

and suggestions. I also thank Dr. Dong Wei from South China University of Technology for

providing the biomass used in my work. I would also thank ARPA-E, NSF-STTR, and USB

for their financial support during my Ph.D. research.

vi
 Finally, I would like to thank my wife Yao Nie who accompanies me and sacrifices her

time taking care of our son Felix at home so I have more time doing research. Thank my

parents and friends for their supports.

vii
 Vita

June 2003………………………………………………………Rizhao No. 1 high school

2003 – 2007………………………………… B.S. Chemcial Engineering & Technology,

Zhejiang University
2007-2010…………………………………………………… M.S. Chemical Engineering,

Zhejiang University

2010-2011……..…..……...…………… Graduate Fellowship, Department of Chemical &

Biomolecular Engineering

The Ohio State University

2011 – present…….....……..… Graduate Research Associate, Department of Chemical &

Biomolecular Engineering

The Ohio State University

Publications

Lu, Congcong; Dong, Jie; Yang, Shang-Tian, Butanol production from wood pulping
hydrolysate in an integrated fermentation-gas stripping process, Bioresource Technology
(2013), 143, 467-475.

Dong, Jie; Wu, Linbo; An, Dong; Li, Bo-Geng; Zhu, Shiping, Stability study of inverse
suspension copolymerization of 1,1,3,3-tetramethylguandium acrylate and N,N'-methylene
bisacrylamide, Journal of Applied Polymer Science (2010), 118(3), 1450-1454.

Fields of Study

Major Field: Chemical & Biomolecular Engineering

viii
 Table of Contents

Abstract ............................................................................................................................... ii
Acknowledgements ............................................................................................................ vi
Vita................................................................................................................................... viii
Table of Contents ............................................................................................................... ix
List of Tables ................................................................................................................... xiii
List of Figures .................................................................................................................. xvi
Chapter 1: Introduction ....................................................................................................... 1
1.1 Project goal and specific tasks: ................................................................................. 5
1.2 Significance and major impacts ................................................................................ 7
1.3 References ................................................................................................................. 9
Chapter 2: Literature Review ............................................................................................ 13
2.1 Acetone-Butanol-Ethanol (ABE) Fermentation ..................................................... 13
2.1.1 Microorganisms and strain improvement ........................................................ 14
2.1.2 Renewable feedstocks ...................................................................................... 16
2.2 Hydrolysis of lignocellulosic biomass .................................................................... 20
2.2.1 Pretreatments.................................................................................................... 20
2.2.2 Enzymatic hydrolysis ....................................................................................... 24
2.3 Detoxification of lignocellulosic hydrolysates ....................................................... 26
2.3.1 Inhibitors to enzymatic hydrolysis ................................................................... 26
2.3.2 Inhibitors to ABE fermentation ....................................................................... 28
2.3.2 Detoxification methods .................................................................................... 29
2.5 References ............................................................................................................... 33
Chapter 3: Effects of lignocellulosic biomass hydrolysate derived inhibitors on acetone-
butanol-ethanol fermentation by Clostridium spp. ........................................................... 59

ix
 3.1. Introduction ............................................................................................................ 60
3.2. Materials and methods ........................................................................................... 62
3.2.1 Strain and inoculum preparation ...................................................................... 62
3.2.2 ABE fermentation in serum bottles.................................................................. 63
3.2.3 Butyraldehyde and butanol dehydrogenase activities ...................................... 63
3.2.4 Analytical methods .......................................................................................... 64
3.3. Results and discussion ........................................................................................... 65
3.3.1 Effects of individual inhibitors on ABE fermentation ..................................... 65
3.3.2 Effects on fermentation kinetics ...................................................................... 68
3.3.3 Effects on butanol dehydrogenase and butyraldehyde dehydrogenase ............ 70
3.4. Conclusion ............................................................................................................. 73
3.5 References ............................................................................................................... 74
Chapter 4: The impact of acid and alkali pretreatments on lignocellulosic biomass and the
following acetone-butanol-ethanol fermentation .............................................................. 89
4.1 Introduction ............................................................................................................. 90
4.2 Materials and methods ............................................................................................ 91
4.2.1 Strain and inoculum preparation ...................................................................... 91
4.2.2 ABE fermentation in serum bottles.................................................................. 91
4.2.3 Hydrolysis of biomass...................................................................................... 92
4.2.4 Detoxification of hydrolysate .......................................................................... 94
4.2.5 Analytical methods .......................................................................................... 94
4.3 Results and discussion ............................................................................................ 95
4.3.1 Dilute acid pretreatment with different concentrations of acids ...................... 95
4.3.2 Detoxification and ABE fermentation using the hydrolysate .......................... 99
4.3.3 Comparison of acid and alkali pretreatment with removing supernatant before
enzymatic hydrolysis .............................................................................................. 101
4.4 Conclusion ............................................................................................................ 105
x
 4.5 Reference .............................................................................................................. 106
Chapter 5: The inhibition mechanism of lignin derived phenolic compounds on the
metabolic pathways of Clostridium spp.......................................................................... 124
5.1 Introduction ........................................................................................................... 124
5.2. Materials and methods ......................................................................................... 126
5.2.1 Strains and inoculum preparation .................................................................. 126
5.2.2 ABE fermentation in serum bottles................................................................ 126
5.2.3 Hydrolysis of biomass.................................................................................... 127
5.2.4 Detoxification of hydrolysate ........................................................................ 127
5.2.5 Analytical methods ........................................................................................ 128
5.3 Results and discussion .......................................................................................... 128
5.3.1 Inhibition of phenolic compounds to Clostridium strains.............................. 128
5.3.2 Corn steep liquor as the nitrogen source for ABE fermentation .................... 130
5.4 Conclusion ............................................................................................................ 132
5.5 Reference .............................................................................................................. 133
Chapter 6: Butanol production from non-lignocellulosic biomass ................................. 145
6.1 Introduction ........................................................................................................... 146
6.2 Materials and methods .......................................................................................... 147
6.2.1 Strains and inoculum preparation .................................................................. 147
6.2.2 ABE fermentation in serum bottles and bioreactors ...................................... 147
6.2.3 Hydrolysis of biomass.................................................................................... 149
6.2.4 Detoxification of hydrolysate ........................................................................ 150
6.2.5 Analytical methods ........................................................................................ 150
6.3 Results and discussion .......................................................................................... 151
6.3.1 Cassava bagasse and Jerusalem artichoke as the carbon source for ABE
fermentation ............................................................................................................ 151
6.3.2 Soy molasses as the carbon source for ABE fermentation ............................ 153
xi
 6.3.3 Soybean meal as carbon and nitrogen sources for ABE fermentation........... 155
6.3.4 Butanol production from soybean meal and soy molasses ............................ 156
6.4 Conclusion ............................................................................................................ 158
6.5 Reference .............................................................................................................. 159
Chapter 7: Conclusions and Recommendations ............................................................. 181
7.1 Conclusions ........................................................................................................... 181
7.1.1 Effects of lignocellulosic biomass hydrolysate derived inhibitors on ABE
fermentation ............................................................................................................ 181
7.1.2 Butanol production from lignocellulosic biomass and agriculture residues .. 182
7.1.3 The inhibition mechanism of phenolic inhibitors to the metabolic pathway of
Clostridia ................................................................................................................. 183
7.2 Recommendations ................................................................................................. 184
7.2.1 Combined Effects of lignocellulosic biomass hydrolysate derived inhibitors184
7.2.2 Optimizations of hydrolysis processes........................................................... 185
7.2.3 The inhibition mechanism of phenolic inhibitors to the metabolic pathway of
Clostridia ................................................................................................................. 186
Bibliography ................................................................................................................... 188
Appendix A. Cost analysis of butanol plants using soybean hull or soy molasses and
soybean meal as substrates.............................................................................................. 223
Appendix B. Analytical procedures of inhibitors by high performance liquid
chromatograph ................................................................................................................ 242

xii
 List of Tables

Table 2.1 Properties of butanol (Hastings, 1978) ............................................................. 47

Table 2.2 Summary of various solventogenic Clostridia with their substrates, products,
fermentation pH and temperature ..................................................................................... 48
Table 2.3 ABE production by solventogenic Clostridia from traditional substrates and
renewable lignocellulosic biomass ................................................................................... 49
Table 2.4 Compositions of different lignocellulosic biomass and their current use ......... 50
Table 2.5 Processes Used for the Pretreatment of Lignocellulosic Biomass .................... 51
Table 2.6 Inhibition effect of phenolic compounds on cellulases .................................... 53
Table 2.7 Inhibition effect of sugars on cellulases (Xiao et al., 2004; Ximenes et al., 2011)
........................................................................................................................................... 54
Table 2.8 Major fermentation inhibitors present in the hydrolysates generated from
lignocellulose degradation ................................................................................................ 55
Table 2.9 Efficiency of different detoxification methods ................................................. 56
Table 3.1 The activities of butanol dehydrogenase and butyraldehyde dehydrogenase with
1.0 g/L inhibitors in the enzyme assay.............................................................................. 82
Table 3.2 The activities of butanol dehydrogenase and butyraldehyde dehydrogenase with
1.0 g/L inhibitors in fermentation medium ....................................................................... 83
Table 4.1 Composition of various lignocellulosic biomass ............................................ 114
Table 4.2 Sugar concentrations and yields from biomass with different pretreatment
conditions a ...................................................................................................................... 115
Table 4.3 Inhibitors present in the biomass hydrolysate with different acid pretreatments
......................................................................................................................................... 116
Table 4.4 Sugar concentrations and yields of the biomass after hydrolysis a ................. 117
Table 4.5 C. acetobutylicum ATCC824 fermentation results after 72 h using different
biomass w/o activated carbon adsorption ....................................................................... 117
Table 4.6 The concentrations of inhibitors present in the biomass hydrolysates before and
after activated carbon absorption .................................................................................... 118

xiii
Table 4.7 The fermentation results using biomass hydrolysate as the carbon source for
three Clostridium strians ................................................................................................. 118
Table 4.8 % loss in cellulose, hemicellulose and lignin in biomass after acid or alkali
pretreatment a .................................................................................................................. 119
Table 4.9 C. acetobutylicum ATCC824 fermentation results with biomass hydrolysates
after acid or alkali pretreatment a or activated carbon detoxification ............................. 120
Table 5.1 The fermentation results of different strains combining lignocellulosic
hydrolysate with corn steep liquor .................................................................................. 141
Table 6.1 The compositions of Cassava bagasse and Jerusalem artichoke .................... 167
Table 6.2 The sugar concentrations of the biomass after the hydrolysis ........................ 167
Table 6.3 The inhibitors‘ concentrations in hydrolysates before and after activated carbon
absorption ........................................................................................................................ 168
Table 6.4 The fermentation results using biomass hydrolysates as the carbon source ... 168
Table 6.5 Carbohydrate content of soybean soluble and soy molasses. ......................... 169
Table 6.6 Butanol and acids production from soy molasses by C. acetobutylicum ATCC
824 and C. tyrobutyricum (Δack, adhE2) after 3 days fermentation .............................. 170
Table 6.7 Hydrolysis of oligosaccharides present in soybean meal by different acid
pretreatments followed with enzymatic hydrolysis with α-galactosidase ...................... 171
Table 6.8 Fermentation results of C. acetobutylicum ATCC 824 using soybean meal
hydrolysates pretreated with various acid concentrations followed with enzymatic
hydrolysis with α-galactosidase ...................................................................................... 171
Table 6.9 Butanol production from soy molasses hydrolysate as the carbon source and
soybean meal hydrolysate as the nitrogen source ........................................................... 172
Table 6.10 Fermentation Parameters based on C. tyrobutyricum(Δack, adhE2) ............ 172
Table 6.11 Hydrolysis parameters of different biomasses .............................................. 173
Table A.1 Executive summary (2013 prices) - Soybean hull ......................................... 227
Table A.2 Major equipment specifications and cost (2013 prices) - Soybean hull ........ 228
Table A.3 Fixed capital estimate summary (2013 prices in $) - Soybean hull ............... 230
Table A.4 Material costs - Soybean hull ......................................................................... 231
xiv
Table A.5 Annual operating cost (2013 prices) - Soybean hull ...................................... 232
Table A.6 Utilities cost (2013 prices) - Soybean hull..................................................... 232
Table A.7 Executive summary (2013 prices) - Soy malasses ......................................... 233
Table A.8 Major equipment specifications and cost (2013 prices) - Soy molasses ....... 234
Table A.9 Fixed capital estimate summary (2013 prices in $) - Soy molasses .............. 236
Table A.10 Materials cost – Soy molasses ..................................................................... 237
Table A.11 Annual operating cost (2013 prices) – Soy molasses .................................. 238
Table A.12 Utilities cost (2013 prices) - Soy molasses .................................................. 238

xv
 List of Figures

Figure 1.1 Overview of project goal and major tasks carried out in this study ................ 12
Figure 2.1 Metabolic pathways in C. acetobutylicum for the acidogenic and
solventogenic phase. ......................................................................................................... 57
Figure 2.2 The endo-exo synergistic action of the cellulolytic enzymes: EGs cleave the
internal b-1, 4 bonds. CBHs attack the reducing/nonreducing ends. β-glucosidase
produces glucose from cellobiose units (Gokhale et al., 2009) ........................................ 58
Figure 2.3 Simplified scheme showing different functional domains of cellobiohydrolase
(CBH) (Gokhale et al., 2009) ............................................................................................ 58
Figure 3.1 Optical densities, butanol and acids production of three Clostridium spp.with
1.0 g/L different inhibitors after 72 hr. ............................................................................. 84
Figure 3.2 The fermentation results of C. beijerinckii BA101-SV6, C. acetobutylicum
ATCC 824 and C. tyrobutyricum ATCC 25755 mutant after 72 h with different
concentrations of phenolic inhibitors. ............................................................................... 85
Figure 3.3 C. beijerinckii BA101 Fermentation Kinetics with different inhibitors. (1.0 g/L
furfural, 1.0 g/L formic acid, 1.0 g/L vanillin, 0.5 g/L ferulic acid) ................................. 86
Figure 3.4 C. acetobutylicum ATCC 824 Fermentation Kinetics with different inhibitors.
(1.0 g/L furfural, 1.0 g/L formic acid, 1.0 g/L vanillin, 0.5 g/L ferulic acid) ................... 87
Figure 3.5 C. tyrobutyricum ATCC 25755 mutant fermentation kinetics with different
inhibitors. (1.0 g/L vanillin, 0.5 g/L ferulic acid) ............................................................. 88
Figure 4.1 The fermentation kinetics of C. acetobutylicum ATCC824 with lignocellulosic
hydrolysates after activated carbon adsorption ............................................................... 121
Figure 4.2 The fermentation kinetics of beijerinckii BA101 with lignocellulosic
hydrolysates after activated carbon adsorption ............................................................... 122
Figure 4.3 SEM images of untreated, acid pretreated or alkali pretreated biomass
structures ......................................................................................................................... 123
Figure 5.1 Chemical structures of phenolic inhibitors .................................................... 142
Figure 5.2 The inhibition of phenolic compounds (1.0 g/L each) to the growth and
solvents and acids production of four Clostridium strains.............................................. 143

xvi
Figure 5.3 The fermentation results of two C. tyrobutyricum ATCC25755 mutants with
different concentrations of corn steep liquor as nitrogen source .................................... 144
Figure 6.1 Hydrolysis of soybean molasses by α-galactosidase (0.1% v/v) at pH 5.0, 50
o
C..................................................................................................................................... 173
Figure 6.2 The fermentation kinetics of different Clostridia with soy molasses and
soybean meal hydrolysates: (a) C. acetobutylicum ATCC 55025; (b) C. tyrobutyricum
(Δack, adhE2); (c) C. acetobutylicum ATCC 824 .......................................................... 174
Figure 6.3 Unit production costs by using different raw materials................................. 175
Figure 6.4 Capital costs by using different raw materials .............................................. 175
Figure 6.5 Operating costs by using different raw materials .......................................... 176
Figure 6.6 Sensitivity analysis of unit production cost for the variation in the cost of raw
materials. ......................................................................................................................... 176
Figure 6.7 Sensitivity analysis of unit production cost for the variation in the yield of
butanol............................................................................................................................. 177
Figure 6.8 Pretreatment of corn starch process ............................................................... 177
Figure 6.9 Pretreatment of lignocellulose hydrolysis process ........................................ 178
Figure 6.10 Pretreatment of cassava bagasse .................................................................. 178
Figure 6.11 Pretreatment of Jerusalem artichoke ........................................................... 179
Figure 6.12 Pretreatment of soy molasses ...................................................................... 179
Figure 6.13 Fermentation Section ................................................................................... 180
Figure 6.14 Separation of acetone, ethanol and butanol ................................................. 180
Figure A.1 The acid pretreatment and hydrolysis processes for soybean hull and soybean
meal ................................................................................................................................. 239
Figure A.2 The pretreatment and hydrolysis processes for soy molasses and soybean meal.
......................................................................................................................................... 240
Figure A.3 Process flowsheet for butanol fermentation and separation. ........................ 241
Figure B.1 HPLC chromatogram of 1.0 g/L hydroquinone at 280 nm ........................... 243
Figure B.2 HPLC chromatogram of 1.0 g/L furfural at 280 nm ..................................... 244

xvii
Figure B.2 HPLC chromatogram of 1.0 g/L HMF at 280 nm ........................................ 244
Figure B.4 HPLC chromatogram of 1.0 g/L syringaldehyde at 280 nm......................... 245
Figure B.5 HPLC chromatogram of 1.0 g/L levulinic acid at 200 nm ........................... 245
Figure B.6 HPLC chromatogram of 1.0 g/L p-coumaric acid at 200 nm ....................... 246
Figure B.6 HPLC chromatogram of 1.0 g/L formic acid at 200 nm ............................... 246

xviii
 Chapter 1: Introduction

The growing scarcity and increasing price of fossil fuels are driving researchers to

find viable substitutes. Butanol is a saturated primary alcohol (CH3CH2CH2CH2OH) and

also a promising biofuel. Compared to ethanol, butanol has a higher energy density and

lower vapor pressure, so it is more convenient and safer for utilization and storage. More

importantly, butanol‘s energy density, air-fuel ratio and heat of vaporization are similar to

those of gasoline. Besides being a potential replacement for fossil fuels, butanol is an

important solvent used in the production of antibiotics, vitamins and hormones. Butanol

is also an intermediate in the manufacturing of butyl acrylate and methacrylate esters

(Jones et al., 1986).

Acetone-Butanol-Ethanol (ABE) fermentation is an important bioprocess that

produces butanol. It was first discovered by Pasteur in 1861 (Jones et al., 1986). During

1900s, ABE fermentation developed very rapidly both in the research field and in

industrial process due to the large demand for acetone in the synthesis of rubber,

especially during World War I (Gabriel, 1928). However, after 1950s, several

petrochemical processes were developed for butanol production, and ABE fermentation

was no longer economically competitive due to the high cost of substrates (Kumar et al.,

2011). Recently, because the oil price is increasing very quickly and the crude oil is

depleting, more and more research is returning to ABE fermentation. In order to reduce
1
the cost of ABE fermentation and make it competitive to petrochemical process, a lot of

research is being done on increasing the butanol production by metabolic engineering and

reducing the substrate cost by using cheaper lignocellulosic biomass or agriculture

residues as feedstocks.

Solventogenic Clostridia species are usually the microorganisms in ABE

fermentation. There are mainly 5 metabolic products in ABE fermentation, two acids

(acetic acid and butyric acid) and three solvents (acetone, ethanol and butanol). In most

solventogenic Clostridia, the solvent (acetone/butanol/ethanol) ratio is 3:6:1. The total ABE

titer is 15-18 g/L with 10-13 g/L butanol. ABE fermentation by Clostridia usually has two

phases, acidogenesis and solventogenesis (Lee et al., 2008). In the acidogenesis phase,

glucose or other sugars are converted into acids such as acetate acid and butyrate acid,

which leads to rapid decrease in pH. Under certain critical pH, Clostridia shifts to the

solventogenesis phase during which it consumes acids to produce solvents (acetone,

ethanol and butanol). The most important factor that triggers the shift from acidogenesis

to solventogenesis is external and internal pHs (Jones et al., 1986). Some metabolically

engineered Clostridia do not have this shift mechanism (Yu et al., 2011). Extensive

metabolic engineering work has been done to enhance butanol production in Clostridia

(Dürre, 1998; Ezeji et al., 2004; Qureshi et al., 2008). This is one way to reduce the cost of

ABE fermentation.

Another way to reduce the cost of ABE fermentation is looking for cheaper

substrates. Over 50% of ABE fermentation cost is due to the cost of substrates. Maize

and molasses, which are the current substrates used in ABE fermentation, make up a
2
large percentage of the processing costs. Based on maize, butanol production cost was

projected to be $0.55/kg and substrate cost accounted for over 56% ($0.31/kg butanol) of

the production cost (Ezeji et al., 2008). It is therefore necessary to find other, more

economical substrates in order to reduce the cost of producing butanol by ABE

fermentation. Lignocellulosic biomass, including wheat straw, barley straw, maize stover,

switchgrass, etc., is generally much cheaper than starch and sugar based substrates. Their

prices ranged from $26.5 to $66.1/ton as opposed to $256.3/ton for maize in 2009

(Zverlov et al., 2006). So they are more economical substrates for ABE fermentation.

The main components of lignocellulosic biomass are cellulose (30%-60%),

hemicellulose (5%-30%) and lignin (5%-30%). Most solventogenic Clostridium species

cannot use these carbon sources directly. Therefore, the lignocellulosic biomass needs to

be hydrolyzed into sugars before ABE fermentation. The hydrolysis of lignocellulosic

biomass usually contains three steps: pretreatment, enzymatic hydrolysis and

detoxification. The most important part is the enzymatic hydrolysis, in which cellulose is

hydrolyzed by cellulase enzymes. Cellulase is being investigated and combined with

other enzymes to increase its activities and efficiencies in lignocellulosic biomass

(Henrissat, 1994; Duarte, 2012). In order to facilitate the contact between cellulose and

cellulase, some pretreatments to break down the rigid structure of lignocellulosic biomass

are necessary. Extensive research has been done on different pretreatment methods to

increase the sugar yield from lignocellulosic biomass (Kumar et al., 2011). The acid

pretreatment was investigated in detail in this research because its process is relatively

simple and cheap and it can hydrolyze the hemicellulose part of lignocellulosic biomass.

3
 During the pretreatment, some inhibitory compounds are produced due to the

degradation of sugars and lignin. These inhibitory compounds can inhibit the growth of

Clostridia severely and also block butanol production in ABE fermentation. There are

three main groups: furan derivatives (Furfural and 5-hydroxymethyl furfural (HMF)),

weak acids (formic acid, and levulinic acid) and phenolics (p-coumaric acid, ferulic acid,

vanillin, hydroquinone, and syringaldehyde). Furan derivatives and weak acids come

mainly from the degradation of monosaccharides, while phenolics are generated from

lignin degradation. Although the inhibition mechanism is still unclear, their inhibition

effects to Clostridia are obvious (Ezeji et al., 2008; Lee et al., 2011). Therefore, these

inhibitors must be removed or at least reduced by some detoxification methods before

ABE fermentation. Various methods of detoxification have been studied, such as

overliming (Mussatto et al., 2010), electrodialysis (Martinez et al., 2010), adsorption

(Qureshi et al., 2007), filtration (Qureshi et al., 2008) and enzymatic reactions (Larsson et

al., 1999).

Lignocellulosic biomass needs a long treatment process before being used in ABE

fermentation, including pretreatment, enzymatic hydrolysis and detoxification. These

treatments increase the operating cost of ABE fermentation. Therefore, other non-

lignocellulosic biomass has also been studied as feedstocks for ABE fermentation

(Qureshi et al., 1995; Ezeji et al., 2007; Cho et al., 2009; Larsson et al., 1999). Non-

lignocellulosic biomass includes agriculture residues and byproducts from bioprocessing,

which usually do not need the three steps of treatment as for lignocellulosic biomass.

4
1.1 Project goal and specific tasks:

The goals of this research were to find economical substrates for butanol

production and to investigate the effects of these substrates on ABE fermentation. The

most economical substrates for ABE fermentation are lignocellulosic biomass, such as

cotton stalk, sugarcane bagasse, soybean hull and corn fiber, which are composed mainly

of cellulose, hemicellulose, and lignin. In general, no solventogenic Clostridium can

utilize these polysaccharides directly. So for ABE fermentation to take place, one or more

hydrolysis processes are necessary to decompose cellulose and hemicellulose into single

sugars. The hydrolysis processes were also studied and optimized in this research. An

overview of this study is provided in Figure 1.1. The specific objectives and major tasks are

described below.

Task 1: Effects of lignocellulosic biomass hydrolysate derived inhibitors on ABE

fermentation

The effects of 9 inhibitors (furfural, HMF, formic acid, levulinic acid,

hydroquinone, syringaldehyde, p-coumaric acid, vanillin and ferulic acid) derived from

the degradation of carbohydrate and lignin on the fermentation kinetics of several

solventogenic C. beijerinckii, C. acetobutylicum and C. tyrobutyricum strains were

investigated at various concentration levels. The effects of these inhibitors on butanol

and butyraldehyde dehydrogenase activities were also studied. Among the 9 inhibitors

studied, four lignin-derived inhibitors (syringaldehyde, ferulic acid, vanillin, and p-

coumaric acid) were found to strongly inhibit cell growth and butanol and butyraldehyde

dehydrogenases (Chapter 3).

5
Task 2: Butanol production from lignocellulosic biomass

Four different kinds of lignocellulosic biomass, cotton stalk, corn fiber, soybean

hull and sugarcane bagasse, were pretreated with acid and then hydrolyzed by enzymes.

The effects of different acid concentrations on sugar yields and inhibitors production

were studied. The main inhibitors after acid pretreatment were formic acid and ferulic

acid. The obtained hydrolysates were further detoxified by adsorption with activated

carbon to remove the inhibitors. The detoxified hydrolysates were then used as carbon

sources in ABE fermentation of Clostridium. All hydrolysates gave high butanol

production. The acid and alkali pretreatments followed with removing the liquors

containing the dissolved inhibitors were also studied and compared for their effects on

biomass compositions and subsequent fermentation (Chapter 4).

Task 3: The inhibition mechanism of lignin-derived phenolic compounds

The inhibition mechanism of four lignin-derived phenolic compounds was

investigated. Four phenolic inhibitors, syringaldehyde, ferulic acid, vanillin, and p-

coumaric acid, were tested in C. tyrobutyricum mutants. They were found to be very

toxic to the mutant overexpressing ctfAB and adhE2 genes, but not to the mutant

overexpressing only adhE2 gene. Therefore, the phenolic compounds are inhibitors to

CoA-transferase expressed by ctfAB. This was further confirmed by their toxicity to C.

beijerinckii and C. acetobutylicum. This inhibition mechanism was also found to exist

when corn steep liquor was used as nitrogen source in ABE fermentation (Chapter 5).

Task 4: Butanol production from non-lignocellulosic biomass

6
 Four agriculture residues were investigated: cassava bagasse, Jerusalem artichoke,

soy molasses and soybean meal. The main composition of cassava bagasse is starch. It

did not need the acid pretreatment. After hot water pretreatment and enzymatic

(glucoamylase) hydrolysis, a high sugar yield was obtained. Its hydrolysate contained no

toxic inhibitors and performed very well in the ABE fermentation. Jerusalem artichoke

contains plenty of inulin. It did not need enzymatic hydrolysis. After acid pretreatment,

most sugars were released. Its hydrolysate contained a high concentration of ferulic acid.

Soy molasses contain mainly oligosaccharides, which can be hydrolyzed by α-

galactosidase. But in the ABE fermentation using soy molasses hydrolysate, butanol

production was partly inhibited. Soybean meal was also proved to be a good nitrogen

source, after acid hydrolysis, for ABE fermentation (Chapter 6).

1.2 Significance and major impacts

n-Butanol is currently an important chemical and solvent in industry. The main

application of n-butanol currently is to synthesize butyl acrylate and butyl methacrylate.

Butyl acrylate and butyl methacrylate are further polymerized to esters, which are widely

used for latex paints, coatings, binders and adhesives (Mascal, 2011). In 2011, the global

market size for n-butanol was 2.8 million metric tons. In USA, the consumption of

butanol is 740,000 metric tons in 2011 with 2.2% growth rate (Mascal, 2011). So there is

already a big market for n-butanol. Moreover, as a potential biofuel, the market would be

much larger. The Energy Independence and Security Act of 2007 (EISA) set a revised

Renewable Fuels Standard (RFS). The revised RFS mandates the volume of renewable

7
fuels sold in the USA must be 36 billion gallons by 2022, of which 16 billion gallons

must be cellulosic biofuels.

This study provided a better understanding of the inhibitors present in the biomass

hydrolysates and their effects on solventogenic clostridia. The inhibition mechanism of

phenolic compounds on CoA-transferase was revealed, and this finding can guide the

detoxification process in the hydrolysis of lignocellulosic biomass. This study also

showed the diversity and feasibility of feedstocks in butanol production. Four different

kinds of lignocellulosic biomass and three different agricultural residues were proved to

be good substrates for ABE fermentation. With proper treatments, they all could be

converted to butanol at a high concentration suitable for industrial application. The

hydrolysis of lignocellulosic biomass was successfully optimized. The acid pretreatment

was demonstrated to be an efficient pretreatment for lignocellulosic biomass. With

activated carbon detoxification, the yield of butanol from all four lignocellulosic biomass

can be as high as that from pure glucose. The detailed capital and operating costs of large

scale butanol plants were also analyzed. The large scale cellulosic butanol was proved to

be profitable and competitive with petrochemically synthesized butanol. This also means

that a promising sustainable fuel, butanol, can be produced from economical renewable

resources.

8
1.3 References

Annous B. A. and Blaschek H. P. Isolation and Characterization of Clostridium

acetobutylicum Mutants with Enhanced Amylolytic Activity, Applied and

Environmental Micorbiology, 1991, 57:2544-2548

Cho, D. H., Lee Y.J, Um Y., Sang B.I., Kim Y. H., Detoxification of model phenolic

compounds in lignocellulosic hydrolysates with peroxidase for butanol production

from Clostridium beijerinckii. Appl Microbiol Biotechnol 2009, 83: 1035–1043.

Duarte, GC, Moreira LR, Jaramillo PM, Filho EX, Biomass-Derived Inhibitors of

Holocellulases, Bioenerg. Res. 2012, 5:768–777

Dürre P. New insights and novel developments in clostridial acetone/ butanol/

isopropanol fermentation. Appl. Microbiol. Biotechnol., 1998, 49, 639-648.

Ezeji T.C., N. Qureshi, and H.P. Blaschek. Butanol fermentation research: upstream and

downstream manipulations. The Chemical Record, 2004, 4, 305-314.

Ezeji H., Blaschek H. P. Fermentation of dried distillers‘ grains and solubles (DDGS)

hydrolysates to solvents and value-added products by solventogenic clostridia,

Bioresource Technology, 2008,99: 5232–5242.

Gabriel, C.L. Butanol fermentation process. Ind. Eng. Chem., 1928, 20, 1063-1067.

Hastings, J.H.J. Acetone-butyl alcohol fermentation, In A. H. Rose (ed.), Economic

microbiology, Primary products of metabolism. Academic Press, Inc. New York

1978, 2: 31-45

9
Henrissat, B. Cellulose, 1994, 1:169–196

Jones D.T., Woods D.R. Acetone-butanol fermentation revisited. Microbiological

Reviews 1986, 50:484–524

Kumar M., Gayen K. Developments in biobutanol production: New insights, Applied

Energy 2011, 88: 1999–2012

Larsson, S., Reimann A., Nilvebrant N.O., Jonsson L. Comparison of Different Methods

for the Detoxification of Lignocellulose Hydrolyzates of Spruce. Applied

Biochemistry and Biotechnology 1999, 77: 91-103

Lee S.Y., Park J. H., Jang S.H., Nielsen L. K., Kim J., Jung K.S. Fermentative Butanol

Production by Clostridia, Biotechnology and Bioengineering, 2008, 101: 209-228

Mascal M. Chemicals from biobutanol: technologies and markets, 2011, 6: 483-493

Martinez A., Rodriguez, M.E., Wells, M.L., York, S.W., Preston, J.F., Ingram, L.O.,

Detoxification of dilute acid hydrolysates of lignocellulose with lime,

Biotechnology Progress 2001, 17: 287-293.

Meinita M.N., Yong-Ki Hong, Gwi-Taek Jeong, Detoxification of acidic catalyzed

hydrolysate of Kappaphycus alvarezii (cottonii), Bioprocess Biosyst Eng 2012,

35:93–98

Mussatto S.I., Roberto, I.C., Hydrolysate detoxification with activated charcoal for

xylitol production by Candida guilliermondii. Biotechnol. Lett. 2001, 23: 1681–

1684.
10
Qureshi, N. and T.C. Ezeji. Butanol, ‗a superior biofuel‘ production from agricultural

residues (renewable biomass): recent progress in technology. Biofuels, Bioprod.

Bioref.,2008, 2, 319-330.

Qureshi N. Agricultural residues and energy crops as potentially economical and novel

substrates for microbial production of butanol (a biofuel), Perspectives in

Agriculture, Veterinary Science, Nutrition and Natural Resources 2010, 5: 1-8

Qureshi N. and Blaschek H. P. Recent advances in ABE fermentation: hyper-butanol

producing Clostridium beijerinckii BA101, Journal of Industrial Microbiology &

Biotechnology, 2001, 27: 287 –291

Yu, M.-R.; Zhang, Ya-Li; Tang, I.-Ching; Yang, Shang-Tian, Metabolic engineering of

Clostridium tyrobutyricum for n-butanol production, Metabolic Engineering, 2011,

13(4), 373-382.

Zverlov, V.V., Berezina O., Velikodvorskaya G.A., Schwarz W.H. Bacterial acetone and

butanol production by industrial fermentation in the Soviet union: use of

hydrolyzed agricultural waste for biorefinery, Applied Microbiology and

Biotechnology 2006, 71: 587-597.

11
 Project Goal
High butanol production from
lignocellulosic biomass and
agriculture residues

Task 1 Task 2 Task 3 Task 4

Effects of Butanol The inhibition Butanol

lignocellulosic production mechanism of production
lignin derived from non-
biomass from
phenolic lignocellulosic
hydrolysate lignocellulosic compounds biomass
derived biomass
inhibitors on hydrolysates
ABE (Chapter 6)
(Chapter 4) (Chapter 5)
fermentation

(Chapter 3)
(Chapter 4)

Figure 1.1 Overview of project goal and major tasks carried out in this study

12
 Chapter 2: Literature Review

2.1 Acetone-Butanol-Ethanol (ABE) Fermentation

The production of butanol in fermentation was first discovered by Pasteur in 1861

(Jones et al., 1986). In 1905, Schardinger found the fermentation could also produce

acetone. Due to the shortage of natural rubber and the research of synthetic rubber,

butanol fermentation was further studied as butanol can be converted into butadiene .

Butadiene is the monomer to synthesize rubber. Between 1912 and 1914, Chaim

Weizmann (Hastings et al., 1978) isolated a bacterium which could produce a large

amount of butanol and acetone. This strain was named as Clostridium acetobutylicum by

McCoy et al. (1926). During World war I, the large demand of acetone further promoted

the development of ABE fermentation. Weizmann developed an ABE fermentation

process to produce acetone and directed a large pilot-scale work (Dürre, 1998). From

1920 to 1980, many ABE fermentation plants were built all over the world. Acetone and

butanol were the major products. The main application of acetone and butanol was to

synthesize rubber. However, after 1950s, several petrochemical processes were

developed for butanol production, and ABE fermentation was no longer economically

competitive due to the high cost of substrates (Kumar et al., 2011).

Over the past few decades, because the oil price is increasing very quickly and the

crude oil is depleting, more and more research is returning to ABE fermentation because
13
butanol is a promising biofuel (Demain, 2009; Dürre, 1998; 2007; Lee et al., 2008;

Qureshi et al., 2008; Swana et al., 2011; Weber et al., 2010; Zheng et al., 2009).

Compared to ethanol, butanol has a higher energy density and lower vapor pressure, so it

is more convenient and safer for utilization and storage. More importantly, butanol‘s

energy density, air-fuel ratio and heat of vaporization are similar to those of gasoline

(Table 2.1).

2.1.1 Microorganisms and strain improvement

Clostridia species are usually the microorganisms used in ABE fermentation.

Clostridia are rod-shaped, spore-forming, gram-positive and obligate anaerobic bacteria,

belonging to the Firmicutes. Besides C. acetobutylicum, three other species, C.

beijerinckii, C. saccharoperbutylacetonicum and C. saccharobutylicum (Lee et al., 2008),

were also identified as butanol producers and they gave high butanol production and

yields. Some other Clostridia strains including C. carboxidivorans, C.butylicum, C.

aurantibutyricum and C. pasteurianum were also investigated (Bruant et al., 2010; Ezeji

et al., 2008; Somrutai et al., 1996; Ahn et al., 2011). Their substrates, products,

fermentation pH and temperature were summarized in Table 2.2. Almost all strains can

use a variety of hexose and pentose sugars as substrates. Most strains can use

disaccharides such as sucrose and cellobiose and even polysaccharides such as starch.

Some strains can even use novel substrates. C. carboxidivorans can use syngas and C.

pasteurianum can ferment glycerol.

14
 Among the solventogenic Clostridia, C. acetobutylicum and C. beijerinckii are the

most widely studied butanol producing strains. Researchers studied a large variety of

mutants from these strains, including C. acetobutylicum P262, C. beijerinckii P260, 8052,

BA101, and LMD 27.6 (Huang et al., 2004; Parekh et al., 1998; Qureshi et al., 2006, 2010;

Kumar et al., 2011). All of these strains have similar characteristics for ABE fermentation.

For example, they produce total ABE in the range of 10 g/L-30 g/L and the typical ratio

of acetone, butanol and ethanol is about 3:6:1.

ABE fermentation by C. acetobutylicum and C. beijerinckii has two phases,

acidogenesis and solventogenesis (Lee et al., 2008) (Figure 2.1). At first, the bacterium

will convert glucose into acids such as acetate acid and butyrate acid. This is called the

acidogenesis phase and leads to rapid decrease of pH. Under certain critical pH, the

bacterium will shift to the solventogenesis phase during which it will consume acids to

produce ethanol and butanol. The most important factor that triggers the shift from

acidogenesis to solventogenesis is external and internal pH (Jones et al., 1986). One

shortage of Clostridia is degeneration phenomenon. They gradually lose the ability to

produce solvent during the cultivation. In C. acetobutylicum, this degeneration

phenomenon is due to the loss of megaplasmid pSOL1 (Lee et al., 2008). Another

problem lies that butanol is high toxic to Clostridia itself. 0.1–0.15M butanol caused 50%

inhibition of both cell growth and sugar uptake rate by negatively affecting the ATPase

activity (Jones et al., 1986). So the production of butanol ceases when the concentration

of solvent reaches around 20g/L. This solvent toxicity highly inhibits the butanol yields

of Clostridia.

15
 C. tyrobutyricum was studied in our group (Yu et al., 2011). Different from

solventogenic Clostridia, the Wild type C. tyrobutyricum only produces acetate acid and

butyric acid, so its metabolic pathway doesn‘t contain the right side in Figure 2.1. Our lab

has engineered C. tyrobutyricum ATCC 25755 to overexpress aldehyde/alcohol

dehydrogenase 2 (adhE2) and CoA-transferase (ctfAB). Then this mutant can convert

acetate/butyrate into ethanol/butanol, but it doesn‘t have the shift mechanism from

acidogenesis to solventogenesis. Butanol production from glucose by these mutants was

high with 20 g/L butanol titer. Moreover, C. tyrobutyricum showed good butanol

tolerance. It still can reach >80% and ~60% relative growth rate at 1.0% and 1.5% (v/v)

butanol, respectively. C. tyrobutyricum is a promising butanol production strain that may

be used in the future.

2.1.2 Renewable feedstocks

Over 50% of ABE fermentation cost is due to the cost of substrates. Maize and

molasses, which are the current substrates used in ABE fermentation, make up a large

percentage of the processing costs. Based on maize, butanol production cost was

projected to be $0.55/kg and substrate cost accounted for over 56% ($0.31/kg butanol) of

the production cost (Ezeji et al., 2008). It is therefore necessary to find other, more

economical substrates in order to reduce the cost of producing butanol by ABE

fermentation. Lignocellulosic biomass, including wheat straw, barley straw, maize stover,

switchgrass, etc., is generally much cheaper than starch and sugar based substrates. Their

prices ranged from $26.5 to $66.1/ton as opposed to $256.3/ton for maize in 2009

(Zverlov et al., 2006). So they are more economical substrates for ABE fermentation.
16
ABE fermentation using traditional substrates and lignocellulosic feedstocks was listed in

Table 2.3.

Lignocellulose is the main component of plant cell walls. Lignocellulose biomass

is mainly composed of cellulose, hemicellulose, and lignin. The composition of these

three constituents can vary from one plant to another. For one plant species, it can even

vary from one season to another. For instant, wood usually contains more cellulose,

whereas wheat straw and corn cobs have more hemicelluloses. The composition and

current use of some lignocellulosic biomass were listed in Table 2.4. Most lignocellulosic

biomass are just burnt or landfilled. Some of them can be used as animal feed. So they

are waste materials from industrial bioprocessing with extremely low price. If they can

be used as feedstocks in ABE fermentation, the cost of ABE fermentation will be reduced.

The cellulose and hemicellulose contents in lignocellulosic biomass can be

converted into sugars and used as substrates in ABE fermentation. Cellulose which is

usually in a fibrous structure in cell walls is a linear polymer consists of D-glucose

subunits linked to each other by β-(1,4)-glycosidic bonds. Hydrogen and van der Waals

bonds link those cellulose long chains together to form microfibrils. Hemicellulose is also

a long chain polysaccharide but has branches with short chains consisting of some other

monosaccharides, such as pentoses (xylose, rhamnose, and arabinose), hexoses (glucose,

mannose, and galactose). The backbone of hemicellulose is usually linked by β-(1,4)-

glycosidic bonds and occasionally by β-(1,3)-glycosidic bonds. Lignin is a complex

polymer of phenolic monomers which is cross-linked with each other. It is the safeguard

of plant cell wall, which is impermeable and resistant against most microbial attack. The
17
monomers of lignin are coniferyl alcohol (guaiacyl propanol), coumaryl alcohol (p-

hydroxyphenyl propanol), and sinapyl alcohol (syringyl alcohol) which are linked by

alkyl-aryl, alkyl-alkyl, and aryl-aryl ether bonds. Usually, woods have the highest lignin

contents, whereas grasses have the lowest (Howard et al., 2003; Saha, 2003; Reddy and

Yang, 2005; Qureshi et al., 2007; Kumar et al., 2009; Sun and Cheng, 2002).

C. acetobutylicum and C. beijerinckii are capable of fermenting a variety of sugars,

including hexoses (glucose, galactose and mannose) and pentoses (xylose, arabinose).

They can even utilize more complex carbohydrates such as dextrin, starch and inulin.

This flexibility makes a lot of biomass as potential substrates to produce butanol (Table

2.2). Cane molasses has been used in the production of butanol (Qureshi et al., 2005).

Researchers have also investigated whey permeate (Maddox et al., 1993), maize starch

and soy molasses (Qureshi et al., 2001) as substrates. For these substrates, the yield is

high because these substrates contain a large quantity of carbohydrates that can be

utilized by the solventogenic clostridia mentioned above. However, some of these

substrates are also used as animal feed, which leads to a higher price. Improved food

processes also reduce the sugar concentrations in those molasses.

Other, less expensive substrates have also been studied (Table 2.2). These

substrates can be divided into two main categories: lignocelluloses and starch.

Lignocelluloses, like barley straw, wheat straw, corn fiber and corn stover, are mainly

composed of cellulose, hemicelluloses, lignin and a little ash. The solventogenic

clostridia cannot utilize these polysaccharides directly, so lignocelluloses must be

hydrolyzed into single sugars before fermentation. On the other hand, starch, such as sago,
18
extruded corn and cassava can be directly used as carbon source by the solventogenic

clostridia; therefore no hydrolysis is needed before fermentation (Table 2.2). Corn fiber

is a by-product of the maize wet milling process. Qureshi et al studied it as a substrate to

produce butanol after hydrolysis (Qureshi et al., 2007), 9.3 g/L total ABE (Acetone,

butanol and ethanol) was produced. Qureshi et al also used wheat straw as feedstock to

produce butanol, 25.0g/L ABE was obtained (Qureshi et al., 2010a). Moreover, Qureshi

et al investigated barley straw (Qureshi et al., 2010b), maize stover and switchgrass

(Zverlov et al., 2006). Thaddeus et al chose distillers dry grains and soluble (DDGS) as a

substrate and compared it with different mixed streams of pure sugars (Ezeji et al., 2008).

The highest ABE yield in Thaddeus et al‘s research is 12.1 g/L. Instead of feed-grade

biomass, Zverlov et al investigated agriculture wastes, such as hemp waste, corn cobs and

sunflower shells (Qureshi et al., 2008).

Although all of these substrates can be utilized by solventogenic clostridia, the

yield of butanol is much lower than the control which uses pure glucose as substrates.

Most maximum ABE concentration is only around 25 g/L and ABE yield ranged from

0.24-0.44. One possible reason for the low yield is that the total sugar concentration is

relative low after hydrolysis; another possible reason is that some fermentation inhibitors

that are toxic to strains may be contained in those hydrolysates. Therefore, further

research is needed to analyze the optimized hydrolysis conditions to get more sugars and

proper methods to remove inhibitors in hydrolysates, in order to get high yields of ABE,

especially butanol.

19
2.2 Hydrolysis of lignocellulosic biomass

2.2.1 Pretreatments

Most solventogenic clostridia can‘t use lignocellulosic biomasses directly.

Therefore, hydrolysis is always needed to decompose the lignocelluloses. Before the

enzymatic hydrolysis, some pretreatments are usually used to break up the rigid structure

of lignocelluloses so that the enzymes can easily get contact with them in the following

process.

The main goal of the pretreatment process is to reduce the crystallinity of cellulose

and increase the porosity of these materials. Some methods can even remove some parts

of lignin and degrade hemicelluloses. Pretreatment can improve the formation of

monosaccharides or the abilities to form sugars by hydrolysis. Pretreatment also needs to

avoid the further degradation monosaccharides and the formation of byproducts that are

inhibitory to the subsequent fermentation processes. Table 2.5 listed those processes used

to pre-treat lignocellulosic biomass. They can be roughly divided into five categories:

physical (mechanical comminution, pyrolysis); physicochemical (steam explosion, AFEX,

CO2 explosion); chemical (ozonolysis, acid hydrolysis, alkaline hydrolysis, organosolv);

biological; other methods (pulsed electrical field).

Mechanical comminution is usually combined with other pretreatments and used to

reduce the size of biomass particles before further pretreatments. It is used to reduce

cellulose crystallinity. After milling or grinding, the biomass particle size can be reduced

to 0.2-2 mm (Sun et al., 2002). The energy consumption in milling is the major concern

20
in large scale process. Smaller particle size means much higher energy input. If the final

particle size is 3-6 mm, the energy consumption can be kept below 30 kWh per tons of

biomass (Cadoche et al, 1989).

Steam explosion is the most common method to pretreat lignocellulosic biomass.

Biomass is immersed in high pressure saturated steam (0.7-5.0 MPa, 160-260 oC) for

several minutes, and then the pressure is suddenly reduce to atmospheric pressure

(McMillan et al., 1994). The explosive decompression causes fragmentation of cellulose

fiber which increase the surface area to contact with cellulase enzymes. The high

temperature also hydrolyzes hemicellulose and partly removes lignin which also increase

the contact surface between cellulose and cellulase. The efficiency of enzymatic

hydrolysis of polar chips increased to 90% after steam explosion, compared to 15% in

untreated polar chips (Grous et al., 1986). Ammonia fiber explosion (AFEX) is very

similar to steam explosion. It only replaces the saturated steam with liquid ammonia. The

temperature is much lower than steam explosion (~90 oC) and the residence time is much

longer (0.5-1.5 hr). Almost no hemicellulose and lignin are removed during AFEX. But

the biomass structure becomes more porous and water holding capacity is increased.

After AFEX, 90% of cellulose and hemicellulose in switchgrass and bagasse was

hydrolyzed into sugars (Holtzapple et al., 1991). CO2 explosion is to replace ammonia in

AFEX with supercritical CO2. Supercritical CO2 needs lower temperature than ammonia

and reduces the cost further. CO2 can form carbonic acid with water in the biomass and

the carbonic acid can hydrolyze hemicellulose. CO2 molecule has similar size with water

and ammonia. So it can penetrate the biomass structure as well as water or ammonia. For

21
pretreatment of recycled paper mix and sugarcane bagasse, CO2 explosion was more

cost-effective compared with steam and ammonia explosion (Zheng et al., 1998).

Dilute acid pretreatment is also a common method to pretreat lignocellulosic

biomass. The most obvious advantage of dilute acid pretreatment is that it can hydrolyze

and recover almost all hemicellulose as dissolved sugars in the solution. High

temperature is favorable (>120 oC) to increase the hydrolysis efficiency of hemicellulose.

Dilute H2SO4 (below 4 wt %) is most commonly used in the dilute acid pretreatment

(Mosier et al., 2005). HNO3, HCl and H3PO4 were also tested (Brink et al., 1993;

Israilides et al., 1978; Goldstein et al., 1983). Plenty of biomass were examined by dilute

acid pretreatment, such as legume byproducts, reed canary grass, corn cobs and stover,

hardwood bark from aspen, polar and sweet gum (Kumar et al., 2009). Pilot-scale dilute

H2SO4 pretreatment of corn stover was also tested in a vertical reactor. The temperature

was 180-200 oC, the solid loading was 25%-35%, the acid loading was 0.03-0.06 g/g dry

biomass and the residence time is ~1 min (Ishizawa et al., 2007).

Bases can also be used for pretreatment of lignocellulosic biomass which is called

alkali pretreatment. The temperatures and pressures used in alkali pretreatment are much

lower than the explosion and dilute acid pretreatment. It can be carried out at room

temperature and atmospheric pressure, but takes longer time from several hours to days.

NaOH, KOH, Ca(OH)2 and NH3 ·H2O are all suitable bases for alkali pretreatment.

Among them, NaOH was studied the most (Elshafei et al., 1991; Soto et al., 1994; Fox et

al., 1989). But Ca(OH)2 pretreatment is more cost effective because it is less expensive

and easy to be precipitated by CO2 from solution and recovered. Alkali pretreatment
22
induces less sugar degradation than dilute acid pretreatment. It only removes part of

lignin and hemicellulose which increases the crystallinity index of cellulose. So after

alkali pretreatment, the initial enzymatic hydrolysis rate will be slow. But it has no effects

on the final sugar yields (Chang et al., 2000). Lime was successfully used to pretreat corn

stover (Kim et al., 2006), wheat straw (Chang et al., 1998), poplar wood (Chang et al.,

2001) and swithgrass (Chang et al., 2001).

Other than the above three common pretreatment methods, there are some other

novel pretreatments that show promising characters. Ozone can be used to reduce the

lignin and hemicellulose contents in many lignocellulosic biomass and doesn't produce

toxic inhibitors (Quesada et al., 1999). Organic solvents mixed with acids can also be

used to break lignin and hemicellulose in biomass. Methanol, ethanol, acetone and

ethylene glycol are the common solvent used in this organosolvation pretreatment

(Thring et al., 1990). It involves both the hydrolysis of hemicellulose and delignification

of biomass supported by organic solvents. In biological pretreatment, fungi such as

brown, white and soft rots are used to degrade lignin and hemicellulose in biomass.

White-rot fungi are the most effective to degrade lignin because it has lignin degrading

enzymes such as peroxidase and laccase (Lee et al., 2007).

Each process has its own advantages and disadvantages (Table 2.5). Therefore,

sometimes two or more pretreatment processes are combined together to use their

advantages and make up their disadvantages. It‘s hard to say which process is the best. It

depends on the lignocellulosic biomass, like their compositions and possible products

produced in the pretreatment process. We are inclined to use dilute acid pretreatment
23
because it can hydrolyze hemicelluloses sugars and its cost is not very high. However,

some components that are inhibitory to the fermentation process may be generated in the

acid hydrolysis process. How to minimize these inhibitors is one of our tasks.

2.2.2 Enzymatic hydrolysis

After the pretreatment, enzymes such cellullase and xylanase are used to further

degrade the lignocelluloses. The main work of hydrolysis is to beak the β-(1,4)-glycosidic

bonds existed either in cellulose or hemicelluloses. These bonds can be broken either by

acids or by enzymes. However, concentrated acids are usually needed to degrade

cellulose due to its microfibril structure, which can induce a lot of side reactions and may

further degrade those monosaccharides. Those byproducts produced in the concentrated

acid hydrolysis are usually very toxic to the ABE fermentation process. So enzymes such

as cellulase and xylanase are usually preferred to be used to degrade celluloses and

hemicelluloses into monosaccharides.

Cellulase is a class of enzymes that catalyze cellulysis which is the hydrolysis of

cellulose. Many organisms can produce cellulase, such as fungi, bacteria, protozoans and

even some termites. Cellulase breaks the 1,4-β-D-glycosidic linkages in cellulose. There

are mainly three types of cellulases: endocellulase, exocellulase and β-glucosidase.

Although three types all work on the hydrolysis of 1,4-β-D-glycosidic linkages, their

mechanisms are a little different from each other. Endocellulases (EGs) randomly cleave

glycosidic linkages at amorphous sites and chains with different length (molecular weight)

are generated in this process. Exocellulase cleaves mainly two glucose units from one end

24
of cellulose chains and produces cellubiose. So there are two main types of exocellulases

(cellobiohydrolases, CBHs): CBHI works from the reducing end, and CBHII works from

the nonreducing end. β-glucosidase breaks cellubiose into single sugars. When

lignocellulosic biomass are hydrolyzed, all these three celluloses can work together in a

synergistic mechanism to get a better production of monosaccharides (Figure 2.2).

Trichoderma reesei which is the Industrial strain of cellulase production can produce up

to five types of EGs and two types of CBHs (CBH I and CBH II). There are other two

types of cellulases that are not very common, oxidative cellulases and cellulose

phosphorylases.

Although EGs, CBHs and β-glucosidase have different functions in the

breakdown of cellulose, they share very similar molecular structures. They mainly have

two functional domains: a cellulose binding domain (CBD) and a catalytic domain (CD).

CBD binds to the cellulose substrate while CD cleaves the glycosidic bonds (Henrissat,

1994). These two domains are linked together by a peptide sequencer (Figure 2.3).

The hydrolysis of celluloses and hemicelluloses is a crucial step in the fermentation

to produce biofuels because lignocellulosic biomasses are more promising and

economical substrates. But most yeast or bacteria strains cannot use celluloses or

hemicelluloses directly. So they must be hydrolyzed before fermentation. Cellulases

catalyze this key hydrolysis process. However, it‘s hard to use cellulases directly to

hydrolyze celluloses due to the rigid structure of plant cell walls. There are mainly three

components in the plant cell wall: cellulose, hemicelluloses and lignin. Lignin has a three

dimensional network of p-hydroxyphenylpropane units and its main function is to protect

25
celluloses from breakdown or hydrolysis. It is entangled with or even chemically linked

to celluloses (Siqueira et al., 2010). Cellulose crystallization is another resistance to the

hydrolysis. So before cellulase hydrolysis, biomasses need to be treated in some way that

destroy the rigid structure between celluloses and lignin and also reduce the degree of

cellulose crystallinity. Different methods and materials have been used in this

pretreatment process as described in the previous section, such as liquid hot water, steam

explosion, dilute acid, etc. Although these pretreatments can facilitate the contact

between celluloses and cellulases, they may generate some toxic components that can

inhibit the cellulase activity. The cellulose inhibitors derived in the biomass treatment

processes need to be removed before enzymatic hydrolysis.

2.3 Detoxification of lignocellulosic hydrolysates

2.3.1 Inhibitors to enzymatic hydrolysis

Although the pretreatments of lignocellulosic biomass can facilitate the contact

between cellulose and cellulase, they can also generate or release some components that

can inhibit the cellulase activity.

First inhibitor group comes from the partly degradation of lignin in the pretreatment

process. The partly degradation of lignin can generate some phenolic compounds that

may be very toxic to cellulases. Table 2.6 lists some of these lignin derived phenolic

compounds and their effects on cellulases. Tannic acid is the most toxic inhibitor to

cellulases. 300 μg condensed tannins/mL can completed inhibit the activity of

endoglucanases (Bae et al., 1993). Tannic acid can precipitate β-glucosidases, which is

26
one reason for its high toxicity. Sinapic acid is another very toxic compound. The

production of cellulases was completely inhibited in the presence of 3 μmol cm−3 of

sinapic acid (Sineiro et al., 1997). Simple and oligomeric phenolics can absorb onto

cellulose and thus prevent the contact between cellulase and its substrate. Simple

phenolics can also form complex with cellulases and deactivate them (Tejirian et al.,

2011). Insoluble lignin also has some negative effects on the hydrolysis process because

it can absorb cellulases. Therefore, it‘s better to use some separation methods to remove

those inhibitors before the hydrolysis using cellulases.

Second inhibitor group comes from the products-monosaccharides. Table 2.7 lists

the main monosaccharides and cellobiose produced in the hydrolysis process and their

inhibition effects on cellulases. Glucose has a significant inhibition effect on cellulases.

Glucose strongly inhibited the cellulase activity from T. reesei QM 9414, in which 2.5%

glucose inhibited 50% of the reaction. Trichoderma β-glucosidase activity was inhibited

by 50% in the presence of 0.2–0.4 g/L glucose (Takagi, 1984). Cellobiose, which is the

main product of exocellulases, is considered to be a powerful inhibitor of cellulases and

inhibits this enzyme 14 times more than glucose (Duarte, 2012). Therefore, it‘s better to

continuously remove these monosaccharides in the hydrolysis process so that this kind of

production inhibition can be limited.

There are some other components that may be generated in the hydrolysis process

and inhibit cellulase activity. Acetic acid, formic acid and sodium gluconate are all strong

inhibitors to cellulase activities (Takagi, 1984). Acetic acid and formic acid are products

from the degradation of glucose or other pentose sugars. So they may exist in the medium
27
after pretreatment. Sodium gluconate can be produced in the pH adjustment process,

especially after dilute acid pretreatment when pH adjustment is very necessary.

2.3.2 Inhibitors to ABE fermentation

During hydrolysis process, some inhibitors that could inhibit fermentation process

may be generated due to the complex components of lignocelluloses (celluloses,

hemicelluloses and lignin). Table 2.8 shows the most common potential inhibitors after

hydrolysis process (Ezeji et al., 2008; Lee et al., 2011). There are three main groups:

furan derivatives (Furfural and 5-hydroxymethyl furfural (HMF)), weak acids (glucuronic

acid, formic acid, acetic acid and levulinic acid) and phenolics (p-coumaric acid, ferulic

acid, vanillin, hydroquinone, and syringaldehyde). Furan derivatives come mainly from

degradation of monosaccharide molecules, while phenolics are generated by lignin

degradation or monosaccharide degradation during acid hydrolysis. HMF‘s further

breakdown could form some weak acids like formic acid and levulinic acid (Ezeji et al.,

2008; Lee et al., 2011).

Their toxicities vary with different mechanisms. Furfural and HMF can even

simulate the growth of C. beijerinckii BA101 (Cho et al., 2009). Ferulic acid and p-

coumaric acid are the most toxic compounds to most solventogenic clostridia. The

growth of C. beijerinckii BA101 can be totally inhibited when their concentration reaches

1.0g/L. Ferulic and p-coumaric acid can damage the hydrophobic sites of bacterial cells

and increase membrane permeability, which causes leakage of cellular contents.

Syringaldehyde don‘t inhibit the growth of cells but decrease the production of ABE

dramatically by affecting in the glycolytic pathway and alcohol dehydrogenase enzymes

28
secretion (Ezeji et al., 2008). Moreover, at the same concentration, combinations of these

inhibitors may become even more toxic than single one due to synergistic effects. For

example, furfural and HMF can severely block the xylose metabolic pathway (Vogel-

Lowmeier et al., 1998).

Therefore, these inhibitors must be removed before fermentation. Some other

inhibitors can be brought in by pretreatment (Qureshi et al., 2007; Martinez et al., 2001).

One such inhibitor is sodium sulfate, which is produced by dilute sulfuric acid

pretreatment (Cho et al., 2009). So proper pretreatment methods must be chosen to

reduce the generation of inhibitors. For example, dilute hydrochloric acid pretreatment

can avoid the production of sodium sulfate in the hydrolysis process. Proper choose of

lignocellulosic biomass can also reduce the concentrations of inhibitors. In the three main

components of lignocelluloses: celluloses, hemicelluloses and lignin. Only lignin

contains these phenolic structures in the inhibitors (Table 2.8). Possibly, these inhibitors

come from partial hydrolysis of lignin. Hence, utilizing lignocelluloses with less lignin

could reduce the possibility of producing inhibitors.

2.3.2 Detoxification methods

During hydrolysis process, some inhibitory components may be produced which

can severely inhibit the butanol production. So after hydrolysis, different methods of

removing inhibitors which are called detoxification will be employed, such as overliming

(Mussatto et al., 2010), electrodialysis (Martinez et al., 2010), and adsorption (Qureshi et

al., 2007), filtration (Qureshi et al., 2008) and enzymatic reactions (Larsson et al., 1999).

The effectiveness of some detoxification methods are shown in Table 2.9.

29
 Evaporation, usually under vacuum, can reduce the volatiles in the hydrolysate,

such as furfural, formic acid, acetic acid and vanillin (Larsson et al., 1999; Rodrigues et

al., 2001) while the sugar was concentrated. But other non-volatile inhibitors can also be

concentrated during evaporation. Therefore the detoxification effects of evaporation

largely depends on the types of inhibitors in the hydrolysate. The concentration of green

liquor wood extracts resulted in increased inhibition to E. coli KO11 due to the

concentration of sodium (Walton et al., 2010).

pH adjustment, often called overliming because Ca(OH)2 is used in the process, is

the most widely studied in the detoxification of hydrolysate (Mussatto et al., 2010). The

pH is usually raised to ~10 by some base, usually Ca(OH)2, and then reduced to the

fermentation level. Although the mechanism of overliming is still unclear, it's already an

effective way to detoxify hydrolysate, especially spruce hydrolysate (Persson et al., 2002).

pH=10 tends to be a optimized condition for Ca(OH)2. lower pH gives little improvement

in detoxification and higher pH increases the degradation of lignin and induce higher

concentration of phenolic inhibitors. For corn stover hydrolysate, the detoxification of

NH3 · H2O adjustment was more effective than Ca(OH)2 (Jennings et al., 2011).

Ammonia treatment resulted in the change of phenolic inhibitors.

Adsorption is another effective detoxification method. Activated carbon is a

sorbent with broad spectrum, so it can remove almost all inhibitors but only partly

(Qureshi et al., 2007a). And activated carbon is also very cheap. Control of pH during the

adsorption can minimize the sugar reduction (Berson et al., 2005). After activated carbon

treatment, furfural, acetic acid and phenolic in soybean hull hydrolysate were reduced by
30
95%, 37% and 75% respectively (Schirmer-Michel et al., 2008). XAD-4, a styrene-based

polymer adsorbent, was also used to detoxify corn fiber hydrolysate, decrease the

concentration of furfural in it and increase its fermentability (Weil et al., 2002).

Ion exchange resins are the most effective detoxification method to many

hydrolysates, especially anion exchange resins because most toxic inhibitors are anions.

But they can also partly remove sugars. DeMancilha and Karim (2003) investigated a

weak base anion exchange resin on the detoxification of corn stover hydrolysate and

found it removed almost all HMF, furfural, acetic acid and only 6% of xylose. For

different types of resins, their detoxification efficiency followed the pattern: anion

exchange >plain resin >cation exchange (Nivebrandt et al., 2001). Another base resin,

Dowex MWA-1, eliminated 2-furancarboxylic acid, 2-furanacetic acid, 5-

hydroxymethyl-2-furancarboxylic acid, and ferulic acid which are potential inhibitors in

the hydrolysate of green hybrid poplar (Luo et al., 2002). Horvath et al. (2004) tested

three strong base (Dowex 1X4, Dowex 2X8, IRA458) and three weak base anion

exchangers (IRA67, IRA92 and Duolite A7). And the strong base resins were the most

effective in improving fermentation. However, the disadvantage of resins is also obvious.

The resins are expensive and the cost to recycle them is also high.

The idea of biodetoxification is to use certain microorganism in removing

inhibitors in the hydrolysate. The microorganism must be able to use the inhibitors as

substrates or at least convert the inhibitors to non-toxic compounds. Coniochaeta lignaria,

a fungus isolated from soil, can successfully removed furfural and HMF (Lopez et al.,

2004). C. ligniaria NRRL30616, a bacteria isolated by Nichols et al. (2008), can almost
31
eliminite all furfural and HMF, and reduce 20% of acetate in hydrolysate. S. cerevisiae

YGSDC was able to metabolize acetic acid but no sugars (Schneider, 1996). So it can be

used to remove acetate which is a common inhibitor in hydrolysate due to its high

concentration. Ureibacillus thermosphaericus, a thermophilic bacterium, can oxidize

furfural and HMF to 2-furancarboxylic acid and 5-hydroxymethyl furancarboxylic acid.

The latter two were less toxic than furfural and HMF to yeast (Okuda et al., 2008).

However, most microorganisms also consume sugars. So during the biodetoxification, a

large portion of sugars is also used. One possible solution is to find some microorganisms

that don't consume sugars like S. cerevisiae YGSDC. Another possible solution is to

extract the useful enzymes and only use the enzymes to detoxify hydrolysate. Another

problem is that all new isolated microorganisms works on furan inhibitors. No

microorganism has be detected to have the capability of removing toxic phenolic

compounds.

Some other detoxification methods were also studied. Liquid-liquid extraction is

very effective in extracting phenolic compounds but the solvent residues may be harmful

to fermentation (Um et al., 2010). Lignin fragments in hydrolysate were usually

negatively charged nanoparticles (200-300nm). Cationic flocculating agent can

precipitate the lignin fragments (Hasan et al., 2011). Electrodialysis can be used to

remove ions in hydrolysate, especially the high concentration of salt ions due to the pH

adjustment (Lynd et al., 2001).

32
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46
 Table 2.1 Properties of butanol (Hastings, 1978)

Properties Butanol

Melting point(℃) -89.3 Butanol structure

Boiling point(℃) 117.7

Ignition temperature(℃) 35

Flash point(℃) 365

Density at 20℃ (g/mL) 0.8098

Critical pressure(hPa) 48.4

Critical temperature(℃) 287

Properties of fuels

Butanol Gasoline Ethanol Methanol

Energy density(MJ/L) 29.2 32 19.6 16

Air-fuel ratio 11.2 14.6 9 6.5

Heat of vaporization(MJ/kg) 0.43 0.36 0.92 1.2

Research octane number 96 91-99 129 136

Motor octane number 78 81-89 102 104

47
 Table 2.2 Summary of various solventogenic Clostridia with their substrates, products, fermentation pH and temperature

Species / strain Substrates Products pH Temp. (oC) References

C. acetobutylicum Glucose, xylose, arabinose,
P262 cellobiose, mannose,
galactose
Starch
ATCC 824 Lactose
Sucrose, fructose, lactose,
maltose, cellobiose
C. carboxidivorans Syngas (H2, CO, CO2)
P7
H2, CO
C. Glucose, starch, maltose
saccharoperbutylacetonicum Molasses, starch
N1-4
C. saccharobutylicum Glucose, xylose, arabinose,
262 cellobiose, mannose,
galactose
C. butylicum Glucose, xylose, arabinose,
NRRL 592 cellobiose, mannose,
48
Ezeji and Blaschek, 2008
Acetone, butanol,
ethanol, acetate, butyrate, 5.5 – 6.5 35 ±1 Madihah et al., 2001
H2, CO2 Qureshi and Maddox, 2005
Servinsky et al., 2010
5.0 - 6.0 Bruant et al., 2010
Acetate, ethanol,
6.2 37 ±1 Liou et al., 2005
butyrate, butanol
5.7-5.8 Rajagopalan et al., 2002
Acetone, butanol, 6.2 Thang et al., 2010
ethanol, acetate, butyrate, 30 Hipolito et al., 2008
5.6-5.9
H2, CO2
Acetone, butanol, Ezeji and Blaschek, 2008
ethanol, acetate, butyrate, 5.5 – 6.5 35
H2, CO2
Acetone, butanol, Ezeji and Blaschek, 2008
ethanol, acetate, butyrate, 5.5 – 6.5 35

galactose H2, CO2

C. beijerinckii Glucose, xylose, arabinose, Ezeji and Blaschek, 2008
BA101 cellobiose, mannose, 5.5 – 6.5 35 Ezeji et al., 2007b
galactose,
Acetone, butanol,
Starch 6.8 36 Jesse et al., 2002
ethanol, acetate, butyrate,
Sucrose, fructose 6.8 35 Qureshi et al., 2001b
H2, CO2
NCIMB 8052 Maltodextrin 6.5 33-35 Formanek et al., 1997
Glucitol (sorbitol), Mitchell, 1996
N/A 37
mannitol
C. aurantibutyricum Glucose, , xylan, starch, Somrutai et al., 1996
Acetone, butanol,
ATCC 17777 pectin, arabinose, xylose, 5.5 – 6.8 37
isopropanol, acetate,
galactose, mannose
butyrate
NCIB 10659 Glucose 6.8 35 George et al., 1983
C. pasteurianum Butanol, ethanol, 1,3- 5.0 – 7.0 37 Ahn et al., 2011
DSM 525 Glycerol propanediol, acetate, 4.5 – 7.5 35 Biebl, 2001
butyrate, lactate 7.0 35 Taconi et al., 2009
 Table 2.3 ABE production by solventogenic Clostridia from traditional substrates and renewable lignocellulosic biomass

Pretreatment and ABE titer ABE yield Productivity

Feedstock Strain References
hydrolysis (g/L) (g/g) (g/L.h)
Glucose with corn C. beijerinckii NCIMB 8052 19.2 0.40 38.0
None Parekh et al., 1999
steep water C. beijerinckii BA101 23.6 0.40 36.0
Liquefied corn starch None C. beijerinckii BA101 18.4 0.41 0.15 Ezeji et al., 2007c
Packing peanuts None C. beijerinckii BA101 21.7 0.37 0.2 Jesse et al., 2002
Cassava starch 21.0 0.41 0.44
Corn starch None C. saccharoperbutylacetonicum 20.7 0.48 0.31
Thang et al., 2010
Sago starch N1-4 19.6 0.43 0.27
Cassava chips Enzyme 19.4 0.38 0.40
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 +
C. beijerinckii P260 22.2 0.41 0.55 Qureshi et al., 2008b
enzymes
49

C. acetobutylicum 260
Dilute acid C. acetobutylicum 824
Ezeji and Blaschek,
Liquid hot water C. saccharobutylicum 262 4.9-12.9 0.30-0.35 N/A
Distiller‘s dried 2008
AFEX + enzyme C. butylicum 592
grains and solubles
C. beijerinckii BA101
Alkaline electrolyzed
C. acetobutylicum P260 16.9 N/A N/A Wang et al., 2009
water + enzyme
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
 Table 2.4 Compositions of different lignocellulosic biomass and their current use

Composition (%, dry basis)

Current use Reference
Cellulose Hemicellulose Lignin Starch
Cassava
(Total fiber) 15- 51 41-64 Landfill, burnt 3, 4, 5, 7
bagasse
Corn fiber 15 23- 64 8 12-32 1, 5, 6
Corn cob 45 35 15 --- 5, 6
Animal feed,
Corn stover 38- 40 25- 28 7- 21 --- 5, 6, 7
burnt as fuel,
Rice straw 28- 36 23- 28 12- 14 --- 5, 6, 7
compost,
Wheat straw 35- 40 20- 30 17- 19 --- 5, 7, 8
soil conditioner
Sorghum
27 25 11 --- 5, 7
stalks
Fresh bagasse 33.4 30 18.0 --- Burnt as fuel 5
Sugarcane Burnt as fuel,
40- 50 24- 25 25 --- 2, 5, 6
bagasse landfill
Grass 25- 40 25- 50 10- 30 --- Burnt 5, 9
Hardwood
40-55 24-40 18-25 --- 5, 10
stems Soil conditioner,
Softwood burnt
45-50 25-35 25-35 --- 5, 10
stems
Partially
Newspapers 40- 55 25- 40 18-30 --- 5, 9
recycled
Waste papers Reused in pulp
from chemical 60-70 10-20 5-10 --- and board 5
pulps industry as fuel
(References: 1. Dien et al., 1997; 2. Pandey et al., 2000a; 3. Pandey et al., 2000b; 4.
Sriroth et al., 2000; 5. Howard et al., 2003; 6. Saha, 2003; 7. Reddy and Yang, 2005; 8.
Qureshi et al., 2007; 9. Kumar et al., 2009 ; 10. Sun and Cheng, 2002)

50
 Table 2.5 Processes Used for the Pretreatment of Lignocellulosic Biomass

pretreatment process advantages limitations and disadvantages

mechanical comminution reduces cellulose power consumption usually

crystallinity higher than inherent biomass
energy

steam explosion causes hemicellulose destruction of a portion of the

degradation and lignin xylan fraction; incomplete
transformation; cost- disruption of the lignin-
effective carbohydrate matrix; generation
of compounds inhibitory to
microorganisms

Ammonia Fiber Explosion increases accessible not efficient for biomass with
(AFEX) surface area, removes high lignin
lignin and content
hemicellulose to an
extent; does not
produce inhibitors for
downstream processes

CO2 explosion increases accessible does not modify lignin or

surface area; cost- hemicelluloses
effective; does not
cause formation of
inhibitory compounds

ozonolysis reduces lignin content; large amount of ozone required;

does not produce toxic expensive
residues

acid hydrolysis hydrolyzes high cost; equipment corrosion;

hemicellulose to formation
xylose and other of toxic substances
sugars; alters lignin
structure

Continued
51
Table 2.5 Continued

alkaline hydrolysis removes hemicelluloses and long residence times required;

lignin; irrecoverable
increases accessible surface salts formed and incorporated
area into
biomass

organosolv hydrolyzes lignin and solvents need to be drained from

hemicelluloses the reactor, evaporated,
condensed, and recycled; high
cost

pyrolysis produces gas and liquid high temperature; ash production

products

pulsed electrical field ambient conditions; disrupts process needs more research
plant cells; simple equipment

biological degrades lignin and rate of hydrolysis is very low

hemicelluloses; low energy
requirements

(Kumar et al., 2009)

52
 Table 2.6 Inhibition effect of phenolic compounds on cellulases

Compound Inhibition effect Ref.

Ferulic acid Deactivation effect on β-glucosidases Ximenes et al.,

2011

Tannic acid cause significant inhibition of the hydrolysis Tejirian et al., 2011
by cellulase

Sinapic acid Strong inhibition of commercial cellulase Sineiro et al., 1997

Substituted phenols Moderately active against certain fungal Mandels et al.,

(chlorophenols, cellulases 1965
saligenin,
orthiphenyl phenol,
and chlorophenyl)

Gallic, hydroxy- Cause 20–80% deactivation of cellulases Ximenes et al.,

cinnamic, and/or β-glucosidases after 24 h of pre- 2011
4-hydroxybenzoic incubation
acids, and vanillin

53
Table 2.7 Inhibition effect of sugars on cellulases (Xiao et al., 2004; Ximenes et al., 2011)

Compound Inhibition effect

Glucose Notably strong inhibition of for cellulase activities

Cellobiose Moderate inhibition of cellulase activities

Xylose Significantly inhibitory to cellulase activity

More inhibitory than xylose or D-glucuronic acid to the hydrolysis of

Arabinose
cellobiose

Galactose Significant inhibition effect on cellulase activity

54
 Table 2.8 Major fermentation inhibitors present in the hydrolysates generated from

lignocellulose degradation

Categories Fermentation inhibitors, source of origin

Sugar degradation products Furfural (from xylose)
5-hydroxymethyl furfural (HMF) (from hexose)
Formic acid (from furfural and HMF)
Levulinic acid (from HMF)
Lignin degradation products Vanillin, vanillic acid (from guaiacylpropane units)
Syringaldehyde, syringic acid (from syringyl propane
units)
Hydroquinone (1,4-di-hydroxybenzene), 4-
hydroxybenzoic acid
Catechol (1,2-di-hydroxybenzene)
p-Coumaric acid
Ferulic acid
Glucuronic acid
Coniferyl aldehyde
Lignocellulose structure Acetic acid (from the acetyl groups present in the
degradation product hemicellulose)
(References: Ezeji et al., 2007b; Cho et al., 2009; Mussatto and Roberto, 2004a;

Palmqvis and Hahn-Hagerdal, 2000b; Zautsen et al., 2009)

55
 Table 2.9 Efficiency of different detoxification methods

Method Inhibitors removed Inhibition reduction Sugar retention Ref.

Evaporation, Volatiles Fair, Very good Larsson et al., 1999;
flashing can also exacerbate Rodrigues et al., 2001
Walton et al., 2010
pH adjustment, Aldehydres, Effective Good Mussatto et al., 2010
overliming phenols Persson et al., 2002
Jennings et al., 2011
Activated carbon, Furans, Fair Good Qureshi et al., 2007
polymer adsorption phenolics, Berson et al., 2005
acids Schirmer-Michel et al., 2008
Weil et al., 2002
Liquid-liquid extraction Organic acids, Moderate Fair Um et al., 2010
phenolics
56

Ion exchange Organic acid anions, Effective Fair DeMancilha and Karim, 2003)
metal ions Nivebrandt et al., 2001
Luo et al., 2002. Horvath et al., 2004
Dialysis Salts, Effective Good Martinez et al., 2010
organic ions Hasan et al., 2011
Polymer flocculation Lignin, Not demenstrated Good Hasan et al., 2011
lignin derivatives
Biodetoxification Organics Effective Poor Lopez et al., 2004
Nichols et al., 2008,
Schneider, 1996
Okuda et al., 2008
 Figure 2.1 Metabolic pathways in C. acetobutylicum for the acidogenic and

solventogenic phase.

Enzymes are indicated by numbers as follows: (1) enzymes including in glycolysis

process (2) pyruvate–ferredoxinoxidoreductase; (3) acetaldehyde dehydrogenase; (4)
ethanol dehydrogenase; (5) phosphate acetyltransferase (phosphotransacettylase); (6)
acetate kinase; (7) thiolase (acetyl-CoA acetyltransferase); (8) 3-hydroxybutyryl-CoA
dehydrogenase; (9) acetoacetyl-CoA: acetate/butyrate:CoA-transferase; (10) acetoacetate
decarboxylase; (11) crotonase; (12) butyryl-CoA dehydrogenase; (13) phosphate
butyltransferase (phosphotransbutyrylase); (14) butyrate kinase; (15) butyraldehyde
dehydrogenase; (16) butanol dehydrogenase; (17) hydrogenase (Kumar et al., 2011)

57
 Figure 2.2 The endo-exo synergistic action of the cellulolytic enzymes: EGs cleave the

internal b-1, 4 bonds. CBHs attack the reducing/nonreducing ends. β-glucosidase

produces glucose from cellobiose units (Gokhale et al., 2009)

Figure 2.3 Simplified scheme showing different functional domains of cellobiohydrolase

(CBH) (Gokhale et al., 2009)

58
 Chapter 3: Effects of lignocellulosic biomass hydrolysate derived inhibitors on

acetone-butanol-ethanol fermentation by Clostridium spp.

Abstract

Lignocellulosic biomass is abundant and more economical for use in fermentation

to produce butanol, one promising biofuel. However, lignocelluloses need to be

thermochemically pretreated and enzymatically hydrolyzed into monosaccharides before

fermentation. The pretreatment and hydrolysis processes usually produce some inhibitory

compounds that could severely inhibit cell growth and butanol production in acetone-

butanol-ethanol (ABE) fermentation. Therefore, the inhibitors in the hydrolysate must be

reduced or removed by certain detoxification processes before fermentation. To make the

detoxification process more efficient, a better understanding of these inhibitors and their

inhibition effects is needed. In this study, the effects of 9 inhibitors derived from the

degradation of carbohydrate and lignin on the fermentation kinetics of several

solventogenic C. beijerinckii and C. acetobutylicum strains were investigated at various

concentration levels. The inhibitors' effects on butanol and butyraldehyde dehydrogenase

activities were also investigated. Among the 9 inhibitors studied, four lignin derived

inhibitors (syringaldehyde, ferulic acid, vanillin, and p-coumaric acid) were found to

strongly inhibit cell growth and butanol and butyraldehyde dehydrogenases.

59
Keywords: lignocellulose; ABE fermentation; butanol dehydrogenase; butyraldehyde

dehydrogenase; Clostridium; inhibitors

3.1. Introduction

Butanol is an important intermediate (Jones et al., 1986) and solvent in the

chemical industry. Compared to ethanol, butanol has a higher energy density and lower

vapor pressure (Lee et al., 2008), and is thus considered a preferred fuel additive and a

potential replacement for gasoline. As the oil price rises, the cost of producing butanol

from petrochemical processes also increases dramatically. Therefore, more and more

research is being done on ways to produce butanol from fermentation of renewable

biomass. It is necessary to reduce the cost of fermentation-derived butanol in order to

make it competitive with petrochemically produced butanol. A number of Clostridium

mutant strains have been developed to enhance butanol production (Hastings, 1978;

Kumar et al., 2011; Qureshi, 2010; Annous et al., 1991; Qureshi et al., 2001). Another

most influential economy factor in producing butanol by fermentation is the cost of

substrate (Ezeji et al., 2008). Much cheaper substrates were tested for butanol

fermentation (Qureshi et al., 2001, 2005, 2007, 2008, 2010a,b; Maddox et al., 1993;

Zverlov et al., 2006). Lignocellulosic biomass (Tashiro et al., 2005; Liew et al., 2005;

Badr et a;., 2001; Ezeji et al., 2007; Lin et al. 1983; Tran et al., 2010; Qureshi et al., 1995;

Cho et al., 2009), such as sugarcane bagasse, soybean hull, cotton stalk and corn fibers,

are byproducts in the agricultural processing industry and much cheaper than the current

industrial substrates such as molasses and corn mesh. However, lignocellulosic biomass

cannot be used by the solventogenic Clostridia directly and needs to be hydrolyzed first.

60
The hydrolysis process usually produces some inhibitory compounds that could severely

inhibit cell growth and butanol production (Ezeji et al., 2008). Therefore, the inhibitors in

the hydrolysate must be reduced or removed by certain detoxification processes

(Martinez et al., 2001; Mussatto et al., 2001; Qureshi et al., 2008) before fermentation.

To make the detoxification process more efficient, a better understanding of these

inhibitors and their effects on acetone-butanol-ethanol (ABE) fermentation is needed.

The most common potential inhibitors after hydrolysis process (Ezeji et al., 2008;

Lee et al., 2010; Lowmeier et al., 1998) can be divided into three main groups: furan

derivatives (furfural and 5-hydroxymethyl furfural (HMF)), weak acids (glucuronic acid,

formic acid, and levulinic acid), and phenolics (p-coumaric acid, ferulic acid, vanillin,

hydroquinone, and syringaldehyde). Furan derivatives come mainly from the degradation

of monosaccharides, while phenolics are generated from lignin degradation or

monosaccharide degradation during acid hydrolysis. HMF‘s further breakdown could

form some weak acids like formic acid and levulinic acid (Lee et al., 2011). Ezeji et al.

studied several inhibitors for their effects on C. beijerinckii BA101 (Ezeji et al., 2008).

They found that furfural and HMF stimulated the growth of C. beijerinckii BA101, while

ferulic acid and p-coumaric acid were the most toxic compounds to C. beijerinckii

BA101. Zhang et al. (2012) found that furfural and HMF could be transformed to furfuryl

alcohol and HMF alcohol by C. acetobutylicum ATCC 824. Cao et al. (2010) studied the

effects of furfural, HMF, vanillin, and syringaldehyde on one Thermoanaerobacterium

strain and reported that the effects depended on the inhibitor's concentration. The

61
inhibitors present in wood pulping hydrolysate have also been analyzed in a previous

study (Lu et al., 2013).

The goal of this study was to understand the inhibition levels and mechanism of

main inhibitors derived from lignocellulosic biomass hydrolysates. First, the effects of 9

potential inhibitors on cell growth and butanol production from glucose by several

Clostridia strains were investigated in serum bottles and fermentors. These inhibitor's

influence on butanol and butyraldehyde dehydrogenase activities were also tested. Then

these inhibitors' concentrations in 6 biomass hydrolysates were determined and evaluated.

After detoxification by activated carbon, these hydrolysates were used in the ABE

fermentation.

3.2. Materials and methods

3.2.1 Strain and inoculum preparation

Spores of C. beijerinckii, C. acetobutylicum, C. tyrobutyricum were stored in a

refrigerator at 4 oC in the Clostridia Growth medium (CGM). Spores (2 ml) were heat-

shocked at 80oC for 3 min and transferred to 50 ml Reinforced Clostridia Medium (RCM)

(Difco, Becton, Dickinson and Company, MD, USA) in a 125 ml serum bottle. The

medium was nitrogen-purged for 8 min to remove oxygen. The serum bottle was tightly

capped with a rubber stopper and aluminum seal. The mixture was autoclaved at 121 oC

for 30 min followed by cooling to 37 oC. The heat-shocked spores were incubated at 37
o
C for 12-16 h until cells were highly active. The active culture (5% inoculum) was used

as seed culture in fermentation studies.

62
3.2.2 ABE fermentation in serum bottles

ABE fermentation was studied in serum bottles each containing 50 ml medium to

evaluate the effects of various detoxification methods on butanol production. Unless

otherwise noted, all fermentation studies were carried out with the P2 medium containing

one type of inhibitors, glucose (60 g/L), yeast extract (2 g/L), buffer (0.5 g/L KH2PO4

and 0.5 g/L K2HPO4), 2.2 g/L ammonium acetate, vitamins (0.001 g/L para-amino-

benzoic acid (PABA), 0.001g/L thiamin and 10-5 g/L biotin), and mineral salts (0.2 g/L

MgSO4∙7H2O, 0.01 g/L MnSO4∙H2O, 0.01 g/L FeSO4∙7H2O, 0.01 g/L NaCl). The carbon

source solution (containing inhibitors) and P2 stock solution (containing yeast extract,

ammonium acetate and buffer, 10-fold concentrated) were autoclaved separately at 121
o
C and 15 psig for 30 min for sterilization. Minerals and vitamins were prepared at 200-

fold and 1000-fold concentration, respectively, and were filter-sterilized through 0.2 μm

membrane filters (25 mm syringe filter, Fisher brand, NJ, USA). Before sterilization, the

P2 solutions in serum bottles were purged with nitrogen for 8 min. After sterilization, an

appropriate amount of the P2 stock solution was aseptically added into the serum bottle,

followed by adding minerals and vitamins. To ensure the medium pH maintained around

5.0 throughout the fermentation, 2 g/L of CaCO3 was also included in the medium.

Actively grown cells were inoculated at 5% (v/v). Batch fermentation was performed at

37 oC with no agitation. Samples were taken periodically for analysis of ABE production.

3.2.3 Butyraldehyde and butanol dehydrogenase activities

63
 The activities of butyraldehyde dehydrogenase and butanol dehydrogenase were

measured by monitoring NADH consumption at 365 nm according to the method

described before (Yu et al., 2011) with some modifications. Cells were collected from 50

mL fermentation broth by centrifugation at 4,000 rpm for 10 min using tightly sealed

centrifuge tubes purged by nitrogen gas. The cell pellet was washed with Tris-HCl buffer

(0.1 M, pH 7.5) once and resuspended in 2.5 mL of the same Tris-HCl buffer. Cell lysis

was carried out using Mini BeadBeater (Biospec) with 0.1 mm disruption beads

(Zirconia/Silica) for 6 min, stopped every 30 s to cool down the medium on ice.

Supernatant was collected by centrifugation at 13,000 rpm for 10 min and used for

enzyme activity assay. Enzyme activity was calculated on the basis of a molar NADH

extinction coefficient of 3.4 cm-1mM-1. One unit of enzyme activity was defined as the

amount of enzyme converting 1 mmol NADH per minute under the reaction conditions.

Protein concentration in cell extract was determined using the Bio-Rad protein assay kit

with bovine serum albumin as standard.

3.2.4 Analytical methods

The fermentation products, acetone, butanol, ethanol, acetic acid, and butyric acid

were analyzed with a gas chromatograph (GC, Shimadzu GC-2014) equipped with a

flame ionization detector and a 30-m fused silica column (0.25 m film thickness and

0.25 mm ID, Stabilwax-DA). The carrier gas was nitrogen at 1.47 ml/min (linear velocity:

35 cm/s). Samples were diluted 20 times with an internal standard buffer solution

containing 0.5 g/L isobutanol, 0.1 g/L isobutyric acid and 1% phosphoric acid (for

acidification), and injected (1 μL each) by using an auto-injector (AOC-20i Shimadzu).

64
The column temperature was held at 80 oC for 3 min, raised to150 oC at a rate of 30
o
C/min, and held at 150 oC for 3.7 min. Both the injector and detector were set at 250 oC.

Cell density was analyzed by measuring the optical density (OD) of cell suspension

at 600 nm using a spectrophotometer (Shimadzu, Columbia, MD, UV-16-1). The

inhibitors including furfural, HMF, levulinic acid, ferulic acid, p-coumaric acid, and

phenolic compounds (syringaldehyde) were analyzed by high performance liquid

chromatography (HPLC) using Rezex ROA-Organic Acid H+ (8%) column (Phenomenex)

at 40 oC and a UV/UIS detector (SPD-20AV, Shimadzu). The eluent was 0.01 N H2SO4

at 0.6 ml/min. Furfural, HMF and phenolic compounds were detected at 280 nm.

Syringaldehyde was used as the standard for phenolic compounds. levulinic acid, ferulic

acid, p-coumaric acid were detected at 200 nm.

3.3. Results and discussion

3.3.1 Effects of individual inhibitors on ABE fermentation

Each inhibitor was added at 1.0 g/L into the P2 medium to test its effect on ABE

fermentation of C. beijerinckii strains BA101 and BA101-SV6. Figure 3.1 shows that all

tested inhibitors more or less inhibited the growth of C. beijerinckii. Among them,

syringaldehyde, ferulic acid, vanillin, and p-coumaric acid had significantly higher

inhibition effects. O.D. was only about 2.0 compared to around 9.0 in the control. These

four inhibitors also obviously blocked butanol production (Figure 3.1). Almost no

butanol was produced under the influence of 1.0 g/L of these inhibitors. Only 3.0~5.0 g/L

acids was produced (Figure 3.1). These four inhibitors were derived from partly

65
breakdown of lignin (Ezeji et al., 2008; Mussatto et al., 2001). According to these results,

it would be better to remove lignin from biomass before hydrolysis. The concentration of

furan derivatives is usually under 1.0 g/L in lignocellulosic hydrolysates (Lee et al.,

2011). Under this concentration, furfural and HMF did not inhibit C. beijerinckii.

Therefore, it is unnecessary to remove these two compounds in the detoxification

processes. Weak acids (formic acid and levulinic acid) also showed no obvious inhibition

effects at 1.0 g/L. The most toxic inhibitors for C. beijerinckii were phenolics

(syringaldehyde, ferulic acid, vanillin, and p-coumaric acid). Then, further studies were

done on different concentrations of these four toxic inhibitors (Figure 3.2). Even when

the concentration was as low as 0.25 g/L, syringaldehyde, ferulic acid, and vanillin could

still inhibit cell growth and butanol production (Figure 3.2). A possible explanation of the

toxicity of these inhibitors to cells is that they may destroy cell membrane permeability

(Ezeji et al., 2008). Therefore, these phenolic inhibitors were the focus in the following

detoxification processes.

Then, these inhibitors‘ effects in the fermentation of C. acetobutylicum strains

were also tested (Figure 3.3). C. acetobutylicum ATCC 824 showed more tolerance to

these 4 lignin-degraded inhibitors. Almost all OD values were close to the level of

control, around 5.0. Only formic acid and vanillin inhibited C. acetobutylicum ATCC 824

severely. However, C. acetobutylicum strains produced more acids than C. beijerinckii

strains (Figure 3.1). Usually, more than 3.0 g/L total acids could be produced by C.

acetobutylicum strains. Syringaldehyde and ferulic acid did not show severe inhibition to

butanol production compared to C. beijerinckii strains. Therefore, C. acetobutylicum

66
strains are more tolerant to these lignin-derived inhibitors than C. beijerinckii strains. So

C. acetobutylicum strains may perform better in ABE fermentation using lignocellulosic

hydrolysate as substrates. Further lowering the concentrations of those 4 lignin derived

inhibitors was tested on C. acetobutylicum ATCC 824 (Figure 3.2). For syringaldehyde,

ferulic acid and p-coumaric acid, when the concentration decreased, OD and butanol titer

increased. When the concentration was 0.25 g/L, there was almost no obvious inhibition

for these three inhibitors. But vanillin was still toxic to C. acetobutylicum ATCC 824

even at 0.25 g/L.

These 9 inhibitors effects on one mutant of C. tyrobutyricum ATCC 25755 (Yu et

al., 2011) were also investigated. This strain is engineered to overexpress

aldehyde/alcohol dehydrogenase 2 (adhE2) and CoA-transferase (ctfAB). adhE2 converts

butyryl-CoA to butanol while ctfAB converts acetate/butyrate to acetyl-CoA/butyryl-CoA.

The metabolic pathway of C. tyrobutyricum doesn‘t contain the shift from acidogenesis

phase to solventogenesis phase like C. acetobutylicum and C. beijerinckii. Figures 3.1

shows both C. acetobutylicum and C. beijerinckii still produced a lot of acids when their

butanol production was inhibited by some inhibitors. Therefore, these inhibitors may only

block the shift process from acidogenesis phase to solventogenesis phase. C.

tyrobutyricum is used to check this presumption since it doesn‘t contain this shift process.

Figure 1 shows the results. Its character is very similar to that of C. beijerinckii. Those

four lignin derived inhibitors, syringaldehyde, ferulic acid, vanillin and p-coumaric acid,

are still the most toxic to both cell growth and butanol production. Formic acid did not

inhibit cell growth but reduced butanol production. Compared to C. beijerinckii, C.

67
tyrobutyricum showed some resistance to ferulic acid and p-coumaric acid. It can still

produce about 2.0 g/L butanol in the presence of 1.0 g/L of each inhibitor. So those four

most toxic inhibitors may work on a very complex mechanism. They don‘t just inhibit the

shift process, but they can inhibit both cell growth and butanol production while they

have little effect on the acid production.

3.3.2 Effects on fermentation kinetics

The inhibitors' effects on fermentation kinetics were further investigated. Figure

3.3 shows the fermentation kinetics of C. beijerinckii BA101 with different inhibitors.

The kinetics with 1.0 g/L furfural or 1.0 g/L formic acid was very similar to the control

without any inhibitor. The OD increased very rapidly to about 6.5 within the first 24 h,

became relatively stable and then decreased a little in the end of the fermentation. The

OD with 1.0 g/L formic acid was a little lower than the control. It could only reach

around 5.5, indicating that 1.0 g/L formic acid was a little toxic to cells and growth.

Glucose was consumed about 35 g/L in the first 48 h and then leveled off.

Correspondingly, butanol increased very fast in the first 48 h and then leveled off. 1.0

g/L furfural gave a higher butanol titer than the control. 1.0 g/L vanillin and 0.5 g/L

ferulic acid were very toxic to C. beijerinckii BA101. The fermentation ended very early.

The OD almost didn't increase after 24 h and could only reach to ~3.0, less than 50% of

the control. The consumption of glucose also leveled off after 24 h. The concentrations of

acids and solvents became stable very fast and did not change afterward which means C.

beijerinckii BA101 cannot detoxify these two inhibitors by itself during the fermentation.

68
 For C. acetobutylicum ATCC 824, the fermentation kinetics with 1.0 g/L furfural

is similar to the control (Figure 3.4) and gave a slightly higher butanol titer. Different

from C. beijerinckii BA101, 1.0 g/L formic acid (Figure 7c) was toxic to C.

acetobutylicum ATCC 824. The OD leveled off at around 3.5 after 24 h, only 70% of the

control (5.0). The butanol titer could only reach 7.0 g/L, also about 70% of the control

(10.5 g/L). Similar to C. beijerinckii BA101, with 1.0 g/L vanillin, all parameters, such as

the OD, the glucose concentration and the product concentrations, became stable after 24

h. There was no sign of self-detoxification. But C. acetobutylicum ATCC 824 could

tolerate 0.5 g/L ferulic acid very well. The OD increased to around 3.5 after 24 h. From

24 h to 48 h, the OD was relatively stable. From 48 h to 72 h, the OD increased again to

around 6.0 and then leveled off again. There was also a large increase in the butanol

concentration between 48 h and 72 h. So C. acetobutylicum ATCC 824 could tolerate 0.5

g/L ferulic acid, but it needs time to build up the tolerance. The fermentation kinetics of

C. tyrobutyricum ATCC25755 mutant with 1.0 g/L vanillin and 0.5 g/L ferulic acid were

also studied. They inhibited cell growth and butanol production from very beginning of

the fermentation. The concentrations of solvents and acids leveled off and did not change

after 48 h, which means the fermentation stopped due to the toxicity of these inhibitors.

With 1.0 g/L vanillin, the OD of C. tyrobutyricum ATCC25755 mutant was only about

3.0. The final butanol titer was less than 1.0 g/L. About 2.0 g/L butyric acid and 1.0 g/L

acetic acid were produced at the end of the fermentation. Less than 10 g/L glucose was

consumed during the fermentation. With 0.5 g/L ferulic acid added in the medium, the

OD of C. tyrobutyricum ATCC25755 mutant was only about 4.0. The final butanol titer

69
was about 2.0 g/L. About 3.8 g/L butyric acid and 1.7 g/L acetic acid were produced at

the end of the fermentation. About 30 g/L glucose was consumed during the fermentation.

This indicates that C. tyrobutyricum ATCC25755 mutant has some tolerance to 0.5 g/L

ferulic acid, but has almost no resistance to 1.0 g/L vanillin.

The fermentation kinetics confirm the end-point results of the first part. Lignin

degraded inhibitors (vanillin and ferulic acid) were the most toxic to C. acetobutylicum,

C. beijerinckii and C. tyrobutyricum strains. They inhibited the whole fermentation

process from the very beginning of the fermentation.

3.3.3 Effects on butanol dehydrogenase and butyraldehyde dehydrogenase

From the fermentation results in the first two parts, the lignin derived phenolic

inhibitors (syringaldehyde, ferulic acid, vanillin, and p-coumaric acid) are the most toxic

ones to these three Clostridium strains. C. beijerinckii BA101 and C. tyrobutyricum

ATCC25755 mutant could hardly grow with 1.0 g/L each of these four inhibitors. But

some cells could still survive under this condition. For C. beijerinckii BA101, the ODs

were 20% of the control; For C. tyrobutyricum ATCC25755 mutant, the ODs were 45%

of the control. These cells produced a lot of acids, 3.0-5.0 g/L acetic acid and butyric acid.

But the butanol titers were very low, less than 2.0 g/L. Therefore, the metabolic pathway

of acidogenesis (from glucose to acetyl-CoA and butyryl-CoA, then to acetic acid and

butyric acid) was fine in these survived cells. But the metabolic pathway of

solventogenesis (from acetyl-CoA and butyryl-CoA to ethanol and butanol) was partly

blocked. So these inhibitors may inhibit the activities of two dehydrogenases that convert

70
butyryl-CoA to butanol. These two dehydrogenases are butanol dehydrogenases and

butyraldehyde dehydrogenase.

In this part, the effects of individual inhibitors on these two dehydrogenases were

investigated. Total six inhibitors were studied, two sugar degraded inhibitors (furfural

and formic acid) and four lignin derived inhibitors (syringaldehyde, ferulic acid, vanillin,

and p-coumaric acid). First, 1.0 g/L of each inhibitor was added directly into the enzyme

assays to test their in-vitro effects (due to the pH, corresponding sodium salts were added

for those acid). The results are shown in Table 3.1. Among those three Clostridium

strains, C. beijerinckii BA101 and C. tyrobutyricum ATCC25755 mutant had higher

butanol dehydrogenase activity (0.060 U/mg). The butanol dehydrogenase activity in C.

acetobutylicum ATCC824 was lower (0.022 U/mg). When 1.0 g/L furfural or formic acid

was added, the butanol dehydrogenase activity was only a little lower than the control.

Among those three strains, the butanol dehydrogenase activity of C. beijerinckii BA101

decreased the most, only 50% of the control. When those four lignin degraded inhibitors

were added, no butanol dehydrogenase activity was detected in all three strains, which

means these four inhibitors can strongly inhibit butanol dehydrogenase. For

butyraldehyde dehydrogenase, similar trends can be found. When furfural or formic acid

was added, the butyraldehyde dehydrogenase activities were slightly lower than the

control. There was one exception. No butyraldehyde dehydrogenase activity was detected

in C. acetobutylicum ATCC824 when 1.0 g/L formic acid was added. So 1.0 g/L formic

acid can strongly inhibit butyraldehyde dehydrogenase in C. acetobutylicum ATCC824.

This can explain why 1.0 g/L formic acid inhibited butanol production severely during

71
fermentation. When the four lignin derived inhibitors were added, almost no

butyraldehyde dehydrogenase activities was detected in those three strains. There was

also one exception. Butyraldehyde dehydrogenase still showed activity (0.25 U/mg, 63%

of the control) in C. beijerinckii BA101 with 1.0 g/L p-coumaric acid.

During the in-vitro tests, the enzyme protein was directly exposed to the inhibitor

at 1.0 g/L. But in the real fermentation, due to the protection of cell membrane and other

metabolic pathways, even 1.0 g/L inhibitor was added in the medium, the

dehydrogenases may not be exposed to 1.0 g/L inhibitor. Therefore, the enzymes were

extracted after 24 h fermentation with inhibitors and the in-vivo effects of inhibitors on

the two dehydrogenases were investigated. The results are shown in Table 3.2. With 1.0

g/L furfural or formic acid, the butanol and butyraldehyde dehydrogenase activities were

almost the same as the control in all three strains. But for C. acetobutylicum ATCC 824,

there was still no butyraldehyde dehydrogenase activity with formic acid, which further

proves that formic acid can strongly inhibit the dehydrogenase activity in C.

acetobutylicum ATCC 824. For those four lignin-degradation inhibitors, better than

those in Table 1, some strains still showed butanol and butyraldehyde dehydrogenase

activities. For C. beijerinckii BA101, most of them still totally inhibited butanol and

butyraldehyde dehydrogenase activities. But with ferulic acid or p-coumaric acid,

butyraldehyde dehydrogenase still showed 10% of the activity. For C. acetobutylicum

ATCC 824, three of them (syringaldehyde, ferulic acid and p-coumaric acid) showed

almost no effects on butanol dehydrogenase except for vanillin which still totally

inhibited the activity. This is one of the reason why C. acetobutylicum ATCC 824

72
showed better tolerance to the lignin-degradation inhibitors than the other two. However,

syringaldehyde and vanillin still totally inhibited butanol dehydrogenase while ferulic

acid and p-coumaric acid only partly inhibited butanol dehydrogenase. For C.

tyrobutyricum ATCC 25755 mutant, all of them still showed butanol dehydrogenase and

butyraldehyde dehydrogenase activities, but lower than the control. Syringaldehyde

inhibited butyraldehyde dehydrogenase most (25% of the control). p-Coumaric acid and

vanillin inhibited butanol dehydrogenase most (23% of the control). Butanol

dehydrogenase and butyraldehyde dehydrogenase have better tolerance to lignin-derived

inhibitors, maybe because they are overexpressed enzymes and unregulated by the cell.

3.4. Conclusion

Sugar-degradation inhibitors (furfural, HMF, formic acid and levulinic acid) are

not toxic to C. beijerinckii, C. acetobutylicum and C. tyrobutyricum strains. On the

contrary, lignin derived phenolic inhibitors (syringaldehyde, vanillin, ferulic acid and p-

coumaric acid) are very toxic to Clostridia strains. Their toxicity has two aspects. First,

they can destroy the cell membrane's permeability and lead to cell death. The very low

OD in the fermentation is due to the death of cells. Second, they can strongly inhibit

butanol and butyraldehyde dehydrogenases. So the Clostridia strains can only produce a

lot of acids, but cannot convert them to butanol.

73
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 Table 3.1 The activities of butanol dehydrogenase and butyraldehyde dehydrogenase with 1.0 g/L inhibitors in the enzyme

assay

Butanol dehydrogenase (U/mg) Butyraldehyde dehydrogenase (U/mg)

Inhibitor
BA101 824 C. tyro BA101 824 C. tyro

None 0.060±0.016 0.022±0.011 0.060±0.006 0.46±0.15 0.032±0.016 0.040±0.014

Furfural 0.045±0.012 0.018±0.011 0.055±0.009 0.40±0.11 0.020±0.008 0.029±0.009

Formic acid 0.032±0.008 0.011±0.004 0.045±0.013 0.35±0.09 - 0.031±0.003

Syringaldehyde - - - - - -
82

Ferulic acid - - - - - -

p-Coumaric acid - - - 0.25±0.09 - -

Vanillin - - - - - -
 Table 3.2 The activities of butanol dehydrogenase and butyraldehyde dehydrogenase with 1.0 g/L inhibitors in fermentation

medium

Butanol Dehydrogenase (U/mg) Butyraldehyde Dehydrogenase (U/mg)

Inhibitor
BA101 824 C. tyro BA101 824 C. tyro

None 0.040±0.015 0.016±0.006 0.060±0.005 0.20±0.10 0.016±0.010 0.020±0.010

Furfural 0.050±0.010 0.017±0.008 0.050±0.009 0.30±0.15 0.018±0.008 0.020±0.015

Formic acid 0.038±0.011 0.014±0.005 0.055±0.008 0.15±0.08 - 0.015±0.010

Syringaldehyde - 0.022±0.005 *0.023±0.011 - - 0.005±0.003

Ferulic acid - 0.010±0.003 *0.034±0.005 0.016±0.010 0.009±0.003 0.008±0.004

p-Coumaric acid - 0.017±0.001 *0.014±0.006 0.016±0.009 0.008±0.005 0.009±0.005

83

Vanillin - - *0.014±0.008 - - 0.018±0.005

* Significantly different from the control (no inhibitor)

 10
BA101
8 824
C.tyro
6
O.D.
4

12
Butanol (g/L)

6
Acids (g/L)

Figure 3.1 Optical densities, butanol and acids production of three Clostridium spp.with

1.0 g/L different inhibitors after 72 hr.

84
 10
BA101 824 C. tyro
8
1.0 g/L
0.5 g/L
O.D.

6
0.25 g/L
4

12
10
8
Butanol (g/L)

6
4
2
0

6
Acids (g/L)

Figure 3.2 The fermentation results of C. beijerinckii BA101-SV6, C. acetobutylicum

ATCC 824 and C. tyrobutyricum ATCC 25755 mutant after 72 h with different

concentrations of phenolic inhibitors.

85
 60 10 60 12

Products (g/L), OD600

Products (g/L), OD600

50 50 10
8
Control Furfural
Glucose (g/L)

Glucose (g/L)
40 40 8
6
30 30 6
4
20 20 4

2
10 10 2

0 0 0 0
0 40 80 120 0 40 80 120
Time (h) Time (h)

60 10 60 10

Products (g/L), OD600

Products (g/L), OD600

50 50
8 8
Formic acid Vanillin
Glucose (g/L)

Glucose (g/L)
40 40
6 6
30 30
4 4
20 20

2 2
10 10

0 0 0 0
0 40 80 120 0 40 80 120
Time (h) Time (h)

60 10
Products (g/L), OD600

50
8
Ferulic acid
Glucose (g/L)

40
6
30
4
20

2
10

0 0
0 40 80 120
Time (h)

Figure 3.3 C. beijerinckii BA101 Fermentation Kinetics with different inhibitors. (1.0 g/L

furfural, 1.0 g/L formic acid, 1.0 g/L vanillin, 0.5 g/L ferulic acid)

86
 60 12 60 12

Products (g/L), OD600

Products (g/L), OD600

50 10 50 10
Control
Furfural
Glucose (g/L)

Glucose (g/L)
40 8 40 8

30 6 30 6

20 4 20 4

10 2 10 2

0 0 0 0
0 40 80 120 0 40 80 120
Time (h) Time (h)

60 12
Products (g/L), OD600 60 12

Products (g/L), OD600

50 10 50 10
Formic acid Vanillin
Glucose (g/L)

40 8 Glucose (g/L) 40 8

30 6 30 6

20 4 20 4

10 2 10 2

0 0 0 0
0 40 80 120 0 40 80 120
Time (h) Time (h)

60 12
Ferulic acid
Products (g/L), OD600

50 10
Glucose (g/L)

40 8

30 6

20 4

10 2

0 0
0 40 80 120
Time (h)

Figure 3.4 C. acetobutylicum ATCC 824 Fermentation Kinetics with different inhibitors.

(1.0 g/L furfural, 1.0 g/L formic acid, 1.0 g/L vanillin, 0.5 g/L ferulic acid)

87
 60 10 60 10
Control

Products (g/L), OD600

Products (g/L), OD600

50 50
Glucose (g/L) 8 8

Glucose (g/L)
40 40
6 6
30 30
Vanillin
4 4
20 20

2 2
10 10

0 0 0 0
0 40 80 120 0 40 80 120
Time (h) Time (h)

60 10

50
Products (g/L), OD600
8
Ferulic acid
Glucose (g/L)

40
6
30
4
20

2
10

0 0
0 40 80 120
Time (h)

Figure 3.5 C. tyrobutyricum ATCC 25755 mutant fermentation kinetics with different

inhibitors. (1.0 g/L vanillin, 0.5 g/L ferulic acid)

88
 Chapter 4: The impact of acid and alkali pretreatments on lignocellulosic biomass

and the following acetone-butanol-ethanol fermentation

Abstract
Butanol is a promising biofuel that has a higher energy density than ethanol and

similar octane number with gasoline. Producing butanol from microbial fermentation is a

sustainable energy generating strategy. But one challenge is to find more economical

substrates to reduce the total cost of butanol fermentation. Lignocellulosic biomass is a

type of carbon source that's abundant in nature. The cellulose and hemicellulose contents

in it can be hydrolyzed into sugars. So it's a potential cheap substrate for butanol

fermentation. In this work, four lignocellulosic biomass, cotton stalk, corn fiber, soybean

hull and sugarcane bagasse, were pretreated with acid and then hydrolyzed by enzymes.

The obtained hydrolysates were further detoxified to remove the inhibitors in them. The

detoxified hydrolysates were then used as carbon source in Acetone-Butanol-Ethanol

(ABE) fermentation of Clostridium. All hydrolysates gave very high butanol production.

The acid and alkali pretreatments were also compared. Their effects on biomass

compositions and subsequent fermentation were also studied.

Keywords: butanol; lignocellulosic biomass; acid pretreatment; detoxification; inhibitors

89
4.1 Introduction

Butanol, a four carbon alcohol, can be used as fuel or fuel additives. It has higher

energy than ethanol and can be directly used by most automobile engines without any

modification (Jones et al., 1986). Moreover, butanol can be produced from microbial

fermentation which is a more sustainable way to generate energy. However, the cost of

butanol fermentation needs to be reduced in order to make it competitive with

petrochemical fuels. The substrate cost accounted for over 56% of the butanol production

cost (Ezeji et al., 2008). Therefore, choosing cheaper substrates can dramatically reduce

the production cost. Lignocellulosic biomass, such as wood, grass, and agriculture

residues, are very cheap and abundant in nature. So it is a promising substrate for butanol

fermentation.

Extensive research has been done to evaluate lignocellulosic biomass as feedstock

for fermentation. Corn fiber is a by-product of the maize wet milling process. Qureshi et

al studied it as a substrate to produce butanol after hydrolysis (Qureshi et al., 2007), and

obtained 9.3 g/L total ABE (Acetone, butanol and ethanol) was produced. Qureshi et al

also used wheat straw as feedstock to produce butanol, and 25.0 g/L ABE was obtained

(Qureshi et al., 2010a). Moreover, Qureshi et al investigated barley straw (Qureshi et al.,

2010b), maize stover and switchgrass (Zverlov et al., 2006). Thaddeus et al (2008) chose

distillers dry grains and soluble (DDGS) as substrate and compared it with different

mixed streams of pure sugars. The highest ABE yield in Thaddeus et al‘s work was 12.1

g/L. Instead of feed-grade biomass, Zverlov et al investigated agriculture wastes,

including hemp waste, corn cobs and sunflower shells (Qureshi et al., 2008).

90
 In this study, four different kinds of lignocellulosic biomass were investigated.

Acid pretreatments were first applied to these biomass. The effects of different acid

concentrations on the final sugar yield were studied. Inhibitors in the hydrolysates were

also measured. Detoxification by activated carbon was also used to remove the inhibitors

in the hydrolysates. Finally, acid and alkali pretreatments followed with removing the

supernatant before enzymatic hydrolysis were compared, including their influence on

biomass compositions and subsequent fermentation.

4.2 Materials and methods

4.2.1 Strain and inoculum preparation

Spores of C. beijerinckii BA101, C. acetobutylicum ATCC 824, C. tyrobutyricum

were stored in a refrigerator at 4 oC in the Clostridia Growth medium. Spores (2 ml) were

heat-shocked at 80 oC for 3 min and transferred to 50 ml RCM growth medium (Difco

Reinforced Clostridia Medium, Becton, Dickinson and Company, MD, USA) in a 125 ml

serum bottle. The medium was nitrogen-purged for 8 min to remove oxygen. The serum

bottle was tightly capped with a rubber stopper and aluminum seal. The mixture was

autoclaved at 121 oC for 30 min followed by cooling to 37 oC. The heat-shocked spores

were incubated at 37 oC for 12-16 h until cells were highly active. The active culture (5%

inoculum) was used as seed culture for all fermentations studied.

4.2.2 ABE fermentation in serum bottles

ABE fermentation was studied in serum bottles each containing 50 ml medium to

evaluate the effects of various detoxification methods on butanol production. Unless

91
otherwise noted, all fermentation studies were carried out with the P2 medium containing

one type of inhibitors, glucose (60 g/L), yeast extract (2 g/L), buffer (0.5 g/L KH2PO4

and 0.5 g/L K2HPO4), 2.2 g/L ammonium acetate, vitamins (0.001 g/L para-amino-

benzoic acid (PABA), 0.001g/L thiamin and 10-5 g/L biotin), and mineral salts (0.2 g/L

MgSO4∙7H2O, 0.01 g/L MnSO4∙H2O, 0.01 g/L FeSO4∙7H2O, 0.01 g/L NaCl).The carbon

source solution (glucose or lignocellulose hydrolysates) and P2 stock solution (containing

yeast extract, ammonium acetate and buffer, 10-fold concentrated) were autoclaved

separately at 121oC and 15 psig for 30 min for sterilization. Minerals and vitamins were

prepared at 200-fold and 1000-fold concentration, respectively, and were filter-sterilized

through 0.2 μm membrane filters (25mm syringe filter, Fisher brand, NJ, USA). Before

sterilization, the P2 solutions in serum bottles were purged with nitrogen for 8 min. After

sterilization, an appropriate amount of the P2 stock solution was aseptically added into

the serum bottle, followed by addition of minerals and vitamins. To ensure the medium

pH maintained around 5.0 throughout the fermentation, 2 g/L of CaCO3 was also

included in the medium. Actively grown cells were inoculated at 5% (v/v). Batch

fermentation was performed at 37oC with no agitation. Samples were taken periodically

for analysis of ABE production.

4.2.3 Hydrolysis of biomass

Four lignocellulosic biomasses were hydrolyzed: cotton stalk, sugarcane bagasse

(Obtained from South China University of Technology), soybean hull (Minnesota Soybean

Processors) and corn fiber (Cargill Inc.). 50 g or 100 g solid particles were suspended in

1.0 L in different concentrations of acid solution (HCl or H2SO4) or just distilled water.
92
Autoclave at 121 oC, 15 psig for 30 min. Then adjust their pH to 5.0 by NaOH. Add 3.0

g CTec2 (Novozymes) and hydrolyze at 50 oC for 72 h. Centrifuge at 7000 rpm, 25 oC for

15 min to remove the undissolved solid. The supernatant hydrolysate was concentrated

by vacuum rotary evaporation at 60 oC to remove ~670 mL water. The concentrated

hydrolysate was filter-sterilized through 0.2 μm membrane filters (25 mm syringe filter,

Fisher brand, NJ, USA) and stored at 4 oC for further use.

For the processes that remove the acid pretreatment supernatant, 50 g solid particles

were suspended in 1.0 L in 0.04 N HCl or H2SO4. Autoclave at 121 oC, 15 psig for 30

min. Centrifuge at 7000 rpm, 25 oC for 15 min and discard the supernatant. Resuspend

the solid in 1.0 L distilled water. Centrifuge again at 7000 rpm, 25 oC for 15 min and

discard the supernatant. Repeat this washing process twice until the pH of the supernatant

had reached about 5.0. Then resuspend the solid in 330 mL distilled water. Take out 5

mL samples for composition analysis. Add 3.0 g CTec2 (Novozymes) into the rest and

hydrolyze at 50 oC for 72 h. Centrifuge at 7000 rpm, 25 oC for 15 min to remove the

undissolved solid. The supernatant hydrolysate was filter-sterilized through 0.2 μm

membrane filters (25 mm syringe filter, Fisher brand, NJ, USA) and stored at 4 oC for

further use.

For the processes that remove the alkali pretreatment supernatant, 50 g solid

particles were suspended in 1.0 L in 0.04 N NaOH. Keep at 50 oC for 60 min under

stirring. Then wash the solid three times by the same method in the acid pretreatment

until the pH of supernatant reached about 8.0. Resuspend the solid in 330 mL distilled

93
water, adjust the pH to about 5.0 with H2SO4. The enzymatic hydrolysis was the same as

in the above acid pretreatment.

4.2.4 Detoxification of hydrolysate

The pH of hydrolysate was first adjusted to 2.0 with H2SO4. Then 2% (w/w)

activated carbon was added into the hydrolysate and incubated at pH 2.0, 80 oC for 60

min under stirring. Remove activated carbon by filter paper. Adjust pH back to 6.5 by

NaOH. Centrifuge them at 8000 rpm, 25 oC for 15 min. Remove the sediments and keep

the clear hydrolysate at 4 oC for further use.

4.2.5 Analytical methods

The compositions of biomass were determined by the methods described by Foster

et al. The fermentation products, acetone, butanol, ethanol, acetic acid, and butyric acid

were analyzed with a gas chromatograph (GC, Shimadzu GC-2014) equipped with a

flame ionization detector and a 30-m fused silica column (0.25 µm film thickness and

0.25 mm ID, Stabilwax-DA). The carrier gas was nitrogen at 1.47 ml/min (linear velocity:

35 cm/s). Samples were diluted 20 times with an internal standard buffer solution

containing 0.5 g/L isobutanol, 0.1 g/L isobutyric acid and 1% phosphoric acid (for

acidification), and injected (1 μL each) by using an auto-injector (AOC-20i Shimadzu).

The column temperature was held at 80 oC for 3 min, raised to150 oC at a rate of 30
o
C/min, and held at 150 oC for 3.7 min. Both the injector and detector were set at 250 oC.

Cell density was analyzed by measuring the optical density (OD) of cell suspension

at 600 nm using a spectrophotometer (Shimadzu, Columbia, MD, UV-16-1). The

94
inhibitors including furfural, HMF, levulinic acid, ferulic acid, p-coumaric acid, and

phenolic compounds (syringaldehyde) were analyzed by high performance liquid

chromatography (HPLC) using Rezex ROA-Organic Acid H+ (8%) column (Phenomenex)

at 40 oC and a UV/UIS detector (SPD-20AV, Shimadzu). The eluent was 0.01 N H2SO4

at 0.6 ml/min. Furfural, HMF and phenolic compounds were detected at 280 nm.

Syringaldehyde was used as the standard for phenolic compounds. levulinic acid, ferulic

acid, p-coumaric acid were detected at 200 nm.

Acid and alkali pretreated and washed biomass were dried at 120 oC for 24 h. Then

the biomass were coated with gold under vacuum. SEM images were taken with Quanta

200.

4.3 Results and discussion

4.3.1 Dilute acid pretreatment with different concentrations of acids

The compositions of cotton stalk, sugarcane bagasse, soybean hull and corn fiber

were first analyzed. These four are typical lignocellulosic biomass, their main

compositions are cellulose, hemicellulose and lignin. Cellulose accounted for 30%-44%,

hemicellulose was 9%-18% and lignin was 13%-28%. Among these four, cotton stalk

had the highest cellulose content (44%), lowest hemicellulose (9%) and lignin (13%)

content. The composition of soybean hull was very similar to cotton stalk, but it

contained much more lignin (28%) than cotton stalk. Compared to cotton stalk and

soybean hull, sugarcane bagasse and corn fiber had less cellulose (~30%), but more

hemicellulose (18%). Sugarcane bagasse contained more lignin (28%) than corn fiber

95
(18%). Cellulose and hemicellulose can be converted into monosaccharides, which can

be used as carbon source in the fermentation. Therefore, cellulose and hemicellulose are

the useful carbon sources for us. Ideally, these four lignocellulosic biomasses can release

48%-53% total sugars. Lignin cannot be used by Clostridium strains. Its degradation

may even produce some inhibitory compounds to Clostridium strains. So high content

of lignin (sugarcane bagasse and soybean hull) may be bad in the following hydrolysis

and fermentation processes.

Cotton stalk, sugarcane bagasse, soybean hull and corn fiber were first pretreated

with different concentrations of acids and then hydrolyzed by cellulase enzyme (Ctec2,

Novozymes). The sugar yields are shown in Table 4.2. All four have similar trends.

Increase the HCl concentration from 0 to 0.3 N, the sugar yields gradually increased.

Only hemicellulose can release xylose, while cellulose can only produce glucose.

Therefore, those that contained more hemicellulose (sugarcane bagasse and corn fiber)

released more xylose. Cotton stalk ideally can release 53% total sugars. Increasing HCl

concentration only increased the sugar yield from 0.35 g/g to 0.40 g/g. Increasing the HCl

concentration helped break down more hemicellulose and release more xylose. The

glucose and xylose ratio decreased from 6:1 to 4:1. HCl and H2SO4 showed no difference

on sugar yield at 0.04 N. Sugarcane bagasse ideally can release 48% total sugars.

Increasing HCl concentration increased the sugar yield from 0.20 g/g to 0.43 g/g, almost

reaching its highest yield 0.48 g/g. Increase HCl concentration from 0.04 N to 0.3 N, the

glucose production also increased from 21.7 g/L to 36.0 g/L, but xylose production

decreased from 11.4 g/L to 6.8 g/L. The glucose and xylose ratio also increased from 2:1

96
to 6:1. This means xylose began to degrade at high HCl concentration. HCl and H2SO4

showed great difference on sugar yield at 0.04 N. The yield with H2SO4 (0.23 g/g) was

much lower than that with HCl (0.33 g/g). For corn fiber, the sugar yield decreased a

little bit (from 0.60 g/g to 0.58 g/g) with increasing HCl concentration. The glucose and

xylose ratio was around 1:1. The xylose degradation was not as bad as in sugarcane

bagasse. The xylose production decreased from 29.1 g/L to 25.0 g/L. HCl and H2SO4

showed a little difference on sugar yield at 0.04 N. The yield with H2SO4 (0.53 g/g) was

lower than that with HCl (0.60 g/g). For soybean hull, increasing HCl concentration also

increased the total sugar yield from 0.50 g/g to 0.57 g/g. The glucose and xylose ratio

was around 3:1. The xylose degradation was not obvious in soybean hull. The xylose

production still increased from 11.6 g/L to 15.0 g/L with increasing the HCl

concentration. 0.04 N HCl gave much better sugar yield than 0.04 N.

The inhibitors' concentrations after acid pretreatments are shown in Table 4.3.

There were 6 types of inhibitors produced after acid pretreatments. Three came from

sugar degradation: furfural, HMF and formic acid; the other three came from lignin

degradation: ferulic acid, p-coumaric acid and other phenolic compounds (Ezeji et al.,

2008; Lee et al., 2011). Generally, increase the acid concentration, the concentrations of

these 6 inhibitors also increased. But the concentrations of these 6 inhibitors varied with

different biomass. Formic acid was the most easily released inhibitor. It was generated

even just using hot water pretreatment. Compared 0.04 N HCl with 0.04 N H2SO4, 0.04

N HCl tended to produce more formic acid except in soybean hull. The previous study

(Chapter 3) showed that furfural, HMF and formic acid didn't inhibit the ABE

97
fermentation of Clostridium strains under 1.0 g/L. On the other hand, ferulic acid and p-

coumaric acid are very toxic to Clostridium strains, they can strongly inhibit the ABE

fermentation even at 0.25 g/L. Therefore, when choosing which acid and which

concentration are best for each biomass, both Table 4.2 and 4.3 need to be considered.

Higher sugar yield is better because this can reduce the substrate cost, but lower

inhibitors' level is also needed to have a good butanol production. For cotton stalk, the

ferulic acid level exceeded 0.25 g/L even at 0.04 N HCl and the sugar yield didn't

increase a lot by increasing HCl concentration (from 0.35 g/g to 0.40 g/g). So it's better

just use hot water pretreatment for cotton stalk. For sugarcane bagasse, the ferulic acid

level didn't reach 0.25 g/L at 0.04 N HCl. At higher HCl concentrations or 0.04 N H2SO4 ,

ferulic acid all exceeded 0.25 g/L level. On the other hand, the sugar yield with hot water

pretreatment was too low (0.20 g/g). So 0.04N HCl was chosen to pretreat sugarcane

bagasse later. For corn fiber, the concentrations of formic acid and ferulic acid were very

high even at 0.04 N HCl, 1.72 g/L and 2.38 g/L respectively. On the contrary, with 0.04

N H2SO4, there was no ferulic acid detected. The sugar yield with 0.04 N H2SO4 (0.63 g/g)

was similar to that with 0.04 N HCl (0.60 g/g). So 0.04 N H2SO4 was chosen to pretreat

corn fiber. For soybean hull, the p-coumaric acid level exceeded 0.25 g/L at 0.1 N HCl.

Among hot water, 0.04 N HCl and 0.04 N H2SO4, 0.04 N HCl has the highest sugar yield

(0.50 g/g). Therefore, 0.04 N HCl was used as the pretreatment for soybean hull in the

following research. Different biomass released different inhibitor levels even under the

same pretreatment conditions. The main reason may be that they have different lignin

98
compositions and structures. For these four biomass, 0.04 N acid level was enough to

obtain high sugar yields with low inhibitors' concentration.

4.3.2 Detoxification and ABE fermentation using the hydrolysate

The hydrolysates obtained by the optimized pretreatments were to be used in ABE

fermentation. In order to increase the sugar concentration further, the hydrolysis process

was modified. Initial 50 g/L biomass loading was used instead of 100 g/L, then the

hydrolysate was further concentrated to remove 2/3 water. So the equivalent initial dry

biomass loading was 150 g/L. The sugar yields are shown in Table 4.4. The sugar yield

was the same as in Table 4.2, which means there was no sugar degradation during the

vacuum concentration process. The initial sugar concentrations were in the range of 52.7

g/L-84.4 g/L, enough to provide sufficient carbon source in the ABE fermentation. The

fermentation results of C. acetobutylicum ATCC824 using these hydrolysates as carbon

sources are shown in Table 4.5. Only sugarcane bagasse and cotton stalk gave relatively

good butanol production, 7.3 g/L and 8.4 g/L, respectively. Soybean hull only gave 3.0

g/L butanol titer. The butanol production in corn fiber hydrolysate was totally blocked.

But soybean hull and corn fiber still produced a lot of acids, 14.1 g/L and 20.9 g/L,

respectively. Only butanol production was inhibited. The inhibitors' concentrations in

these hydrolysates were also tested and are given in Table 4.5. Due to the post-hydrolysis

concentration, some inhibitors' levels increased to above 0.25 g/L. Ferulic acid

concentrations in sugarcane bagasse and soybean hull were 0.29 g/L and 0.32 g/L,

respectively. This can explain why butanol production in sugarcane bagasse and soybean

hull were partly blocked and lower than the control using pure glucose. In corn fiber

99
hydrolysate, no ferulic acid was detected, but it contained 1.71 g/L formic acid and 0.19

g/L phenolic compounds. These two together might have caused the total block of

butanol production. The inhibitors' levels in cotton stalk hydrolysate were all below the

toxic levels. That's why it had the best butanol production among these four hydrolysates.

Activated carbon adsorption is efficient in removing these toxic inhibitors and

detoxifying the hydrolysates (Lu et al., 2013). After activated carbon adsorption, the

concentrations of inhibitors in the hydrolysates are also shown in Table 4.6. Activated

carbon was not very good at adsorbing formic acid. Formic acid in cotton stalk

hydrolysate even increased a little bit after the adsorption. This might be due to the side

reactions during the adsorption. However, activated carbon worked very well in

adsorbing ferulic acid and other phenolic compounds. It could remove all ferulic acid in

cotton stalk, sugarcane bagasse and soybean hull hydrolysates. All other phenolic

compounds in sugarcane bagasse hydrolysate were also removed. 42% in corn fiber were

also removed. In the following fermentation studies, cotton stalk hydrolysates were used

directly while the other three hydrolysates were detoxified first by activated carbon

absorption.

The fermentation results using these hydrolysates are shown in Table 4.7. Three

strains were tested: C. acetobutylicum ATCC 824, C. beijerinckii BA101 and C.

tyrobutyricum ATCC 25755 mutant (knocking out Ack genes and overexpressing ctfAB

and adhE2 genes). Cotton stalk hydrolysate was the best carbon source. All three strains

produced similar butanol titers to the control and some were even a little higher. Corn

fiber hydrolysate gave the lowest butanol titer (5.4-6.1 g/L) in all three strains. As shown

100
in Table 4.6, corn fiber hydrolysate only contained 1.13 g/L formic acid and 0.11 g/L

phenolic compounds. There were two possible reasons for the low butanol titer; one was

that corn fiber hydrolysate contained some other inhibitors other than those 9 tested; the

other is that the 0.11 g/L phenolic compounds contained some very toxic inhibitors. The

other two hydrolysates had different performances in different strains. Soybean hull

hydrolysate gave very good butanol production in C. beijerinckii BA101 (10.0 g/L), but

the butanol titer in C. acetobutylicum ATCC 824 and C. tyrobutyricum ATCC 25755

mutant was lower (7.6 g/L). For sugarcane bagasse hydrolysate, the butanol titer in C.

acetobutylicum ATCC 824 was 9.6 g/L, but only 7.8 g/L and 7.9 g/L in C. beijerinckii

BA101 and C. tyrobutyricum ATCC 25755 mutant. So ferulic acid is a very toxic

inhibitor to these three strains but may not be the only inhibitor in the hydrolysates. The

fermentation kinetics of C. acetobutylicum ATCC 824 and C. beijerinckii BA101 are

shown in Figure 4.1 and Figure 4.2. In the first 24 h, only glucose was used for cell

growth and almost no xylose was consumed. After 24 h, both glucose and xylose were

consumed for acid and solvent production.

4.3.3 Comparison of acid and alkali pretreatment with removing supernatant before

enzymatic hydrolysis

The inhibitors mentioned above (furfural, HMF, formic acid, ferulic acid, p-

coumaric acid and other phenolic compounds) are mainly produced in the pretreatment

process. During the enzyme digestion, the pH (~5.0) and temperature (50 oC) were mild

so almost no inhibitor should be generated. The concentrations of the inhibitors were all

under 2.0 g/L (Table 4.6), so they could dissolve into water very well. This means the

101
inhibitors were contained in the liquor after the acid pretreatment. If the liquor containing

inhibitors was removed and the biomass solid was washed, then the hydrolysate obtained

after enzyme digestion should contain little or no inhibitor. Another common economical

pretreatment is using alkali (NaOH). In this part, the effects of these two pretreatments

(acid and alkali) on four biomass structures (cotton stalk, sugarcane bagasse, soybean hull

and corn fiber) and subsequent ABE fermentation were studied.

Table 4.8 shows the composition changes after acid or alkali pretreatment. some

of the biomass content was either broken up into short fragments and dissolved into water

or degraded further and produced the inhibitors. From Table 4.8, acid pretreatment could

remove a large portion of hemicellulose, part of lignin and only very small part of

cellulose. The hydrogen ion can catalyze the breakage of glycosidic bonds. It more easily

attacks the amorphous hemicellulose at low concentration. It can even break the

crystalline cellulose into fragments. At a high concentration, the hydrogen ion can break

crystal cellulose into glucose. It can also catalyze the breakage of ether bonds in lignin

and partly break the lignin structures. In this acid pretreatment, only 0.04 N HCl or 0.04

N H2SO4 was used. The concentration was very low so it mainly broke the hemicellulose

part in the biomass. 0.04 N H2SO4 removed 56% of hemicellulose and 31% of lignin in

cotton stalk. 0.04 N HCl removed 78% of hemicellulose and 7% of lignin in sugarcane

bagasse. It could also remove 90% of hemicellulose and 65% of lignin in soybean hull.

28% of cellulose, 61% of hemicellulose and 50% of lignin in corn fiber were removed by

0.04 N H2SO4. So 0.04 N hydrogen ions removed 56%-90% of hemicellulose and 7%-65%

of lignin in the biomass. Among these four biomass, the lignin in sugarcane bagasse was

102
the hardest to be broken, only 7% was removed. And the lignin in soybean hull was the

easiest to be broken, 65% of lignin was dissolved. The result of corn fiber is different

from the others. 28% of cellulose was also dissolved. Possible reason was that the

cellulose in corn fiber was not a rigid crystalline fiber. It has a large portion of

amorphous structures that can be attacked by the dilute acid. Based on the sugar yields,

the acid pretreatment promoted the enzymatic digestion of cellulose and hemicellulose

very well. Almost all cellulose and hemicellulose in the biomass were converted into

monosaccharides. Compared with acid pretreatment, alkali pretreatment dissolved less

hemicellulose, more lignin and almost no cellulose. In cotton stalk, 0.04 N NaOH

removed 44% of hemicellulose and 77% of lignin. It dissolved 44% of hemicellulose and

46% of lignin in sugarcane bagasse. It also removed 80% of hemicellulose and 48% of

lignin in soybean hull, and 16% of cellulose, 56% of hemicellulose and 78% of lignin in

corn fiber. So 0.04 N NaOH dissolved 44%-80% of hemicellulose and 46%-78% of

lignin in the biomass. The lignin in cotton stalk and corn fiber was more soluble in 0.04

N NaOH, which removed 77% and 78%, respectively. Less cellulose in corn fiber was

dissolved in NaOH, 16% compared with 28% in H2SO4. After alkali pretreatment, the

rest solid was digested by the enzyme very well. From the sugar yield, almost all

cellulose and hemicellulose were hydrolyzed into single sugars.

The SEM images of untreated, acid treated and alkali treated biomass were are

compared in Figure 4.3. There were no long fibrous structures in untreated cotton stalk.

That's why it had a relatively high sugar yield even just using hot water pretreatment.

After acid or alkali pretreatment, the structure only became more shattered. Untreated

103
corn fiber had a mesh structure because it contained a lot of hemicellulose (18%). The

hemicellulose formed branches from cellulose fiber and links them to form the grids.

After acid pretreatment, there were less branches and it became more like long fiber.

After alkali pretreatment, the structure was almost the sam. Untreated soybean hull had a

hollow fiber structure. After acid pretreatment, almost no hollow fiber could be seen from

the image. All the fiber structures were destroyed. After alkali pretreatment, some fibers

were still there but with scratches on the fiber, a sign of partly breakage of the fibrous

structure. There were still long fiber structures in untreated sugarcane bagasse. 78% of

hemicellulose was removed after acid pretreatment and the structure became very

amorphous. 44% of hemicellulose and 46% of lignin were removed after alkali

pretreatment, and the structure became more scattered and more powdered. Therefore,

either acid or alkali pretreatment can destroy the fibrous structures of biomass to some

extent, make the structure more fragmented, and promote the enzyme contact in the

hydrolysis process.

Removing the liquor and washing the acid or alkali pretreated biomass also

removed all the inhibitors produced in the pretreatment process. Actually no inhibitors

mentioned above (furfural, HMF, formic acid, ferulic acid, p-coumaric acid and other

phenolic compounds) were detected in the hydrolysate. This means all these inhibitors

were below 0.01 g/L and had almost no effects on the subsequent ABE fermentation.

Then the ABE fermentations of C. acetobutylicum ATCC824 using these hydrolysates as

carbon sources were performed and are compared with the performance using activated

carbon detoxified hydrolysates in Table 4.9. The situation was more complex than

104
expected. For the acid pretreated with removing supernatant, only cotton stalk and

soybean hull hydrolysates gave high butanol titers, 8.2 g/L and 8.4 g/L, respectively.

Butanol production in corn fiber and sugarcane bagasse hydrolysates were only 2.9 g/L

and 2.3 g/L. For the alkali pretreated with removing supernatant, the final butanol titers in

cotton stalk and corn fiber hydrolysates were high, 8.5 g/L and 8.6 g/L. Soybean hull and

sugarcane bagasse hydrolysates did not perform as well as the other two, only 5.1 g/L and

5.7 g/L butanol were produced. Therefore, there should be some components or new

produced chemicals in some of the biomass that can influence butanol production by C.

acetobutylicum ATCC824. Corn fiber hydrolysate only gave good butanol production

after the alkali pretreatment with removing supernatant. The possible reason is that the

alkali pretreatment remove most of lignin in corn fiber (78%) which contained most toxic

potential inhibitors for ABE fermentation.

4.4 Conclusion

Dilute acid pretreatment is an effective method to promote the enzymatic

hydrolysis of cellulose and hemicellulose in biomass. But high concentration of acids

catalyzed the further degradation of biomass components and generated large amount of

inhibitors to ABE fermentation, especially ferulic acid, exceeding the toxic level that

inhibited the butanol production, especially ferulic acid. For corn fiber, soybean hull and

sugarcane bagasse, 0.04 N was the best acid level. Activated carbon absorption was

effective in removing phenolic inhibitors in the hydrolysate, especially ferulic acid. After

the acid and alkali pretreatment, removing the liquor and washing the biomass could

105
removed all the inhibitors dissolved in the water, but there are still some other inhibitors

in the biomass that inhibited butanol production by ABE fermentation.

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113
 Table 4.1 Composition of various lignocellulosic biomass

Biomass Cellulose Hemicellulose lignin

Cotton stalk 44%±5% 9%±5% 13%±2%

Sugarcane bagasse 30%±5% 18%±7% 28%±2%

Soybean hull 40%±4% 10%±6% 23%±2%

Corn fiber 32%±4% 18%±5% 18%±1%

114
 Table 4.2 Sugar concentrations and yields from biomass with different pretreatment

conditions a

Glucose Xylose Yield

Pretreatment
(g/L) (g/L) (g/g dry biomass)
Hot water 29.8±0.5 5.3±0.2 0.35±0.01
0.04N HCl 29.5±0.6 5.5±0.1 0.35±0.02
Cotton Stalk 0.04N H2SO4 29.6±0.6 5.0±0.1 0.35±0.02
0.1N HCl 33.1±0.8 6.7±0.3 0.40±0.03
0.3N HCl 32.0±0.3 8.0±0.1 0.40±0.01
Hot water 15.0±0.4 5.1±0.1 0.20±0.01
0.04N HCl 21.7±0.3 11.4±0.5 0.33±0.03
Sugarcane 0.04N H2SO4 14.7±0.6 8.2±0.7 0.23±0.05
bagasse
0.1N HCl 25.0±0.8 10.2±0.2 0.35±0.02
0.3N HCl 36.0±0.6 6.8±0.5 0.43±0.03
Hot water 14.0±0.5 4.6±0.1 0.19±0.03
0.04N HCl 30.9±0.3 29.1±0.8 0.60±0.02
Corn fiber 0.04N H2SO4 30.8±0.2 22.1±0.3 0.53±0.01
0.1N HCl 31.2±0.1 27.4±0.3 0.59±0.02
0.3N HCl 33.0±0.8 25.0±0.1 0.58±0.02
Hot water 33.4±0.5 2.9±0.1 0.36±0.02
0.04N HCl 38.7±0.6 11.6±0.3 0.50±0.02
Soybean hull 0.04N H2SO4 15.4±0.6 7.2±0.4 0.23±0.04
0.1N HCl 39.0±0.8 12.3±0.2 0.51±0.02
0.3N HCl 41.6±0.2 15.0±0.5 0.57±0.01
a
The initial biomass loading was 100 g/L. The results were after enzymatic hydrolysis

115
 Table 4.3 Inhibitors present in the biomass hydrolysate with different acid pretreatments

Formi Ferulic p-Coumaric Pheno

Furfural HMF
Pretreatments c acid acid acid lic
(g/L) (g/L)
(g/L) (g/L) (g/L) (g/L)
Hot water - - 0.19 0.05 - -
0.04N HCl 0.10 - 0.80 0.51 - -
Cotton 0.04N H SO
2 4 0.07 - 0.75 0.47 0.01 -
Stalk
0.1N HCl 0.13 0.09 3.32 1.57 0.27 0.05
0.3N HCl 0.13 0.21 4.25 1.86 0.30 0.07
Hot water - - 0.11 - - -
Sugarca 0.04N HCl 0.20 - 0.73 0.19 - 0.12
ne 0.04N H2SO4 -- - 0.54 0.47 0.12 0.04
bagasse 0.1N HCl 0.60 0.30 1.02 2.29 0.32 0.08
0.3N HCl 0.59 0.71 1.54 2.71 0.60 0.09
Hot water 0.02 - 0.71 - - -
0.04N HCl 0.04 - 1.72 2.38 0.05 0.13
Corn 0.04N H2SO4 0.09 - 1.14 - - 0.13
fiber
0.1N HCl 0.15 0.84 3.00 2.14 0.44 0.17
0.3N HCl 0.15 1.12 4.45 3.57 0.72 0.17
Hot water - - 0.06 0.02 - -
0.04N HCl 0.06 0.52 0.21 0.16 - -
Soybea
0.04N H2SO4 0.01 - 0.65 0.12 - -
n hull
0.1N HCl 0.12 0.97 0.31 0.18 0.32 0.06
0.3N HCl 0.13 1.42 3.16 1.29 0.55 0.12
a
The initial biomass loading was 100 g/L. The results were after enzymatic hydrolysis.

116
 Table 4.4 Sugar concentrations and yields of the biomass after hydrolysis a

Yield
Biomass Pretreatment Glucose (g/L) Xylose (g/L)
(g/g dry biomass)

Corn fiber 0.04N H2SO4 43.7±2.9 40.7±1.0 0.53±0.03

Cotton stalk Hot Water 44.7±1.9 8.0±2.3 0.35±0.03

Sugarcane bagasse 0.04N HCl 32.5±0.1 17.1±3.5 0.33±0.02

Soybean hull 0.04N HCl 58.0±2.9 17.4±1.6 0.50±0.03

a
The initial biomass loading was 50 g/L, and the hydrolysate was concentrated by 300%.

Table 4.5 C. acetobutylicum ATCC824 fermentation results after 72 h using different

biomass w/o activated carbon adsorption

Pretreatments Butanol (g/L) Acids (g/L)

Activated Activated
Not Not
carbon carbon
detoxified detoxified
treated treated

P2 (control) 9.6±0.1 - 4.5±0.2 -

Soybean Hull 0.04N H2SO4 3.0±0.2 7.6±0.1 14.1±0.2 5.0±0.3

Sugarcane
0.04N HCl 7.3±0.1 9.6±0.3 9.9±0.3 4.7±0.2
Bagasse

Corn Fiber 0.04N H2SO4 - 5.4±0.2 20.9±0.4 11.7±0.2

Cotton Stalk Hot Water 8.4±0.2 10.8±0.2 8.8±0.1 7.8±0.1

117
Table 4.6 The concentrations of inhibitors present in the biomass hydrolysates before and

after activated carbon absorption

Activated Formic Ferulic

Furfural HMF Phenolics
carbon acid acid
(g/L) (g/L) (g/L)
detoxification (g/L) (g/L)
Before - - 0.28±0.09 0.07±0.01 -
Cotton stalk
After - - 0.68±0.06 - -

Sugarcane Before 0.30±0.05 - 1.10±0.05 0.29±0.03 0.18±0.02

bagasse After 0.19±0.08 - 0.68±0.04 - -

Before 0.09±0.02 - 1.45±0.08 0.32±0.07 -

Soybean hull
After - - - - -

Before 0.14±0.04 - 1.71±0.06 - 0.19±0.05

Corn Fiber
After - - 1.13±0.03 - 0.11±0.03

Table 4.7 The fermentation results using biomass hydrolysate as the carbon source for

three Clostridium strians

ATCC 824 BA101 C. tyrobutyricum

Biomass
Butanol Acids Butanol Acids Butanol Acids
(g/L) (g/L) (g/L) (g/L) (g/L) (g/L)
Control 9.6±0.1 4.5±0.2 10.2±0.1 3.0±0.2 9.5±0.3 5.4±0.1

Cotton stalk 10.8±0.2 7.8±0.1 8.9±0.2 3.9±0.2 9.3±0.3 5.9±0.1

Corn fiber 5.4±0.2 11.7±0.2 5.5±0.2 10.7±0.1 6.1±0.2 3.9±0.2

Soybean hull 7.6±0.1 5.0±0.3 10.0±0.3 3.7±0.1 7.6±0.1 4.6±0.3

Sugarcane
9.6±0.3 4.7±0.2 7.8±0.1 2.6±0.2 7.9±0.2 4.3±0.2
bagasse

118
 Table 4.8 % loss in cellulose, hemicellulose and lignin in biomass after acid or alkali pretreatment a

Acid pretreatment Alkali pretreatment

Biomass Sugar Sugar

Hemi Total Hemi Total
Cellulose lignin yield b Cellulose lignin yield b
cellulose loss cellulose loss
(g/g) (g/g)
Cotton
2% 5% 4% 13% 0.47 1% 4% 10% 16% 0.48
stalk
Sugarcane
- 14% 2% 20% 0.34 - 8% 13% 24% 0.41
bagasse
Soybean
3% 9% 15% 30% 0.37 - 8% 11% 22% 0.40
hull
Corn fiber 9% 11% 9% 32% 0.31 5% 10% 14% 31% 0.33
a
Percentage is based on the initial dry biomass weight.
119

b
After enzymatic hydrolysis and based on the dry biomass after removing the supernatant
 Table 4.9 C. acetobutylicum ATCC824 fermentation results with biomass hydrolysates

after acid or alkali pretreatment a or activated carbon detoxification

Acid Alkali Activated

pretreatment pretreatment carbon
Biomass
Butanol Acids Butanol Acids Butanol Acids
(g/L) (g/L) (g/L) (g/L) (g/L) (g/L)
Cotton stalk 8.2 7.0 8.5 6.3 10.8 7.8

Corn fiber 2.9 7.5 8.6 11.2 5.4 11.7

Soybean
8.4 5.0 5.1 6.4 7.6 5.0
hull
Sugarcane
2.3 8.2 5.7 7.1 9.6 4.7
bagasse
a
The liquor containing inhibitors was removed before enzymatic hydrolysis

120
 50 10 50 10
Corn fiber

Products (g/L), OD600

Products (g/L), OD600

40 8 40 8
Glucose (g/L)

Glucose (g/L)
30 6 30 6
Cotton stalk
20 4 20 4

10 2 10 2

0 0 0 0
0 40 80 0 40 80
Time (h) Time (h)

60 10 40 10
Soybean hull Sugarcane bagasse
Products (g/L), OD600

Products (g/L), OD600

50
8 8
30

Glucose (g/L)
Glucose (g/L)

40
6 6
30 20
4 4
20
10
2 2
10

0 0 0 0
0 40 80 0 40 80
Time (h) Time (h)

Figure 4.1 The fermentation kinetics of C. acetobutylicum ATCC824 with lignocellulosic

hydrolysates after activated carbon adsorption

121
 50 10 50 10
Cotton stalk Corn fiber

Products (g/L), OD600

Products (g/L), OD600

40 8 40 8
Glucose (g/L)

Glucose (g/L)
30 6 30 6

20 4 20 4

10 2 10 2

0 0 0 0
0 40 80 0 40 80
Time (h) Time (h)

60 12 40 10
Soybean hull Sugarcane bagasse
Products (g/L), OD600

Products (g/L), OD600

50 10
8
30
Glucose (g/L)

Glucose (g/L)
40 8
6
30 6 20
4
20 4
10
2
10 2

0 0 0 0
0 40 80 0 40 80
Time (h) Time (h)

Figure 4.2 The fermentation kinetics of beijerinckii BA101 with lignocellulosic

hydrolysates after activated carbon adsorption

122
 Untreated Acid pretreated Alkali pretreated

Cotton
stalk

Corn fiber

Soybean
hull

Sugarcane
bagasse

Figure 4.3 SEM images of untreated, acid pretreated or alkali pretreated biomass

structures

123
Chapter 5: The inhibition mechanism of lignin derived phenolic compounds on the

metabolic pathways of Clostridium spp.

Abstract
Lignocellulosic biomass is a more economical substrate for fermentation to

produce butanol, which is a promising biofuel. However, lignocellulosic biomass can not

be directly used by most microbes and needs to be hydrolyzed to generate simple sugars.

In the hydrolysis process, some inhibitors that are toxic to Clostridia may be generated,

especially some phenolic compounds. But the inhibition mechanism is still unclear. Four

phenolic inhibitors were tested in C. tyrobutyricum mutants. They were very toxic to one

mutant overexpressing ctfAB and adhE2 genes, but they had no effect on the other mutant

overexpressing only adhE2 gene. Therefore, the phenolic compounds are inhibitors to

CoA-transferase expressed by ctfAB. This was further confirmed by their toxicity to C.

beijerinckii and C. acetobutylicum. This also explained the toxicity of corn steep liquor as

nitrogen source in the butanol fermentation.

Keywords: phenolic inhibitors; ctfAB; CoA-transferase; corn steep liquor

5.1 Introduction

Lignocellulosic biomass is being studied as substrate in the Acetone-Butanol-

Ethanol fermentation because it is more economical than the current substrates (maize or

124
sugarcane). Extensive research has been done in using lignocellulosic biomass as butanol

fermentation feedstock (Tashiro et al., 2005; Liew et al., 2005; Badr et a;., 2001; Ezeji et

al., 2007; Lin et al. 1983; Tran et al., 2010; Qureshi et al., 1995; Cho et al., 2009).

Lignocellulosic biomass cannot be directly used by Clostridia and the hydrolysis process

is needed. In genreal, pretreatments are also all necessary to break down the rigid

structure (Chapter 4). Many inhibitory compounds may be produced during the

hydrolysis or pretreatment process (Ezeji et al., 2008; Lee et al., 2010; Lowmeier et al.,

1998). However, the inhibition mechanism is still unclear. Ezeji et al. mentioned the

phenolic compounds can destroy the permeability of cell membrane (Ezeji et al., 2008).

Zhang et al. found that furfural and HMF can be transformed to furfuryl alcohol and

HMF alcohol by C. acetobutylicum ATCC 824 (Zhang et al., 2012). Cao et al. studied the

effects of furfural, HMF, vanillin, and syringaldehyde on one Thermoanaerobacterium

strain and reported that the effects depended on the inhibitor's concentration (Cao et al.,

2010). Lowmeier et al. found that furfural and HMF can severely block the xylose

metabolic pathway (Lowmeier et al., 1998).

In this paper, four potential phenolic inhibitors (p-coumaric acid, ferulic acid,

vanillin and syringaldehyde) were tested in four Clostridium strains (C. beijerinckii, C.

acetobutylicum, and two C. tyrobutyricum strains). Their inhibition mechanism was

proposed according the different performances on different strains. Corn steep liquor, a

byproduct of corn wet milling, was also used as the nitrogen source in the fermentation of

Clostridia.

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5.2. Materials and methods

5.2.1 Strains and inoculum preparation

Spores of C. beijerinckii, C. acetobutylicum, C. tyrobutyricum were stored in a

refrigerator at 4 oC in the Clostridia Growth medium (CGM). Spores (2 ml) were heat-

shocked at 80 oC for 3 min and transferred to 50 ml RCM growth medium (Difco

Reinforced Clostridia Medium, Becton, Dickinson and Company, MD, USA) in a 125 ml

serum bottle. The medium was nitrogen-purged for 8 min to remove oxygen. The serum

bottle was tightly capped with a rubber stopper and aluminum seal. The mixture was

autoclaved at 121 oC for 30 min followed by cooling to 37 oC. The heat-shocked spores

were incubated at 37 oC for 12-16 h until cells were highly active. The active culture (5%

inoculum) was used as seed culture for all fermentations studied.

5.2.2 ABE fermentation in serum bottles

ABE fermentation was studied in serum bottles each containing 50 ml medium to

evaluate the effects of various detoxification methods on butanol production. Unless

otherwise noted, all fermentation studies were carried out with the P2 medium containing

one type of inhibitors, glucose (60 g/L), yeast extract (2 g/L), buffer (0.5 g/L KH2PO4

and 0.5 g/L K2HPO4), 2.2 g/L ammonium acetate, vitamins (0.001 g/L para-amino-

benzoic acid (PABA), 0.001g/L thiamin and 10-5g/L biotin), and mineral salts (0.2 g/L

MgSO4∙7H2O, 0.01 g/L MnSO4∙H2O, 0.01 g/L FeSO4∙7H2O, 0.01 g/L NaCl).The carbon

source (glucose or biomass hydrolysate) solution and P2 stock solution (containing yeast

extract, ammonium acetate and buffer, or corn steep liquor and buffer, 10-fold

126
concentrated) were autoclaved separately at 121 oC and 15 psig for 30 min for

sterilization. Minerals and vitamins were prepared at 200-fold and 1000-fold

concentration, respectively, and were filter-sterilized through 0.2 μm membrane filters

(25 mm syringe filter, Fisher brand, NJ, USA). Before sterilization, the P2 solutions in

serum bottles were purged with nitrogen for 8 min. After sterilization, an appropriate

amount of the P2 stock solution was aseptically added into the serum bottle, followed by

addition of minerals and vitamins. To ensure the medium pH maintained around 5.0

throughout the fermentation, 2 g/L of CaCO3 was also included in the medium. Actively

grown cells were inoculated at 5% (v/v). Batch fermentation was performed at 37 oC with

no agitation. Samples were taken periodically for analysis of ABE production.

5.2.3 Hydrolysis of biomass

Four lignocellulosic biomasses were hydrolyzed: cotton stalk, sugarcane bagasse,

soybean hull and corn fiber. 50 g solid particles were suspended in 1.0 L in different

concentrations of acid solution (HCl or H2SO4) or just distilled water. Autoclave at 120
o
C, 20 psig for 30 min. Then adjust their pH to 5.0 by NaOH. Add 3.0 g CTec2

(Novozymes) and hydrolyze at 50 oC for 72 h. Centrifuge at 7000 rpm, 25 oC for 15 min

to remove the undissolved solid. The supernatant hydrolysate was concentrated by

vacuum rotary evaporation at 60 oC to remove 670 mL water. The concentrated

hydrolysate was filter-sterilized through 0.2 μm membrane filters (25 mm syringe filter,

Fisher brand, NJ, USA) and stored at 4 oC for further use.

5.2.4 Detoxification of hydrolysate

127
 The pH of hydrolysate was first adjusted to 2.0 by H2SO4. Then 2% (w/w)

activated carbon was added into the hydrolysate and treated at pH 2.0, 80 oC for 60 min

under stirring. Remove activated carbon by filter paper. Adjust pH back to 6.5 by NaOH.

Centrifuge them at 8000 rpm, 25 oC for 15 min. Remove the sediments and keep the clear

hydrolysate at 4 oC for further use.

5.2.5 Analytical methods

The fermentation products, acetone, butanol, ethanol, acetic acid, and butyric acid

were analyzed with a gas chromatograph (GC, Shimadzu GC-2014) equipped with a

flame ionization detector and a 30-m fused silica column (0.25 mm film thickness and

0.25 mm ID, Stabilwax-DA). The carrier gas was nitrogen at 1.47 ml/min (linear velocity:

35 cm/s). Samples were diluted 20 times with an internal standard buffer solution

containing 0.5 g/L isobutanol, 0.1 g/L isobutyric acid and 1% phosphoric acid (for

acidification), and injected (1 μL each) by using an auto-injector (AOC-20i Shimadzu).

The column temperature was held at 80 oC for 3 min, raised to150 oC at a rate of 30
o
C/min, and held at 150 oC for 3.7 min. Both the injector and detector were set at 250 oC.

Cell density was analyzed by measuring the optical density (OD) of cell

suspension at 600 nm using a spectrophotometer (Shimadzu, Columbia, MD, UV-16-1).

5.3 Results and discussion

5.3.1 Inhibition of phenolic compounds to Clostridium strains

Four phenolic compounds were tested for their inhibition to Clostridium strains

(Figure 5.1) : p-coumaric acid, ferulic acid, vanillin and syringaldehyde. Other than the
128
phenolic group, two of them (p-coumaric acid and ferulic acid) have carboxylic group

and the other two (vanillin and syringaldehyde) have a formyl group. These four are

mainly the products of lignin degradation and potential inhibitors for ABE fermentation.

The effects of 1.0 g/L each of these four inhibitors on O.D., solvent and acid production

are shown in Figure 5.2. All four inhibitors inhibited the growth of C. beijerinckii BA101,

C. acetobutylicum ATCC824 and C. tyrobutyricum ATCC25755 with ctfAB and adhE2.

All of them had a final O.D. of lower than 6.0. On the contrary, these four inhibitors

almost had no effect on the growth of C. tyrobutyricum ATCC25755 with only adhE2,

which had O.D. reached ~16.0. For butanol production, all four inhibitors strongly

inhibited butanol production in C. beijerinckii BA101 and C. tyrobutyricum ATCC25755

(ctfAB-adhE2). The butanol titers were below 2.0 g/L. They were still very toxic even at

0.25 g/L (see Chapter 3). C. acetobutylicum ATCC824 showed some resistance to three

of them (p-coumaric acid, ferulic acid and syringaldehyde). The butanol titer reached up

to 4.0-7.0 g/L. These four inhibitors still had almost no effect on the butanol titer of C.

tyrobutyricum ATCC25755 (adhE2), which gave ~11.0 g/L butanol production in the

presence of these inhibitors. For acids production, all four strains gave relatively high

acid concentrations, 3.0 g/L-7.0 g/L. In 1.0 g/L p-coumaric acid, vanillin or

syringaldehyde, C. acetobutylicum ATCC824 had the highest acid titer, over 5.5 g/L.

The only difference of those two C. tyrobutyricum ATCC25755 mutants was that

one overexpressed both ctfAB and adhE2 genes while the other only overexpressed

adhE2 genes (Yu et al., 2011). Moreover, both C. beijerinckii BA101 and C.

acetobutylicum ATCC824 have ctfAB gene and can express CoA-transferase. Those four

129
phenolic inhibitors only had severe inhibition effects on Clostridium strains with ctfAB

gene that express CoA-transferase. For C. tyrobutyricum ATCC25755 (adhE2) without

ctfAB gene, those four phenolic inhibitors had no toxicity either to the cell growth nor to

butanol production. Therefore, the inhibition mechanism of these phenolic inhibitors

should work on CoA-transferase. The most important function of CoA-transferase is to

convert acetate and butyrate back to acetyl-CoA and butyryl-CoA in the solventogenesis

phase (Jones et al., 1986). So acetyl-CoA and butyryl-CoA can be further convert into

ethanol and butanol. CoA-transferase is critical in the shift from acidogenesis to

solventogenesis phase. Those four inhibitors almost had no effect on acids production in

all four strains, so they didn't affect on the acidogenesis phase. This further proves that

they influenced CoA-transferase. p-Coumaric acid and ferulic acid both have a carboxylic

group, so they are potential substrates for CoA-transferase just like acetic acid or butyric

acid. They may attach to CoA-transferase and work as competitive inhibitors to CoA-

transferase.

5.3.2 Corn steep liquor as the nitrogen source for ABE fermentation

Corn steep liquor is the byproduct of corn wet milling and is rich in protein and

amino acids. So it is a potential economical nitrogen source for butanol production.

When corn steep liquor was used as the only nitrogen source in the fermentation C.

tyrobutyricum ATCC25755 mutants, very obvious difference was seen between the two

mutants. For C. tyrobutyricum ATCC25755 with both ctfAB and adhE2 genes, corn steep

liquor partly inhibited the butanol production even at 0.25% w/v, only 5.7 g/L. Increasing

the concentration of corn steep liquor further decreased the butanol titer. On the other
130
hand, the acid production was not suppressed by corn steep liquor. The acid

concentrations were even higher than the control. For C. tyrobutyricum ATCC25755 with

only adhE2 gene, corn steep liquor didn't inhibit butanol production even at 9% (w/v). It

even increased the butanol titer in the lower concentration. The highest butanol titer was

around 15.0 g/L. This is a similar phenomenon with the previous phenolic inhibitors.

Therefore, corn steep liquor must have contained some of those phenolic inhibitors. But

due to the complex composition of corn steep liquor, it's very hard to determine the

spedific phenolic compounds and their concentrations. However, further proofs were

obtained when the corn steep liquor was combined with lignocellulosic hydrolysate in the

ABE fermentation with three strains (C. beijerinckii BA101, C. acetobutylicum

ATCC824 and C. tyrobutyricum ATCC25755 with ctfAB and adhE2) containing ctfAB

genes (Table 5.1). Therefore, their butanol production should be inhibited by those four

phenolic compounds (p-coumaric acid, ferulic acid, vanillin and syringaldehyde). When

corn steep liquor was used as nitrogen source and glucose as carbon source, butanol

production was all lower than that using yeast extract and ammonium acetate as nitrogen

source. Among those three strains, C. acetobutylicum ATCC824 had the highest

resistance to the toxicity of corn steep liquor, it still gave 8.0 g/L butanol compared to 9.6

g/L in the control. The butanol titers in C. beijerinckii BA101 and C. tyrobutyricum

ATCC25755 with ctfAB-adhE2 were only 50% and 70% of the control. The acids

production when using corn steep liquor was slightly higher. When combining corn steep

liquor with lignocellulosic hydrolysates, the butanol titer was much lower compared with

that with yeast extract and ammonium acetate. Especially in corn fiber, soybean hull and

131
sugarcane bagasse hydrolysates, almost no butanol was produced. Possible reason is that

the phenolic inhibitors in corn steep liquor combined with some other inhibitors in the

lignocellulosic hydrolysates showed stronger toxicity to Clostridium strains. When the

strain was C. tyrobutyricum ATCC25755 with adhE2, combining corn steep liquor and

the lignocellulosic hydrolysates still showed very high butanol production. So the

phenolic inhibitors in corn steep liquor were still the most important factor that affects the

butanol production. It can inhibit the function of CoA-transferase and block butanol

production. So for C. tyrobutyricum ATCC25755 mutant that doesn't contain CoA-

transferase, corn steep liquor is a very good and economical nitrogen source for butanol

production.

5.4 Conclusion

The phenolic compounds (p-coumaric acid, ferulic acid, vanillin and

syringaldehyde) were toxic to Clostridium strains with CoA-transferase but showed

almost no effect on strains without CoA-transferase. These phenolic inhibitors may work

as competitive inhibitors to CoA-transferase and block the solventogenesis phase that

converts acids into solvents. Corn steep liquor is rich in protein and amino acids, and is a

potential nitrogen source for butanol production. But the phenolic inhibitors in it may

severely inhibit butanol production by solventogenic Clostridia containing ctfAB genes.

132
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 Table 5.1 The fermentation results of different strains combining lignocellulosic hydrolysate with corn steep liquor

824 BA101 C. tyro (ctfAB-adhE2)

Biomass
YE, NH4Ac 30 g/L CSL YE, NH4Ac 30 g/L CSL YE, NH4Ac 30 g/L CSL
butanol acids butanol acids butanol acids butanol acids butanol acids butanol acids
Control 9.6 4.5 8.0 5.9 10.2 3.0 5.1 4.9 8.9 3.1 6.2 5.4

cotton stalk 10.8 7.8 5.9 7.7 8.9 3.9 3.9 5.2 9.5 3.9 8.5 4.1

corn fiber 5.4 11.7 2.5 8.8 5.4 9.7 - 2.3 5.9 8.1 - 2.3

soybean hull 7.6 5.0 - 2.3 10.0 3.7 - 3.4 8.2 2.8 3.4 3.7

sugarcane
9.6 4.7 - 2.5 7.8 2.6 - 2.7 6.3 2.9 - 2.7
bagasse
141
 p-coumaric acid Ferulic acid

Vanillin Syringaldehyde

Figure 5.1 Chemical structures of phenolic inhibitors

142
 20
BA101 824 C.tyro(ctfAB-adhE2) C.tyro(adhE2)
15

10
O.D.

15
Butanol (g/L)

10

6
Acids (g/L)

0
Syringaldehyde Ferulic Acid Vanillin p-Coumaric Acid

Figure 5.2 The inhibition of phenolic compounds (1.0 g/L each) to the growth and

solvents and acids production of four Clostridium strains

143
 C. tyro (ctfAB-adhE2)
10

8
Titer (g/L)

Butanol
6
Acids
4

0
control 0.25% 0.50% 1% 2% 3%
Corn steep liquor (wt %)

C. tyro (adhE2)
16
butanol
12 Acids
Titer (g/L)

0
Control 9% 6% 3% 1%
Corn steep liquor (wt %)

Figure 5.3 The fermentation results of two C. tyrobutyricum ATCC25755 mutants with

different concentrations of corn steep liquor as nitrogen source

144
 Chapter 6: Butanol production from non-lignocellulosic biomass

Abstract

The high cost of feedstock is a bottleneck for large-scale butanol fermentation.

More economical substrates are needed to reduce the cost of butanol production.

Lignocellulosic biomass is a potential substrate. Another possibility is some other

agricultural residues. In this study, three agricultural residues were investigated: cassava

bagasse, soy molasses and soybean meal. Cassava bagasse and soy molasses are rich

carbon source, while soybean meal has plenty of proteins and is a potential nitrogen

source. Starch based cassava bagasse only needs enzyme (glucoamylase) hydrolysis. Its

hydrolysate contained no toxic inhibitors and performed very well in ABE fermentation.

Inulin based Jerusalem artichoke was hydrolyzed only by dilute acid. But its hydrolysate

contained high concentration of ferulic acid which is an inhibitor to ABE fermentation.

Soy molasses contain mainly oligosaccharides and can be hydrolyzed by α-galactosidase.

Soybean meal was also proved to be a good nitrogen source after the acid hydrolysis.

ABE fermentation kinetics of three different Clostridium strains using soy molasses as

carbon source and soybean meal as nitrogen source were also studied, and C.

acetobutylicum ATCC 824 gave the highest butanol titer (8.7 g/L). The detailed cost

analysis of large scale butanol plants from different types of biomass were also made, and

butanol from soy molasses had the lowest unit cost ($2.71 /gal).
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Keyword: cassava bagasse; soybean meal; soy molasses; Jerusalem artichoke

6.1 Introduction

The increasing requirement of energy promotes the research on biofuels. Among

the biofuels, butanol has a lot of promising characters, such as high energy density, low

vapor pressure and similar octane number with gasoline. In order to reduce the cost of

butanol production and make it competitive with petrochemical fuels, cheaper substrates

are needed for butanol fermentation (Qureshi et al., 2001, 2005, 2007, 2008, 2010a,b;

Maddox et al., 1993; Zverlov et al., 2006). Lignocellulosic biomass is hot candidate

because it is cheap and abundant in nature (Tran et al., 2010; Qureshi et al., 1995; Cho et

al., 2009). But it has some disadvantages. It cannot be directly used by solventogenic

Clostridia. So cellulase enzyme is needed to hydrolyze the lignocellulosic biomass,

which increases the production cost. Sometimes pretreatments are also necessary to break

down its rigid structures. And the pretreatments can generate inhibitors to Clostridia

(Ezeji et al., 2008; Lee et al., 2010; Lowmeier et al., 1998). So other agricultural resiues

were also studied by researchers (Ezeji et al., 2008; Qureshi et al., 2008; Zverlov et al.,

2006).

In this chapter, four biomass were investigated. Three as carbon sources, cassava

bagasse, Jerusalem artichoke and soy molasses. The other was as nitrogen source,

soybean meal. Cassava bagasse is starch based, Jerusalem artichoke is inulin based

biomass, and main components of soy molasses are oligosaccharides, such as stachyose

and raffinose. These biomass were first hydrolyzed by acids or enzyme and then used as

carbon sources in the ABE fermentation. Soybean meal was also hydrolyzed by acids to
146
release more amino acids and then used to provide nitrogen source during the ABE

fermentation. Cost analysis of large-scale butanol plants from different types of biomass

was also made.

6.2 Materials and methods

6.2.1 Strains and inoculum preparation

Spores of C. beijerinckii BA101, C. acetobutylicum ATCC 824 and ATCC 55025,

C. tyrobutyricum were stored in a refrigerator at 4 oC in the Clostridia Growth medium

(CGM). Spores (2 ml) were heat-shocked at 80 oC for 3 min and transferred to 50 ml

RCM growth medium (Difco Reinforced Clostridia Medium, Becton, Dickinson and

Company, MD, USA) in a 125 ml serum bottle. The medium was nitrogen-purged for 8

min to remove oxygen. The serum bottle was tightly capped with a rubber stopper and

aluminum seal. The mixture was autoclaved at 121 oC for 30 min followed by cooling to

37 oC. The heat-shocked spores were incubated at 37 oC for 12-16 h until cells were

highly active. The active culture (5% inoculum) was used as seed culture for all

fermentations studied.

6.2.2 ABE fermentation in serum bottles and bioreactors

ABE fermentation was studied in serum bottles each containing 50 ml medium to

evaluate the effects of various detoxification methods on butanol production. Unless

otherwise noted, all fermentation studies were carried out with the P2 medium containing

one type of inhibitors, glucose (60 g/L), yeast extract (2 g/L), buffer (0.5 g/L KH2PO4

and 0.5 g/L K2HPO4), 2.2 g/L ammonium acetate, vitamins (0.001 g/L para-amino-

147
benzoic acid (PABA), 0.001 g/L thiamin and 10-5 g/L biotin), and mineral salts (0.2 g/L

MgSO4∙7H2O, 0.01 g/L MnSO4∙H2O, 0.01 g/L FeSO4∙7H2O, 0.01 g/L NaCl).The carbon

source solution (glucose or biomass hydrolysates) and P2 stock solution (containing yeast

extract, ammonium acetate and buffer, 10-fold concentrated) were autoclaved separately

at 121oC and 15 psig for 30 min for sterilization. Minerals and vitamins were prepared at

200-fold and 1000-fold concentration, respectively, and were filter-sterilized through 0.2

μm membrane filters (25mm syringe filter, Fisher brand, NJ, USA). Before sterilization,

the P2 solutions in serum bottles were purged with nitrogen for 8 min. After sterilization,

an appropriate amount of the P2 stock solution was aseptically added into the serum

bottle, followed by addition of minerals and vitamins. To ensure the medium pH

maintained around 5.0 throughout the fermentation, 2 g/L of CaCO3 was also included in

the medium. Actively grown cells were inoculated at 5% (v/v). Batch fermentation was

performed at 37 oC with no agitation. Samples were taken periodically for analysis of

ABE production.

In the 500 mL bioreactor fermentation using soy molasses and soybean meal, only

450 mL soy molasses hydrolysate (250 g/L) and 50 mL soybean meal hydrolysate (100

g/L) were mixed into the bioreactor. No other nutrients were added. The bioreactor was

then autoclaved at 121 oC for 30 min. After autoclave, the bioreactor was cooled down to

37 oC with continuous nitrogen purging. Then 50 mL seed culture was inoculated into the
o
bioreactor. The fermentation was performed at 37 C, pH 5.0 (pH 6.0 for C.

tyrobutyricum) with 150 rpm agitation. Samples were taken periodically for analysis of

ABE production.

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6.2.3 Hydrolysis of biomass

Cassava bagasse

50 g solid particles were suspended in 1.0 L in 0.04N H2SO4 or just distilled water.

Autoclave at 121 oC, 15 psig for 30 min. Then adjust their pH to 5.0 by NaOH. Add 3.0 g

CTec2 (Novozymes) and 0.03 g Distillase® L-400 (glucoamylase, Genencor) hydrolyze

at 50 oC for 72 h. Centrifuge at 7000 rpm, 25 oC for 15 min to remove the undissolved

solid. The supernatant hydrolysate was concentrated by vacuum rotary evaporation at 60

o
C to remove 670 mL water. The concentrated hydrolysate was filter-sterilized through

0.2 μm membrane filters (25 mm syringe filter, Fisher brand, NJ, USA) and stored at 4 oC

for further use.

Jerusalem artichoke

50 g solid particles were suspended in 1.0 L in 0.04 N H2SO4 or just distilled

water. Autoclave at 121 oC, 15 psig for 30 min. Then adjust their pH to 5.0 by NaOH.

Centrifuge at 7000 rpm, 25 oC for 15 min to remove the undissolved solid. The

supernatant hydrolysate was concentrated by vacuum rotary evaporation at 60 oC to

remove 670 mL water. The concentrated hydrolysate was filter-sterilized through 0.2 μm

membrane filters (25 mm syringe filter, Fisher brand, NJ, USA) and stored at 4 oC for

further use.

Soy molasses

200 g soy molasses slurry was suspended in 1.0L distilled water. Autoclave at

121 oC, 15 psig for 30 min. Add 10 mL α-galactosidase (Enzyme Development Inc)

149
hydrolyze at 50 oC for 48 h. Then adjust the pH to 5.0 by NaOH. Centrifuge at 7000 rpm,

25 oC for 15 min to remove the undissolved solid. The supernatant hydrolysate was

stored at 4 oC for further use.

Soybean meal

10 g of soybean meal was suspended in 100 mL dilute acid solution (with various

acid concentrations) and then autoclaving at 121°C, 15 psig for 30 minutes. Then, the pH

was adjusted to 5.0 and 1 mL α-galactosidase was added into the medium. The enzymatic

hydrolysis took 72 h at 50°C under stirring. Centrifuge at 7000 rpm, 25 oC for 15 min to

remove the undissolved solid. The supernatant hydrolysate was stored at 4 oC for further

use.

6.2.4 Detoxification of hydrolysate

The pH of hydrolysate was first adjusted to 2.0 with H2SO4. Then 2% (w/w)

activated carbon was added into the hydrolysate and treated at pH 2.0, 80 oC for 60 min

under stirring. Remove activated carbon by filtering through filter paper. Adjust pH back

to 6.5 by NaOH. Centrifuge them at 8000 rpm, 25 oC for 15 min. Remove the sediments

and keep the clear hydrolysate at 4 oC for further use.

6.2.5 Analytical methods

The compositions of biomass were determined by the methods described by

Foster et al.(2010) The fermentation products, acetone, butanol, ethanol, acetic acid, and

butyric acid were analyzed with a gas chromatograph (GC, Shimadzu GC-2014)

150
equipped with a flame ionization detector and a 30-m fused silica column (0.25 μm film

thickness and 0.25 mm ID, Stabilwax-DA). The carrier gas was nitrogen at 1.47 ml/min

(linear velocity: 35 cm/s). Samples were diluted 20 times with an internal standard buffer

solution containing 0.5 g/L isobutanol, 0.1 g/L isobutyric acid and 1% phosphoric acid

(for acidification), and injected (1 μL each) by using an auto-injector (AOC-20i

Shimadzu). The column temperature was held at 80 oC for 3 min, raised to150 oC at a rate

of 30 oC/min, and held at 150 oC for 3.7 min. Both the injector and detector were set at

250 oC.

Cell density was analyzed by measuring the optical density (OD) of cell

suspension at 600 nm using a spectrophotometer (Shimadzu, Columbia, MD, UV-16-1).

The inhibitors including furfural, HMF, levulinic acid, ferulic acid, p-coumaric acid, and

phenolic compounds (syringaldehyde) were analyzed by high performance liquid

chromatography (HPLC) using Rezex ROA-Organic Acid H+ (8%) column (Phenomenex)

at 40 oC and a UV/UIS detector (SPD-20AV, Shimadzu). The eluent was 0.01 N H2SO4

at 0.6 ml/min. Furfural, HMF and phenolic compounds were detected at 280 nm.

Syringaldehyde was used as the standard for phenolic compounds. levulinic acid, ferulic

acid, p-coumaric acid were detected at 200 nm.

6.3 Results and discussion

6.3.1 Cassava bagasse and Jerusalem artichoke as the carbon source for ABE

fermentation

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 Cassava bagasse is the byproduct of starch production from cassava. Jerusalem

artichoke is a common wild plant native to eastern North America. The compositions of

these two are shown in Table 6.1. Although cassava bagasse is the residue after

extracting starch from cassava, 43% of its content was still starch. It also contained 25%

cellulose and 9% hemicellulose. Jerusalem artichoke is rich in inulin (54%) and it also

contained 15% cellulose. Either starch or inulin can be hydrolyzed and used as carbon

sources in the ABE fermentation. Hot water and dilute acid (0.04 N H2SO4) pretreatments

were compared for cassava bagasse and Jerusalem artichoke (Table 6.2). Inulin in

Jerusalem artichoke was easy to be hydrolyzed, with just hot water pretreatment, which

gave a sugar yield of 0.51 g/g. With 0.04 N H2SO4, the sugar yield was 0.75 g/g. No

enzyme was needed to further hydrolyze Jerusalem artichoke. Since the sugar yield was

much higher for acid treated Jerusalem artichoke, acid pretreatment was used for the

following ABE fermentation. For cassava bagasse, the sugar yields were similar either

with hot water or 0.04N H2SO4, around 0.45 g/g. Cellulase and amylase were needed to

further hydrolyze the cellulose and starch in cassava bagasse. The sugar yields were

almost the same, but acid pretreatment had the high risk of generating inhibitors. So hot

water pretreatment was chosen in the following ABE fermentation. The inhibitors present

in acid pretreated Jerusalem artichoke hydrolysate and hot water pretreated cassava

bagasse hydrolysate are listed in Table 6.3. The cassava bagasse hydrolysate was clean,

only containing 0.64 g/L formic acid, which was not toxic to most Clostridium strains

(Chapter 3). So cassava bagasse hydrolysate did not need to be detoxified before

fermentation. Jerusalem artichoke hydrolysate had more inhibitors. It contained 2.19 g/L

152
formic acid, 0.71 g/L ferulic acid and 0.18 g/L other phenolic compounds. Ferulic acid

above 0.25 g/L is toxic to Clostridium strains (Chapter 3). So Jerusalem artichoke

hydrolysate needs to be detoxified before fermentation. Activated carbon adsorption was

used to remove the inhibitors in Jerusalem artichoke hydrolysate. About 32% of formic

acid, 38% of ferulic acid, and all of other phenolic compounds were removed after

activated carbon adsorption. But the ferulic acid concentration was still 0.44 g/L, above

0.25 g/L toxic level. These two hydrolysates were used as carbon sources in the ABE

fermentation of C. beijerinckii BA101, C. acetobutylicum ATCC 824 and C.

tyrobutyricum ATCC 25755 mutant (Table 6.4). Cassava bagasse hydrolysate performed

very well. All butanol titers were similar to the control, some were even better than the

control, because it almost contained no inhibitors. Butanol production in Jerusalem

artichoke hydrolysate was lower than the control for all strains studied. C. acetobutylicum

ATCC 824 and C. tyrobutyricum ATCC 25755 mutant gave ~80% of butanol production

while C. beijerinckii BA101 only had ~50% butanol titer compared to the control. The

lower butanol production was attributed to the high concentration of ferulic acid (0.44

g/L) in Jerusalem artichoke hydrolysate. In my previous study, C. acetobutylicum ATCC

824 showed more resistance to ferulic acid than C. beijerinckii BA101 (see Chapter 3).

So C. acetobutylicum ATCC 824 had high butanol production than C. beijerinckii BA101

in Jerusalem artichoke hydrolysate. Another detoxification method needs to be developed

for Jerusalem artichoke hydrolysate to further improve the butanol production in the

fermentation.

6.3.2 Soy molasses as the carbon source for ABE fermentation

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 Soy molasses and soybean soluble contain oligosaccharides, mainly stachyose and

raffinose, sucrose, glucose and other monosaccharides including galactose, fructose,

xylose and arabinose. The exact composition varied with the source of the materials and

might change during storage (see Table 6.5). Stachyose (and raffinose) cannot be used by

Clostridium. Earlier efforts using acid pretreatment to hydrolyze soy oligosaccharides

were not successful – only limited amounts of sugars were released but along with

significant amounts of unknown inhibitors that severely inhibited butanol production in

ABE fermentation. Later, it was found that soy oligosaccharides present in the molasses

can be readily hydrolyzed with α-galactosidase. As can be seen in Figure 6.1, it took only

about 0.5 h to hydrolyze all stachyose (and raffinose) present in soybean molasses to

galactose and sucrose, which was further hydrolyzed to glucose and fructose in ~3.5 h. A

total of 24.3 g/L sugars, mainly glucose, galactose and fructose, were obtained from 80

g/L soy molasses. The conversion was 100% with a sugar yield of 0.30 g/g molasses. For

ABE fermentation, 200 g/L initial loading was used, so the sugar concentration at the

beginning was 60 g/L.

Soy molasses was used as carbon source in the ABE fermentation by C.

acetobutylicum ATCC 824 and C. tyrobutyricum (Δack, adhE2), and the results are

shown in Table 6.6. As expected, butanol production from hydrolyzed soy molasses was

much better than that from the untreated one. Also, both corn steep liquor and soybean

meal hydrolysate appeared to be good nitrogen source with butanol production

comparable to that obtained with yeast extract and NH4-acetate. It is noted that the

proteins present in corn steep liquor and soybean meal might adsorb some toxic inhibitors

154
in soy molasses. C. tyrobutyricum with soybean meal as the nitrogen source gave the

highest butanol production and appeared to be better than C. acetobutylicum ATCC 824.

Up to 8 g/L of butyrate were produced in the fermentation with ATCC 824. A large

amount of butyrate was produced, instead of converting into butanol, when the

fermentation pH was above 5.5, in which acids were accumulated because

solventogenesis in C. acetobutylicum usually requires a pH below 5.0. If butyrate can be

fully converted into butanol, butanol production would be much higher.

6.3.3 Soybean meal as carbon and nitrogen sources for ABE fermentation

Similar to soy molasses, soybean meal also contains a large amount of

carbohydrate, mainly oliogsaccharides (stachyose and raffinose), sucrose and some

monosaccharides (galactose, glucose, fructose). The oligosaccharides present in soybean

meal need to be hydrolyzed in order to be utilized by clostridia in butanol fermentation.

Similar enzymatic hydrolysis method with α-galactosidase can be used to hydrolyze

oligosaccharides present in soybean meal. However, a pretreatment with dilute acid is

necessary in order to partially break down the soybean meal solid particles and hydrolyze

the proteins in soybean meal. After treating with α-galactosidase, more than 16 g/L of

sugars (glucose, galactose, and fructose) were obtained. The highest sugar concentration

was 29.2 g/L (pretreated with 0.1 N HCl). If the acid concentration was increased to 0.3

N HCl, the sugars began to degrade and only 20.2 g/L total sugar was obtained. 0.04 N

H2SO4 was slightly inferior to 0.04 N HCl in releasing single sugars. The hydrolysis

results are summarized in Table 6.7

155
 Soybean meal hydrolysate as both carbon and nitrogen sources were tested in

ABE fermentation. No other nutrients were added into the hydrolysates except for some

trace minerals. The fermentation results of C. acetobutylicum ATCC 824 are shown in

Table 6.8. The butanol production was low, with the highest butanol titer of 2.86 g/L, due

to the low sugar content in the hydrolysate. No obvious phenolic compounds or weak

acid inhibitors were detected in the hydrolysate. So the hydrolysate (pretreated with 0.04

N H2SO4) was concentrated to 48 g/L total sugars and then used in the fermentation.

However, the concentrated hydrolysate seemed to be more toxic to the bacteria as less

butanol was produced in the fermentation, indicating the presence of inhibitors in the

concentrated soybean meal hydrolysate. It can be concluded that soybean meal by itself is

not sufficient as both nitrogen and carbon sources for ABE fermentation, although it can

provide both of them. Therefore, soybean meal should be used mainly as nitrogen source

with additional carbon source such as soy molasses for butanol production in ABE

fermentation.

6.3.4 Butanol production from soybean meal and soy molasses

Soy molasses, after hydrolysis to sugars, and soybean meal, which is a good

nitrogen source, were used together without any additional nutrients supplementation for

butanol fermentation. The loading of soy molasses was 200 g/L, and the initial total sugar

concentration was ~54 g/L. Three different loadings of soybean meal and four different

clostridia strains were tested. The results are shown in Table 6.9. It is clear that 10 g/L

soybean meal can provide enough nitrogen source for the fermentation. Increasing the

loading to 30 g/L soybean meal appeared to inhibit butanol production. Among the 4
156
strains studied, C. acetobutylicum ATCC 55025 gave the highest butanol titer (8.7 g/L).

The fermentation kinetics of three different Clostridia strains in 500 mL bioreactors are

shown in Figure 6.2. In the bioreactor, the soy molasses loading was increased to 250 g/L

while the loading of soybean meal was still 10 g/L. In the fermentation of C.

acetobutylicum ATCC 55025, glucose and fructose were consumed faster than galactose

and xylose. After 72 h, the fermentation consumed 78% glucose, 64% fructose, 77%

galactose and 55% xylose. So C. acetobutylicum ATCC 55025 could utilize these four

sugars simultaneously. The xylose consumption was slower than the other three. In the

fermentation of C. tyrobutyricum (Δack, adhE2), only glucose and fructose were

consumed very rapidly. Almost no galactose and xylose were consumed during the

fermentation. After 72 h, 74% glucose, 100% fructose, 7.7% galactose and 27% xylose

were consumed. Therefore, C. tyrobutyricum (Δack, adhE2) preferred to use fructose and

glucose. The consumption rate of fructose was faster than that of glucose. The utilization

of xylose was partly inhibited and the consumption of galactose was almost totally

blocked in C. tyrobutyricum (Δack, adhE2). This can also explain the lower butanol titer

in the fermentation. The sugar consumption in the fermentation of C. acetobutylicum

ATCC 824 showed similar characters as in C. tyrobutyricum (Δack, adhE2). The

consumption of glucose and fructose was much faster than that of galactose and xylose.

After 72 h, 100% glucose, 73% fructose, 62% galactose and 36% xylose were utilized. So

only the metabolism of xylose was partly inhibited. Unlike in C. tyrobutyricum (Δack,

adhE2), the glucose consumption rate was faster than the fructose consumption rate in C.

acetobutylicum ATCC 824. The butanol production was still partly blocked and ~6.0 g/L

157
butyric acid was accumulated. So among there three Clostridia, C. acetobutylicum ATCC

55025 could co-utilize glucose, fructose, galactose and xylose and gave the highest

butanol titer (8.7 g/L).

6.4 Conclusion

Cassava bagasse hydrolysate can be a good carbon source for butanol production.

It had no toxicity to Clostridium. Jerusalem artichoke was easy to be hydrolyzed, but the

hydrolysate contained a higher level of ferulic acid, which partly inhibited butanol

production. Activated carbon can partly adsorb ferulic acid in Jerusalem artichoke

hydrolysate, but not totally removed. Soybean meal with a high protein content (~40%)

can be used as a good nitrogen source, replacing yeast extract and corn steep liquor, in

ABE fermentation to produce butanol from various biomass feedstock. Soybean meal

also has a high carbohydrate content (~30%), which mainly consists of oligosaccharides

(stachyose and raffinose). These oligosaccharides are difficult to be used by Clostridia

and must be pre-hydrolyzed to simple sugars (sucrose, glucose, galactose) preferably by

using α-galactosidase. Soybean hull and soy molasses can be used as low-cost carbon

source for butanol production after acid pretreatment and enzymatic hydrolysis.

Combining the hydrolysates from soybean meal and soybean hull or soy molasses can

provide an economical substrate for butanol production. The butanol unit costs from three

different types of biomass (cassava bagasse, Jerusalem artichoke and soy molasses) were

all lower than the cost from lignocellulosic biomass. Among them, soy molasses had the

lowest unit cost ($2.71 /gal).

158
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166
 Table 6.1 The compositions of Cassava bagasse and Jerusalem artichoke

Biomass Compositions

25%±5% cellulose; 43%±1% starch; 9%±4% hemicellulose;

Cassava bagasse
10%±2% lignin

Jerusalem artichoke 15%±3% cellulose; 54%±5% inulin; 20%±2% lignin

Table 6.2 The sugar concentrations of the biomass after the hydrolysis

Xylose and
Pretreatments Glucose (g/L) Yield (g/g)
fructose(g/L)

Jerusalem Hot water 20.2±0.5 56.4±1.2 0.51±0.01

artichoke H2SO4 24.8±3.1 87.2±0.8 0.75±0.03

Cassava Hot water 59.7±1.5 9.7±3.1 0.46±0.03

bagasse H2SO4 57.6±2.8 5.9±1.0 0.42±0.03

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Table 6.3 The inhibitors‘ concentrations in hydrolysates before and after activated carbon

absorption

Activated Formic Ferulic

Furfural HMF Phenolics
carbon acid acid
detoxification (g/L) (g/L) (g/L)
(g/L) (g/L)

Jerusalem Before - 0.10±0.05 2.19±0.08 0.71±0.08 0.18±0.01

artichoke After - 0.06±0.01 1.48±0.06 0.44±0.04 -

Cassava Before - - 0.64±0.08 - -

bagasse After - - 1.09±0.09 - -

Table 6.4 The fermentation results using biomass hydrolysates as the carbon source

ATCC 824 BA101 C. tyrobutyricum

Biomass Butanol Acids Butanol Acids Butanol Acids
(g/L) (g/L) (g/L) (g/L) (g/L) (g/L)

Control 9.6±0.1 4.5±0.2 10.2±0.1 3.0±0.2 9.5±0.3 5.4±0.1

Cassava bagasse 11.2±0.2 6.7±0.1 9.9±0.1 2.7±0.2 10.1±0.2 5.1±0.3

Jerusalem
7.8±0.2 8.4±0.2 5.7±0.2 3.7±0.3 7.4±0.2 4.9±0.3
artichoke

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 Table 6.5 Carbohydrate content of soybean soluble and soy molasses.

Stachyose Raffinose Sucrose Glucose Others Total

Soybean soluble - 15.7% 5.1% 9.4% 6.7% 36.9%

Soy molasses - 1 9.0% 5.6% 11% 4.4% 5.1% 35.1%

Soy molasses - 2 10.5% - 14.6% 0.5% 1.2% 26.8%

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 Table 6.6 Butanol and acids production from soy molasses by C. acetobutylicum ATCC

824 and C. tyrobutyricum (Δack, adhE2) after 3 days fermentation

Butanol Acetate Butyrate

Treatment N-source
(g/L) (g/L) (g/L)

C. acetobutylicum ATCC 824

No yeast extract, NH4-acetate 0.6 4.1 4.5

Enzymes from

Aspergillus niger yeast extract, NH4-acetate 2.5 6.0 8.0

fermentation broth

α-Galactosidase yeast extract, NH4-acetate 5.0 4.0 4.9

Corn steep liquor (30 g/L) 4.0 5.0 5.9

Soybean meal (10 g/L) 3.6 4.0 5.0

C. tyrobutyricum (Δack, adhE2)

α-Galactosidase Yeast Extract, NH4-acetate 4.5 4.3 4.8

Corn steep liquor (30 g/L) 4.9 4.2 4.6

Soybean meal (10 g/L) 5.2 3.2 3.9

The loading of soy molasses was 200 g/L.

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 Table 6.7 Hydrolysis of oligosaccharides present in soybean meal by different acid

pretreatments followed with enzymatic hydrolysis with α-galactosidase

Pretreatment Glucose (g/L) Galactose + Fructose (g/L) Total sugar (g/L)

0.04 N HCl 8.8 11.1 19.9

0.1 N HCl 13.7 15.5 29.2

0.3 N HCl 7.6 12.6 20.2

0.04 N H2SO4 7.3 9.2 16.5

a
The loading of soybean meal was 100 g/L

Table 6.8 Fermentation results of C. acetobutylicum ATCC 824 using soybean meal

hydrolysates pretreated with various acid concentrations followed with enzymatic

hydrolysis with α-galactosidase

Acid pretreatment Initial sugar Final butanol (g/L) Acids

(g/L) (g/L)

0.04 N HCl 19.9 0.89 5.3

0.1 N HCl 29.2 0.79 4.2

0.3 N HCl 20.2 1.43 4.6

0.04 N H2SO4 16.5 2.86 4.3

0.04 N H2SO4, concentrated 3-fold 48.0 0.12 7.2

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 Table 6.9 Butanol production from soy molasses hydrolysate as the carbon source and

soybean meal hydrolysate as the nitrogen source

Soybean meal
Strain Butanol (g/L) Acids (g/L)
(g/L)

C. acetobutylicum ATCC 824 30 2.2 10.5

20 3.4 9.0

10 3.6 8.9

C. beijerinckii BA101 10 1.7 5.3

C. tyrobutyricum (Δack, adhE2) 10 5.2 7.1

C. acetobutylicum ATCC 55025 10 8.7 4.2

Table 6.10 Fermentation Parameters based on C. tyrobutyricum(Δack, adhE2)

Butanol final titer 20 g/L

Butanolproductivity 1.0 g/L·h

Butanol yield 0.3 g/g sugars

Butanol, Acetone, Ethanol ratio 20:1:2

Annual butanol production ~7.5 Million Gal

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 Table 6.11 Hydrolysis parameters of different biomasses

Temperature Sugar yield

Loading (w/w) Catalyst
(oC) (g/g biomass)
Corn starch 10% 60 No 0.8
120 0.04N HCl
Lignocellulosea 20% 0.5
50 5% cellulase
Cassava bagasse 15% 50 5% glucoamylase 0.5
Jerusalem artichoke 20% 120 0.04N H2SO4 0.7
Soy molasses 20% 50 1% α-galactosidase 0.3b
a
The composition of cotton stalk is used to stand for lignocellulose biomass. And its
hydrolysis contains two steps: HCl pretreatment and cellulose hydrolysis
b
Soy molasses is a thick liquid, so this sugar yield is g sugars/g liquid

Figure 6.1 Hydrolysis of soybean molasses by α-galactosidase (0.1% v/v) at pH 5.0, 50

o
C
173
 30 10 30 14

12
8

Products (g/L), OD600

Products (g/L), OD600
10
20 20
Sugars (g/L)

Sugars (g/L)
6
8

6
4
10 10
4
2
2

0 0 0 0
0 40 80 0 40 80
Time (h) Time (h)
(a) (b)

30 10

8
Products (g/L), OD600

20
Sugars(g/L)

4
10

0 0
0 40 80
Time (h)
(c)

Figure 6.2 The fermentation kinetics of different Clostridia with soy molasses and

soybean meal hydrolysates: (a) C. acetobutylicum ATCC 55025; (b) C. tyrobutyricum

(Δack, adhE2); (c) C. acetobutylicum ATCC 824

174
 5

Butanol unit cost ($/gal)

4

0
Corn starch Lignocellulose Cassava Jerusalem Soy molasses
bagasse artichoke

Figure 6.3 Unit production costs by using different raw materials

(The raw material costs are: Corn starch $150/MT; Lignocellulose $50/MT; Cassava

bagasse $20/MT; Jerusalem artichoke $50/MT; Soy molasses $10/MT)

30
Capital Cost (MM $)

25

20

15

10

0
Corn starch Lignocellulose Cassava Jerusalem Soy molasses
bagasse artichoke

Figure 6.4 Capital costs by using different raw materials

175
 20

Raw Materials
15
Utilities

Operating Cost (MM $) Labor

10

0
Corn starch Lignocellulose Cassava Jerusalem Soy molasses
bagasse artichoke

Figure 6.5 Operating costs by using different raw materials

6
Butanol unit cost ($/gal)

3
Lignocellulose
2 Cassava bagasse

1 Jerusalem artichoke
Soy molasses
0
0 20 40 60 80 100
Raw materials price ($/MT)

Figure 6.6 Sensitivity analysis of unit production cost for the variation in the cost of raw

materials.
176
 7
Lignocellulose

6 Cassava bagasse

Butanol unit cost ($/gal)

Jerusalem artichoke
5 Soy molasses

2
0.15 0.2 0.25 0.3 0.35 0.4
Butanol yield (g/g sugars)

Figure 6.7 Sensitivity analysis of unit production cost for the variation in the yield of

butanol

Figure 6.8 Pretreatment of corn starch process

177
Figure 6.9 Pretreatment of lignocellulose hydrolysis process

Figure 6.10 Pretreatment of cassava bagasse

178
Figure 6.11 Pretreatment of Jerusalem artichoke

Figure 6.12 Pretreatment of soy molasses

179
 Figure 6.13 Fermentation Section

Figure 6.14 Separation of acetone, ethanol and butanol

180
 Chapter 7: Conclusions and Recommendations

7.1 Conclusions

In this study, high butanol production from different types of lignocellulosic

biomass and agriculture residues was successfully obtained. The inhibitory compounds

produced from the hydrolysis process of lignocellulosic biomass were investigated in

detail. The inhibitors' effects on C. beijerinckii, C. acetobutylicum and C. tyrobutyricum

strains were tested at different concentration. Lignin degraded phenolic inhibitors were

the most toxic ones. They can strongly inhibit the activities of butanol and butyraldehyde

dehydrogenases. They also have negative effects on CoA-transferase. Four

lignocellulosic biomass, cotton stalk, sugarcane bagasse, soybean hull and corn fiber, and

three non- lignocellulosic agriculture residues, cassava bagasse, Jerusalem artichoke and

soy molasses were all successfully hydrolyzed and detoxified. The detoxified

hydrolysates gave high butanol production as carbon sources in ABE fermentation. The

important findings in this project were summarized as follows.

7.1.1 Effects of lignocellulosic biomass hydrolysate derived inhibitors on ABE

fermentation

Sugar-degradation inhibitors (furfural, HMF, formic acid and levulinic acid) are

not toxic to C. beijerinckii, C. acetobutylicum and C. tyrobutyricum strains below 1.0 g/L.

181
On the contrary, lignin derived phenolic inhibitors (syringaldehyde, vanillin, ferulic acid

and p-coumaric acid) are very toxic to Clostridia strains even at 0.25 g/L. Their toxicity

has two aspects. First, they can destroy the cell membrane's permeability and lead to cell

death. The very low OD in the fermentation is due to the death of cells. Second, they can

strongly inhibit butanol and butyraldehyde dehydrogenases. So the Clostridia strains can

only produce a lot of acids, but cannot convert them to butanol.

7.1.2 Butanol production from lignocellulosic biomass and agriculture residues

Dilute acid pretreatment is an effective method to promote the enzymatic hydrolysis

of cellulose and hemicellulose in biomass. But high concentration of acids catalyzed the

further degradation of biomass components and generated large amount of inhibitors,

especially ferulic acid to ABE fermentation, exceeding the toxic level that inhibited the

butanol production, especially ferulic acid. For corn fiber, soybean hull and sugarcane

bagasse, 0.04 N was the best acid level. Activated carbon absorption was effective in

removing phenolic inhibitors in the hydrolysate, especially ferulic acid. The acid and

alkali pretreatments were also compared. The acid pretreatment removed 56%-90% of

hemicellulose and 7%-65% of lignin in the biomass at 0.04 N hydrogen ions while 0.04

N NaOH dissolved 44%-80% of hemicellulose and 46%-78% of lignin in the biomass.

Therefore, both acid and alkali pretreatments can remove part of hemicellulose and lignin

in the biomass. The dissolving capability depends on the compositions of the biomass.

Cassava bagasse hydrolysate can be a good carbon source for butanol production. It

had no toxicity to Clostridia. Jerusalem artichoke was easy to be hydrolyzed, but the

182
hydrolysate contained a higher level of ferulic acid, which partly inhibited the butanol

production. Activated carbon can adsorb 38% of ferulic acid in Jerusalem artichoke

hydrolysate, but not totally removed. Soybean meal with a high protein content (~40%)

can be used as a good nitrogen source, replacing yeast extract and corn steep liquor, in

ABE fermentation to produce butanol from various biomass feedstocks. Soybean meal

also has a high carbohydrate content (~30%), which mainly consists of oligosaccharides

(stachyose and raffinose). These oligosaccharides are difficult to be used by clostridia

and must be pre-hydrolyzed to simple sugars (sucrose, glucose, galactose) preferably by

using α-galactosidase. Soy molasses can be used as low-cost carbon source for butanol

production after enzymatic hydrolysis. Combining the hydrolysates from soybean meal

and soy molasses can provide an economical substrate for butanol production.

7.1.3 The inhibition mechanism of phenolic inhibitors to the metabolic pathway of

Clostridia

The phenolic compounds (p-coumaric acid, ferulic acid, vanillin and

syringaldehyde) were toxic to Clostridium strains with CoA-transferase but showed

almost no effects on strains without CoA-transferase. These phenolic inhibitors may work

as competitive inhibitors to CoA-transferase and block the solventogenesis phase that

converts acids into solvents. Corn steep liquor is rich in protein and amino acids, and is a

potential nitrogen source for butanol production. But the phenolic inhibitors in it may

severely inhibit butanol production by solventogenic Clostridia containing ctfAB genes.

183
7.2 Recommendations

Although seven different types of biomass were successfully hydrolyzed, detoxified

and used in ABE fermentation, giving high butanol production, many areas still require

further research before butanol fermentation can be commercialized and competitive with

petrochemically-derived butanol. Some recommendations about further work were listed

as follows.

7.2.1 Combined Effects of lignocellulosic biomass hydrolysate derived inhibitors

In this research, the inhibitors' effects on ABE fermentation were studied. Four

sugar-degradation inhibitors (furfural, HMF, formic acid and levulinic acid) and four

lignin derived inhibitors (syringaldehyde, vanillin, ferulic acid and p-coumaric acid) were

tested on three solventogenic Clostridia (C. beijerinckii, C. acetobutylicum and C.

tyrobutyricum). Some other inhibitors with larger molecule weights like lignin fragments

can also be produced in the hydrolysis of biomass The inhibition effects of these larger

inhibitors need further study. Other Clostridia such as C. saccharobutylicum and C.

aurantibutyricum can also be tested for their tolerance to inhibitors.

Individual inhibitor was used in the inhibition test of ABE fermentation. The

inhibition effects may be different if two or more inhibitors were combined together. The

combined inhibitors will be more like the situations in the real biomass hydrolysate. So

they may be closer to the real inhibition effects in the real biomass hydrolysate. The

combinations of different inhibitors may be huge. So statistical experiment design tools

are necessary. Another approach is that first find the inhibitors' types and concentrations

184
in the real biomass hydrolysate and then only combine the inhibitors existing in the

hydrolysate.

7.2.2 Optimizations of hydrolysis processes

Detailed acid and alkali pretreatments were studied in this research. Other

pretreatments such as steam explosion, ammonia explosion or biological degradation can

also be evaluated. The biomass change after pretreatment is the key to find a better

pretreatment for each biomass. In this study, after removing the acid or alkali

pretreatment supernatant, some of the biomass hydrolysates were still very toxic to

Clostridium strains. What caused this inhibition is still unclear and needs further study.

Other detoxification method can also be investigated. Lignin derived phenolic

inhibitors were found to be the toxic inhibitors to solventogenic Clostridia. So an

effective detoxification method must have the ability be removed all the phenolic

inhibitors. Overliming is the most cost effective detoxification method. But its capability

of removing phenolic compounds is still unclear. Ion exchange resins, especially anion

exchange resins showed promising efficiency in removing phenolic compounds.

Although activated carbon can adsorb the phenolic compounds effectively, it can also

remove certain portion of sugar. So other adsorbents that only remove phenolic

compounds can be tried in the detoxification process. Most inhibitors are produced in the

pretreatments and soluble in water, so another detoxification idea is that the inhibitors can

be removed together with the supernatants after the pretreatments. Removing the

185
pretreatment supernatants and washing the biomass solid can remove most of the soluble

inhibitors.

The compositions of biomass have great impact on the pretreatments and the final

components of hydrolysate. Different types of lignocellulosic biomass also have different

cellulose, hemicellulose and lignin contents. Even for one biomass, its compositions may

vary with different harvest seasons or different places This composition difference

induces different types of inhibitors in the hydrolysate and different detoxification

methods. Therefore, it is necessary to evaluate more biomass with different compositions.

This can further prove the diversity of substrates used in ABE fermentation and

feasibility of large-scale butanol production in different regions.

7.2.3 The inhibition mechanism of phenolic inhibitors to the metabolic pathway of

Clostridia

From this project, the phenolic compounds can inhibit the function of CoA-

transferase. So Clostridium strains that don't contain this CoA-transferase are preferred

when biomass hydrolysate is used as the substrates. Metabolic engineering that knock out

the ctfAB genes which express CoA-transferase or even block the entire acid production

phase is also a possible way to solve the inhibition. Another possible solution is to find an

effective detoxification method to remove the phenolic compounds from biomass

hydrolysate before the fermentation.

Further study about phenolic inhibitors' effects to the metabolic pathway of

solventogenic Clostridia is also needed. The function of phenolic compounds as

186
competitive inhibitors to CoA-transferase needs further proofs. It's better to isolate CoA-

transferase and test the effects of phenolic compounds directly on it. Our study also

showed phenolic compounds can inhibit butanol and butyraldehyde dehydrogenases.

Therefore, it seems phenolic compounds have several different inhibition points in the

metabolic pathway of solventogenic Clostridia. A complete understanding of phenolic

compounds' inhibition mechanism can guide the detoxification process and strain

developments.

187
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with the microbial production of butanol. J. Ind. Microbiol. Biotechnol., 36, 1127-

1138.

Zhu, Y. and S.T. Yang (2003). Adaption of Clostridium tyrobutyricum for enhanced

tolerance to butyric acid in a fibrous bed bioreactor. Biotechnol. Progress, 19, 365-

372.

Zhu, Y., Z. Wu, and S.T. Yang (2002). Butyric acid production from acid hydrolysate of

corn fibre by Clostridium tyrobutyricum in a fibrous bed bioreactor. Process

Biochem., 38, 657-666.

Zverlov, V.V., Berezina O., Velikodvorskaya G.A., Schwarz W.H. Bacterial acetone and

butanol production by industrial fermentation in the Soviet union: use of hydrolyzed

agricultural waste for biorefinery, Applied Microbiology and Biotechnology 2006,

71: 587-597

222
Appendix A. Cost analysis of butanol plants using soybean hull or soy molasses and

soybean meal as substrates

223
 Two plants are designed for butanol production. One uses soybean hull and

soybean meal as substrates, and the other uses soy molasses and soybean meal as

substrates. The process flowsheets for these two plants are shown in Figures A.1-3,

respectively. In general, the plant can be divided into three sections: Pretreatment section,

Fermentation section and Separation section.

In the Pretreatment section (Figure A.1), soybean hull is washed, dispensed in

water (P-2) and grinded into fine powder (P-3). Then, it is pumped into acid pretreatment

reactor (P-4). In this reactor, 90% of hemicellulose in soybean hull is hydrolyzed with

0.04 N HCl at 121 ℃, 15 psig. Then, 90% of cellulose is hydrolyzed by cellulase in the

following reactor (P-5, pH 4.5, 50℃). The hydrolysate is filtered (P-7) to remove ash,

lignin and other undissolved solids. The clear sugar solution is sent to the storage tank (P-

8) for use in the Fermentation section. The treatment of soybean meal is similar. The only

difference is that it doesn‘t need the enzymatic hydrolysis reactor. The pretreatment

process for soy molasses (Figure A.2) is simpler than that for soybean hull. Soy molasses

does not need to be washed, grinded and acid hydrolyzed. It only needs the enzymatic

hydrolysis reactor.

In the Fermentation section (Figure A.3), the seed culture is prepared in the seed

fermentor (P-14) and then pumped into the main fermentor (P-1). The fermentation broth

containing butanol is sent to the storage tank (P-15) for butanol separation. In the

Separation section, distillation is used to separate different solvents. The first two

columns (P-19, P-20) are used to remove acetone and ethanol from the solution. The

224
purification of butanol needs two stages (P-21, P-23) because butanol-water system has

an azeotropic point. At last, the main product is 99.9% butanol.

Cost analysis is based on an annual butanol production of 7.6 MM gal (23,000 ton)

at each plant with the following fermentation parameters: butanol yield, 0.30 g/g sugar;

volumetric productivity, 5.5 g/L·h; butanol titer, 22 g/L. The economic analysis for the

plant using soybean hull and soybean meal is summarized in Table A.1. The total capital

investment is $13.7 MM. The annual operating cost is $29.6 MM. The production cost is

$3.90/gal butanol. The major equipment is listed in Table A.2. The most costly pieces of

equipment are 4 main fermentors (FR-101) and 6 enzyme hydrolysis reactors (R-102).

The Pretreatment section accounts for 43% of the equipment cost, and the Fermentation

section accounts for 22%. Table A.3 lists the other capital investments and Table A.4 lists

the material costs. Soybean meal accounts for 51% of the total cost. Table A.5 lists the

annual operating cost. About 1/3 comes from raw materials, 1/3 from labor and 1/3 from

utilities. Table A.6 shows the utility cost summary. About 73% comes from steam, which

is mainly used in the Separation section. So if the butanol separation method is improved

such as using gas stripping or pervaporation, this steam cost can be significantly reduced.

The steam cost can also be reduced if the plant has its own utility section, which means

burning part of soybean hull to produce steam.

Tables A.7-12 shows the detailed cost analysis for the plant using soy molasses

and soybean meal. Compared to soybean hull plant, this plant has less total capital

investment ($10 MM). One reason is that soy molasses does not need washing, grinding

and acid pretreatment. The other reason is that its enzymatic hydrolysis is much faster
225
than soybean hull, so the total volume of the enzymatic hydrolysis reactors is much

smaller. Soybean hull needs six 200 m3 reactors, while soy molasses only needs two 150

m3 reactors. The operating cost is also reduced because there are fewer enzyme reactors

in the Pretreatment section. The unit product cost is $3.54/gal butanol.

In conclusion, soybean hull, soy molasses and soybean meal can be used as

economical substrates for industrial production of butanol. The projected butanol

production cost from these substrates is significantly lower than the current market price

(~$6.75/gal) and the process should be feasible and economically attractive for industrial

application.

226
 Table A.1 Executive summary (2013 prices) - Soybean hull

Total Capital Investment 13,753,000.00 $

Capital Investment Charged to This Project 13,753,000.00 $

Operating Cost 29,648,000.00 $/yr

Main Revenue 36,855,000.00 $/yr

Other Revenues 1,835,835.00 $/yr

Total Revenues 38,690,000.00 $/yr

Cost Basis Annual Rate 7,605,000 gal(STP) Bu/yr

Unit Production Cost 3.90 $/gal(STP) Bu

Unit Production Revenue 5.09 $/gal(STP) Bu

Gross Margin 23.37 %

Return On Investment 46.87 %

Payback Time 2.13 years

IRR (After Taxes) 27.89 %

NPV (at 7.0% Interest) 48,090,000.00 $

Bu = Total Flow of Stream '>99.9% Butanol'

227
 Table A.2 Major equipment specifications and cost (2013 prices) - Soybean hull

Quantity/

Standby/ Name Description Unit Cost ($) Cost ($)

Staggered

1/0/0 GR-101 Grinder 60,000.00 60,000.00

Size/Capacity = 100000.00

kg/h

1/0/0 GP-101 Gear Pump 12,000.00 12,000.00

Pump Power = 3.78 kW

1/0/0 BF-101 Belt Filter 80,000.00 80,000.00

Belt Width = 31.00 m

1/0/0 GR-102 Grinder 30,000.00 30,000.00

Size/Capacity = 10000.00 kg/h

1/0/0 GP-102 Gear Pump 2,000.00 2,000.00

Pump Power = 0.39 kW

1/0/0 BF-102 Belt Filter 40,000.00 40,000.00

Belt Width = 3.33 m

1/0/0 C-101 Distillation Column 45,000.00 45,000.00

Column Volume = 9481.82 L

2/0/0 C-102 Distillation Column 57,000.00 114,000.00

Column Volume = 11892.87 L

Continued

228
Table A.2 Continued
1/0/0 C-103 Distillation Column 34,000.00 34,000.00

Column Volume = 7319.60 L

1/0/0 V-104 Decanter Tank 38,000.00 38,000.00

Vessel Volume = 1108.79 L

1/0/0 C-104 Distillation Column 31,000.00 31,000.00

Column Volume = 1438.95 L

1/0/0 R-101 Stirred Reactor 80,000.00 80,000.00

Vessel Volume = 52.95 m3

6/0/0 R-102 Stirred Reactor 60,000.00 360,000.00

Vessel Volume = 190.08 m3

1/0/0 R-103 Stirred Reactor 50,000.00 50,000.00

Vessel Volume = 11060.47 L

1/0/0 V-101 Flat Bottom Tank 70,000.00 70,000.00

Vessel Volume = 154.92 m3

1/0/0 SFR-101 Seed Fermentor 48,000.00 48,000.00

Vessel Volume = 15.49 m3

4/0/0 FR-101 Fermentor 70,000.00 280,000.00

Vessel Volume = 154.97 m3

1/0/0 V-102 Flat Bottom Tank 70,000.00 70,000.00

Vessel Volume = 152.48 m3

Unlisted Equipment 361,000.00

229
 Table A.3 Fixed capital estimate summary (2013 prices in $) - Soybean hull

A. Total Plant Direct Cost (TPDC) (physical cost)

1. Equipment Purchase Cost 1,805,000.00

2. Installation 641,000.00

3. Process Piping 632,000.00

4. Instrumentation 722,000.00

5. Insulation 54,000.00

6. Electrical 181,000.00

7. Buildings 812,000.00

8. Yard Improvement 271,000.00

9. Auxiliary Facilities 722,000.00

TPDC 5,839,000.00

B. Total Plant Indirect Cost (TPIC)

10. Engineering 1,460,000.00

11. Construction 2,044,000.00

TPIC 3,504,000.00

C. Total Plant Cost (TPC = TPDC+TPIC)

TPC 9,343,000.00

D. Contractor's Fee & Contingency (CFC)

12. Contractor's Fee 467,000.00

Continued

230
Table A.3 Continued

13. Contingency 934,000.00

CFC = 12+13 1,401,000.00

E. Direct Fixed Capital Cost (DFC = TPC+CFC)

DFC 10,744,000.00

Table A.4 Material costs - Soybean hull

Unit Cost Annual Annual Cost

Bulk Material %
($) Amount ($)

soybean hull 20.00 158,400.00 MT 3,168,000.00 32.85

soybean meal 280.00 15,714.00 MT 4,888,972.00 50.70

HCl 0.10 25.00 MT 2,455.00 0.03

Cellulase 2.00 792.00 MT 1,584,000.00 16.43

TOTAL 9,643,427.00 100.00

231
 Table A.5 Annual operating cost (2013 prices) - Soybean hull

Cost Item $ %

Raw Materials 9,643,000.00 32.53

Labor-Dependent 8,778,000.00 29.61

Facility-Dependent 2,025,000.00 6.83

Laboratory/QC/QA 439,000.00 1.48

Utilities 8,763,000.00 29.56

TOTAL 29,648,000.00 100.00

Table A.6 Utilities cost (2013 prices) - Soybean hull

Unit Cost Annual Ref. Annual Cost

Utility %
($) Amount Units ($)

Std Power 0.05 2,179,998.00 kW-h 109,000.00 1.24

Steam 12.00 535,443.00 MT 6,425,316.00 73.32

Cooling Water 0.05 44,580,414.00 MT 2,229,021.00 25.44

TOTAL 8,763,337.00 100.00

232
 Table A.7 Executive summary (2013 prices) - Soy malasses

Total Capital Investment 10,504,000.00 $

Capital Investment Charged to This Project 10,504,000.00 $

Operating Cost 26,136,000.00 $/yr

Main Revenue 35,736,000.00 $/yr

Other Revenues 1,780,110.00 $/yr

Total Revenues 37,516,000.00 $/yr

Cost Basis Annual Rate 7,374,334.44 gal(STP) Bu/yr

Unit Production Cost 3.54 $/gal(STP) Bu

Unit Production Revenue 5.09 $/gal(STP) Bu

Gross Margin 30.33 %

Return On Investment 72.15 %

Payback Time 1.39 years

IRR (After Taxes) 40.39 %

NPV (at 7.0% Interest) 64,513,000.00 $

Bu = Total Flow of Stream '>99.9% Butanol'

233
 Table A.8 Major equipment specifications and cost (2013 prices) - Soy molasses

Quantity/

Standby/ Name Description Unit Cost ($) Cost ($)

Staggered

1/0/0 BF-101 Belt Filter 80,000.00 80,000.00

Belt Width = 60.00 m

1/0/0 GR-102 Grinder 30,000.00 30,000.00

Size/Capacity = 10000.00 kg/h

1/0/0 GP-102 Gear Pump 2,000.00 2,000.00

Pump Power = 0.39 kW

1/0/0 BF-102 Belt Filter 40,000.00 40,000.00

Belt Width = 3.33 m

1/0/0 C-101 Distillation Column 47,000.00 47,000.00

Column Volume = 10882.94 L

2/0/0 C-102 Distillation Column 61,000.00 122,000.00

Column Volume = 13657.85 L

1/0/0 C-103 Distillation Column 36,000.00 36,000.00

Column Volume = 8318.65 L

1/0/0 V-104 Decanter Tank 38,000.00 38,000.00

Vessel Volume = 1095.06 L

1/0/0 C-104 Distillation Column 32,000.00 32,000.00

Continued

234
Table A.8 Continued

Column Volume = 1556.40 L

2/0/0 R-102 Stirred Reactor 60,000.00 120,000.00

Vessel Volume = 105.13 m3

1/0/0 R-103 Stirred Reactor 50,000.00 50,000.00

Vessel Volume = 11060.47 L

1/0/0 V-101 Flat Bottom Tank 70,000.00 70,000.00

Vessel Volume = 177.11 m3

1/0/0 SFR-101 Seed Fermentor 51,000.00 51,000.00

Vessel Volume = 17.71 m3

4/0/0 FR-101 Fermentor 70,000.00 280,000.00

Vessel Volume = 177.16 m3

1/0/0 V-102 Flat Bottom Tank 70,000.00 70,000.00

Vessel Volume = 174.74 m3

Unlisted Equipment 267,000.00

TOTAL 1,335,000.00

235
 Table A.9 Fixed capital estimate summary (2013 prices in $) - Soy molasses

A. Total Plant Direct Cost (TPDC) (physical cost)

1. Equipment Purchase Cost 1,335,000.00

2. Installation 451,000.00

3. Process Piping 467,000.00

4. Instrumentation 534,000.00

5. Insulation 40,000.00

6. Electrical 134,000.00

7. Buildings 601,000.00

8. Yard Improvement 200,000.00

9. Auxiliary Facilities 534,000.00

TPDC 4,296,000.00

B. Total Plant Indirect Cost (TPIC)

10. Engineering 1,074,000.00

11. Construction 1,503,000.00

TPIC 2,577,000.00

C. Total Plant Cost (TPC = TPDC+TPIC)

TPC 6,873,000.00

D. Contractor's Fee & Contingency (CFC)

12. Contractor's Fee 344,000.00

13. Contingency 687,000.00

Continued

236
Table A.9 Continued

CFC = 12+13 1,031,000.00

E. Direct Fixed Capital Cost (DFC = TPC+CFC)

DFC 7,904,000.00

Table A.10 Materials cost – Soy molasses

Unit
Annual Annual Cost
Bulk Material Cost %
Amount ($)
($)

soybean molasses 0.05 39895306.00 gal(STP) 1,994,765.00 25.98

soybean meal 280.00 15715.00 MT 4,888,972.00 63.67

a-galactosidase 1.00 792.00 MT 792,000.00 10.32

HCl 0.10 24.00 MT 2,376.00 0.03

TOTAL 7,678,113.00 100.00

237
 Table A.11 Annual operating cost (2013 prices) – Soy molasses

Cost Item $ %

Raw Materials 7,678,000.00 29.38

Labor-Dependent 7,783,000.00 29.78

Facility-Dependent 1,490,000.00 5.70

Laboratory/QC/QA 389,000.00 1.49

Utilities 8,796,000.00 33.65

TOTAL 26,136,000.00 100.00

Table A.12 Utilities cost (2013 prices) - Soy molasses

Unit
Annual Ref. Annual Cost
Utility Cost %
Amount Units ($)
($)

Std Power 0.05 1022497.00 kW-h 51125.00 0.58

Steam 12.00 541082.00 MT 6492982.00 73.82

Cooling Water 0.05 45036648.00 MT 2251832.00 25.60

TOTAL 8795939.00 100.00

238
239

To Fermentation
Section

Figure A.1 The acid pretreatment and hydrolysis processes for soybean hull and soybean meal
240

To Fermentation
Section

Figure A.2 The pretreatment and hydrolysis processes for soy molasses and soybean meal.
 From Pretreatment Section
241

Figure A.3 Process flowsheet for butanol fermentation and separation.

Appendix B. Analytical procedures of inhibitors by high performance liquid

chromatograph

242
 The inhibitors including furfural, HMF, levulinic acid, ferulic acid, p-coumaric

acid, and phenolic compounds (syringaldehyde) were analyzed by high performance

liquid chromatography (HPLC) using Rezex ROA-Organic Acid H+ (8%) column

(Phenomenex) at 40 oC and a UV/UIS detector (SPD-20AV, Shimadzu). Samples were

centrifuged at 13.2 g for 5 min in microcentrifuge tubes and diluted 20 times with distilled

water prior to analysis on HPLC. The eluent was 0.01 N H2SO4 at 0.6 ml/min. Furfural,

HMF and phenolic compounds were detected at 280 nm. Syringaldehyde was used as the

standard for phenolic compounds. Levulinic acid, ferulic acid, p-coumaric acid were

detected at 200 nm. 15μL sample was injected by an automatic injector (SIL-10Ai) and the

running time was set at 60 min. Peak height was used to calculate concentration of sugars in

the sample based on the peak height of standard sample. HPLC chromatogram of the 1.0 g/L

standard inhibitor sample is shown in Figure B.1-B.8.

30 30

20 20
mAU

mAU
10 10

0 0

0 5 10 15 20 25 30 35 40 45
Minutes

Figure B.1 HPLC chromatogram of 1.0 g/L hydroquinone at 280 nm

243
 150 150

100 100
mAU

mAU
50 50

0 0

0 5 10 15 20 25 30 35 40 45
Minutes

Figure B.2 HPLC chromatogram of 1.0 g/L furfural at 280 nm

4000 4000
mAU

mAU
2000 2000

0 0

10 15 20 25 30 35 40
Minutes

Figure B.2 HPLC chromatogram of 1.0 g/L HMF at 280 nm

244
 200 200
mAU

mAU
100 100

0 0

0 10 20 30 40 50 60
Minutes

Figure B.4 HPLC chromatogram of 1.0 g/L syringaldehyde at 280 nm

10 10
mAU

mAU
5 5

0 0

0 5 10 15 20 25 30 35 40 45
Minutes

Figure B.5 HPLC chromatogram of 1.0 g/L levulinic acid at 200 nm

245
 40 40

30 30

20 20
mAU

mAU
10 10

0 0

0 5 10 15 20 25 30 35 40
Minutes

Figure B.6 HPLC chromatogram of 1.0 g/L p-coumaric acid at 200 nm

Figure B.6 HPLC chromatogram of 1.0 g/L formic acid at 200 nm

246