fermentation of carbohydrates in ......1-2 ethanol production from sugar or starch feedstock..... 38...
TRANSCRIPT
FERMENTATION OF CARBOHYDRATES IN SWITCHGRASS TO ETHANOL: OPTIMIZING PRETREATMENT AND FERMENTATION CONDITIONS
By
WEI WU
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2017
© 2017 Wei Wu
To my husband, my parents and my two sweet girls who supported me make every progress on my way
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ACKNOWLEDGMENTS
I thank my advisor, Dr. Pratap Pullammanappallil, for allowing me to join his lab. I
thank Dr. Lonnie Ingram for allowing me to do my research in the Stan Mayfield
Biorefinery (Perry pilot plant) and giving me invaluable advice and support. I thank my
committee co-chair Dr. K. T. Shanmugam for his mentorship throughout my graduate
study. I thank my committee members for all their guidance and support. I thank Dr.
Ismael Nieves, Kalvin Weeks, Vanessa Rondon, Dr. Eulogio Castro, Sean York, Joe
Sagues for all the help at the pilot plant. Dr. Ismael Nieves and Kalvin Weeks were
instrumental in carrying out pretreatment of switchgrass. Vanessa Rondon and Dr.
Castro assisted in carrying out fermentations. Sean York was instrumental in creating
the hydrolysate-resistant strain SL100. Joe Sagues gave support by sharing equipment
used in my experiments. I also thank Mr. Fred Circle, FDC enterprises, for providing the
switchgrass used in this study. I thank my parents for their love and support that make
me hold on to my goal and pass each milestone.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 8
LIST OF FIGURES .......................................................................................................... 9
ABSTRACT ................................................................................................................... 11
CHAPTER
1 INTRODUCTION .................................................................................................... 13
History and Background of Biomass Materials for Biofuel and Biochemical ........... 13
Biomass Types, Sources and Availability ......................................................... 17 Dedicated energy crops ............................................................................. 18 Advantages of switchgrass ........................................................................ 18 Environmental benefits .............................................................................. 19
Pretreatment ..................................................................................................... 19 Saccharification ................................................................................................ 23 Microorganisms ................................................................................................ 25
Yeast .......................................................................................................... 25 Escherichia. coli ......................................................................................... 26 Ethanol production improvement ............................................................... 26 Inhibitor reduction ...................................................................................... 27 Cellulases secretion ................................................................................... 28
Fermentation Process Choice .......................................................................... 29 Separate Hydrolysis and Fermentation(SHF) ............................................ 29 Simultaneous Saccharification and Fermentation (SSF) ............................ 30 Comparison between SHF and SSF .......................................................... 31
Proposed Method ................................................................................................... 32 Dilute Phosphoric Acid Catalyzed Steam Explosion Pretreatment ................... 32 Saccharification ................................................................................................ 32 Simultaneous Saccharification co-Fermentation Method (SScF) ..................... 32
Research Objectives ............................................................................................... 33
2 OPTIMIZING STEAM EXPLOSION PRETREATMENT OF SWITCHGRASS WITH DILUTE PHOSPHORIC ACID ...................................................................... 43
Introduction ............................................................................................................. 43 Materials and Methods............................................................................................ 44
Materials ........................................................................................................... 44 Composition Analysis ....................................................................................... 45 Pretreatment ..................................................................................................... 45
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Full Test for Pretreated Switchgrass ................................................................ 46 Characterization of the Hydrolysate ................................................................. 46
Results and Discussion........................................................................................... 47 Effect of Phosphoric Acid as a Catalyst on Hemicellulose Hydrolysis .............. 47 Effect of Temperature on Switchgrass Pretreatment ........................................ 49 Effect of Acid Concentration on Switchgrass Pretreatment .............................. 50 Effect of Pretreatment Time on Switchgrass Hydrolysis ................................... 50
Summary ................................................................................................................ 51
3 OPTIMIZING ENZYMETIC LIQUEFACTION OF ACID PRETREATED SWITCHGRASS SLURRY ...................................................................................... 65
Introduction ............................................................................................................. 65 Materials and Methods............................................................................................ 65
Materials ........................................................................................................... 65 Liquefaction of Pretreated Slurry ...................................................................... 66 Composition of the Slurry before Liquefaction .................................................. 66 Detoxification of Pretreated Slurry for Liquefaction and Downstream
Fermentation ................................................................................................. 66 Results and Discussion........................................................................................... 67
Composition of the Slurry after Pretreatment ................................................... 67 Effect of Pretreatment Temperature on Enzyme Liquefaction .......................... 67 Effect of Acid Concentration during Pretreatment on Enzyme Liquefaction ..... 68 Effect of Pretreatment Time on Enzyme Liquefaction ...................................... 70 Effect of Enzyme Level on Enzyme Liquefaction .............................................. 71 Increase in Solids Concentration on Hydrolysis ............................................... 72
4 ETHANOL PRODUCTION FROM PHOSPHORIC ACID PRETREATED SWITCHGRASS USING INHIBITORY PRODUCTS TOLERANT ESCHERICHIA COLI STRAIN SL100 THROUGH LIQUEFACTION PLUS SIMULTANEOUS SACCHARIFICATION AND CO-FERMENTATION PROCESS .............................................................................................................. 85
Introduction ............................................................................................................. 85 Materials and Methods............................................................................................ 86
Materials ........................................................................................................... 86 Ethanologenic E. coli ........................................................................................ 86 Media, Seed Strain Propagation and Growth Conditions ................................. 87
Medium for cultivation of seed cultures ...................................................... 87 Preparation of seed cultures ...................................................................... 88
Simultaneous Saccharification and co-Fermentation of Liquefied Switchgrass Slurry ........................................................................................ 89
Chemical Analysis ............................................................................................ 90 Results and Discussion........................................................................................... 90
Preparation of Seed Cultures of E. coli Strain SL100 ....................................... 91 Fermentation of Pretreated and Liquefied Switchgrass Slurry.......................... 92 Effect of Pretreatment Temperature on Ethanol Production ............................. 93
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Effect of Acid Concentration in Pretreatment on Ethanol Titer and Yield ......... 94 Effect of Pretreatment Time on Ethanol Titer and Yield ................................... 95 Effect of Enzyme Concentration on Ethanol Production and Yield ................... 96 Effect of Solid Loading Level on Ethanol Production and Yield ........................ 97
5 MASS BALANCE OF ETHANOL PRODUCTION FROM ACID PRETREATED SWITCHGRASS WITH E. COLI SL100 THROUGH L+SScF PROCESS ............. 109
Introduction ........................................................................................................... 109 Result and Discussion .......................................................................................... 109
6 GENERAL CONCLUSION AND FUTURE DIRECTIONS ..................................... 116
LIST OF REFERENCES ............................................................................................. 119
BIOGRAPHICAL SKETCH .......................................................................................... 131
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LIST OF TABLES
Table page 1-1 Composition of different biomass ....................................................................... 34
1-2 Technologies and reaction conditions for biomass pretreatment ........................ 35
1-3 SHF process vs. SSF process applied to corn stover and loblolly pine .............. 36
2-1 Comparison of pretreatment of switchgrass with and without phosphoric acid ... 53
2-2 Effect of pretreatment conditions on the release of sugars from switchgrass ..... 54
2-3 Inhibitors in the hydrolysate of pretreated biomass ............................................ 55
3-1 Composition of switchgrass and slurry from various pretreatment conditions .... 74
3-2 Inhibitors in the slurry after liquefaction of various pretreatment condition ......... 75
3-3 Effect of various pretreatment conditions on enzyme hydrolysis of the slurry during liquefaction .............................................................................................. 76
3-4 Total amount of sugars released by the combined pretreatment and liquefaction steps ................................................................................................ 77
3-5 Effect of enzyme concentration on the amount of sugars released from pretreated switchgrass slurry .............................................................................. 78
3-6 Enzyme-catalyzed release of sugars from pretreated switchgrass slurry at 15% solids loading .............................................................................................. 79
4-1 Ethanol production from the slurry of various pretreatment conditions ............... 99
4-2 Ethanol production from the slurry at various enzyme loading ......................... 100
4-3 Comparison of residue after fermentation of 10% and 15% solids loading ....... 101
5-1 Composition of residue after fermentation ........................................................ 112
5-2 Effect of pretreatment condition on the amount of sugars fermented and remaining after fermentation ............................................................................. 113
5-3 Effect of pretreatment condition on the yield of ethanol from switchgrass ........ 114
5-4 Mass balance from fermentation of switchgrass ............................................... 115
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LIST OF FIGURES
Figure page 1-1 Ethanol production from lignocellulose ............................................................... 37
1-2 Ethanol production from sugar or starch feedstock ............................................ 38
1-3 Major types of inhibitors and their chemical structure ......................................... 39
1-4 Phenolic compounds that may act as inhibitors or deactivators of cellulases ..... 40
1-5 Fermentation pathways in E. coli with an engineered pathway for homoethanol production. .................................................................................... 41
1-6 Schematic representation of SSFF integrated process ...................................... 42
2-1 Process for ethanol production from cellulosic biomass ..................................... 56
2-2 Effect of pretreatment temperature on switchgrass hydrolysis ........................... 57
2-3 Effect of pretreatment temperature on switchgrass hydrolysis ........................... 58
2-4 Effect of acid concentration during pretreatment on switchgrass hydrolysis ....... 59
2-5 Effect of residence time during pretreatment on switchgrass hydrolysis ............ 60
2-6 Sugars in the hydrolysate of slurries from all pretreatments ............................... 61
2-7 Furans and organic acids in the hydrolysate from all pretreatment conditions ... 62
2-8 Water insoluble solids (WIS) from all pretreatment conditions ........................... 63
2-9 Composition of switchgrass solids before and after pretreatment ...................... 64
3-1 Effect of pretreatment temperature on hydrolysis of carbohydrates during enzyme liquefaction. ........................................................................................... 80
3-2 Effect of phosphoric acid concentration during pretreatment on enzyme liquefaction. ........................................................................................................ 81
3-3 Effect of pretreatment time on enzyme liquefaction. ........................................... 82
3-4 Effect of enzyme concentration during liquefaction on the amount of sugars released from a slurry from 190-1-7.5 pretreatment. .......................................... 83
3-5 Effect of solids loading on enzyme liquefaction .................................................. 84
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4-1 Effect of switchgrass hydrolysate concentration on the growth and fermentation of E. coli strain SL100. ................................................................. 102
4-2 Fermentation of pretreated and liquefied switchgrass slurry to ethanol by E. coli strain SL100 ............................................................................................... 103
4-3 Effect of pretreatment temperature on fermentation ......................................... 104
4-4 Effect of pretreatment acid concentration on ethanol production ...................... 105
4-5 Effect of pretreatment time on ethanol production ............................................ 106
4-6 Effect of enzyme concentration on ethanol production from the liquefied slurry from pretreatment condition of 190-1-7.5. ............................................... 107
4-7 Effect of solid loading during fermentation on ethanol production and yield from pretreatment condition 190-1-7.5 ............................................................. 108
6-1 Ethanol production from switchgrass through L+SScF process ....................... 118
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
FERMENTATION OF CARBOHYDRATES IN SWITCHGRASS TO ETHANOL:
OPTIMIZING PRETREATMENT AND FERMENTATION CONDITIONS
By
Wei Wu
August 2017
Chair: Pratap Pullammanappallil Co-chair: Keelnatham T. Shanmugam Major: Agricultural and Biological Engineering
Steam explosion pretreatment of switchgrass (Alamo) with low levels of dilute
phosphoric acid was shown to effectively destabilize the biomass structure.
Pretreatment condition of 190oC with 1% phosphoric acid and steam for 10 minutes,
selected among the combination of three variables including temperature (160-190oC),
acid concentration (0-1%) and pretreatment time (5-10minutes), released highest
amount of sugar and the highest level of inhibitors, though the pretreated slurry was still
fermented successfully by ethanologenic E. coli SL100. The amount of sugar monomers
released by pretreatment of switchgrass increased mostly with increasing temperature
and minimally with acid concentration and time, which was mostly xylose. The inhibitory
side-products also increased with pretreatment severity as well.
After pretreatment, an enzyme liquefaction process (50oC, pH 5) effectively
increased handling and mixing of the slurry before fermentation. The enzymes
continued to hydrolyze biomass solids during co-fermentation of the released sugars
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(37oC, pH 7) even though the temperature and pH were not optimum for the fungal
enzymes.
E. coli SL 100 is not only tolerant to volatile inhibitors such like furfural, but also
to nonvolatile compounds in the acid hydrolysate. With this mutant, liquefied slurry of
acid pretreated switchgrass was successfully fermented without separation of solid fiber
and hemicellulose hydrolysate or removal of toxin. The overall consolidated
bioprocessing steps termed Liquefaction + Simultaneous Saccharification and co-
fermentation (L+SScF) were used on pretreated switchgrass with E. coli SL100 as the
microbial biocatalyst.
Highest ethanol titer obtained with 5% (v/w, 10ml enzyme per 200g dry weight
switchgrass) enzyme loading and 10% solids loading was from pretreatment condition
of 190oC,1% phosphoric acid and 7.5 minutes residence time; 57.8 gallons of ethanol
per ton of dry switchgrass.
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CHAPTER 1 INTRODUCTION
This research is targeting the optimization of a new pretreatment method for
switchgrass to release fermentable sugars, and using an ethanologenic Escherichia coli
strain to co-ferment the hexose and pentose sugars released from switchgrass to
ethanol.
History and Background of Biomass Materials for Biofuel and Biochemical
Worldwide demand for energy is expected to double in 30 years and this
additional requirement cannot be satisfied by crude oil, natural gas, coal and nuclear
energy combined (Hashimoto et al., 2014). Even with new discovery of oil reservoirs,
the world’s economy is still dependent on very limited number of fossil fuels exporting
countries. America’s light-duty transportation fleet constitutes 232 million vehicles, and
these require about 8.1 million barrels of oil to keep them running every day. America’s
transportation sector relies almost exclusively on refined petroleum products, which
account for more than 71% (Davis et al., 2013) of the petroleum used. At the same time
with concerns of climate change, energy independence, finite nature of oil, as well as
the economic security of the nation, developing sustainable sources of energy and
products have become an immediate objective of several countries, including U.S.A.
Renewable energy therefore holds the attention of the world as a substitute for
fossil-based liquid transportation fuels and bioproducts. Biomass is an energy resource
comprising photosynthetic material such as agricultural residues, forest resources,
perennial grasses, woody energy crops, algae, municipal solid waste, etc. It is unique
among renewable energy resources that it can be converted to carbon-based fuels,
chemicals or power.
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To combat the energy problem, in 2005, President George W. Bush signed the
Energy Policy Act into law that provided tax incentives and loan guarantees for
renewable energy production of various types. In 2007, the Energy Independence and
Security Act (EISA) set aggressive goals to reduce the nation’s dependence on fossil
fuels and reduce greenhouse gas emissions from the transportation sector by
increasing the supply of renewable transportation fuels to 36 billion gallons by 2022
(Independence, 2007). Half of the 36 billion gallons of liquid fuel, such as ethanol, is
expected to be derived from cellulosic biomass. The United States has the capacity to
produce more than one billion tons of sustainable biomass per year and this can be
used to produce the required cellulosic biofuels with the development of an effective
technology for the conversion of lignocellulosic biomass to fuels.
Although petroleum based fuel is also biomass-derived, fixation of CO2 from the
atmosphere to produce this biomass and its utilization as fuel today are separated by
millions of years. This time differential in production of biomass and consumption of
petroleum is the leading cause of current environmental damage. By coupling
production and consumption of biomass, a sustainable cyclic process with minimal
environmental impact can be developed.
Ethanol is a readily available liquid fuel produced with sugars from sugarcane or
sugar beet, or starch found in the grain of cereal crops. Ethanol has a relatively high
octane number and high heat of vaporization which makes it easy to be blended into
petroleum fuel. It is an excellent fuel for advanced flex-fuel vehicles (Hahn-Hagerdal et
al., 2006). The manufacture of ethanol is generally associated with rural employment
and diversification of rural economies (Hillring, 2002; Evans, 1997). Widespread use of
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ethanol-blended gasoline will increase energy security due to independence from
imported oil.
In the U.S.A and Canada, corn is the dominant commercial feedstock used by
the ethanol industry (Mabee et al., 2011). Corn is a well-suited feedstock for ethanol
production as it has a relatively high starch content of about 72% by mass of the grain
kernel (Huntington, 1997). With the development of the plant as well as the technology
of tilling, irrigation and harvesting, the yield of corn is high at about 395 bushels/ha and
increasing (WAOB, 2015). Using a simplified assumption of 70% starch on a dry-weight
basis, this production corresponds to between 6.8 and 7.2 t/ha of starch that can be
utilized in the bioconversion process (McAloon et al., 2000). Conversion technology of
cornstarch to ethanol is relatively simple and the infrastructure for planting, harvesting,
and processing is already in place.
The sugar or starch based bioethanol is not a long-term solution to meet the
energy demand, since it has three serious limitations. First, corn, sugarcane, or sugar
beet are all food crops, and their use in biofuel production will compete with food and
animal feed, which will increase the food price potentially leading to hunger, especially
in developing countries. Second, the use of corn or sugar cane for fuel production will
indirectly compete with other food crops for fertile soil and force the use of marginal
lands for food production that will lower the crop yield per hectare. Third, the effective
savings of CO2 emission and fossil fuel consumption are limited by the energy needed
to grow the crop and convert it to biofuel.
To overcome these limitations and expand biofuel production, research is
focused on utilization of lignocellulosic materials for biofuels production in a sustainable
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way (Davis et al., 2013; Chaturvedi and Verma, 2013). Lignocellulose, a combination of
three polymers (cellulose, hemicellulose and lignin) interlinked in a dense matrix, is
present in all biomass, including residues from agricultural and forest operations.
Cellulose is a straight chain polymer consisting of units of glucose connected via
(1,4)-β linkages. It has high molecular weight and held rigidly together as bundles of
fibers to provide material strength. Different plant cells have various cellulose
component percentages.
Hemicellulose is a heterogeneous co-polymer made up of five sugars, including
glucose, galactose, mannose, xylose, and arabinose. Hemicellulose polymer chain is
shorter than cellulose and functions like glue to bundle together well organized cellulose
into crystalline pattern.
Lignin consists of a tri-dimensional polymer of propyl-phenol that is imbedded in
and bound to the hemicellulose, which provides rigidity to the structure. Although lignin
is not fermented by microorganisms, the phenolic compounds present in this polymer
upon release may act as inhibitors of fermentation (Lange, 2007).
To be used as feedstock in a biofuel industry, lignocellulose has to satisfy these
elements: availability of large amount, sustainable supplies of regionally available
biomass, cost-effective feedstock infrastructure, equipment and systems for harvesting,
collection, storage, preprocessing and transportation.
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Biomass Types, Sources and Availability
Agricultural feedstock and residues, particularly bagasse, are likely to offer some
of the lowest cost lignocellulosic feedstock available in significant quantities (Kline et al.,
2008). Since bagasse and wood process residue are relatively concentrated at the
processing location, the collecting cost is low. Other agriculture residues like straw have
to be collected from harvesting sites and separated from grains, which add to cost of
feedstock.
The variability of biomass is another main characteristic that affect bioethanol
production. For instance, cereal straws from both Europe and North America are
characterized by a composition of cellulose between 35-40% of total oven dry weight,
hemicellulose between 26-27%, and lignin between 15- 20% (Misra, 1993). The balance
of the mass is made up of non-organic ash and silica, which for straw can vary between
10-20% (higher in rice straw than other cereal straws) and 2-5% for wood. Even within a
single cereal species, some variation occurs within the specified ranges due to both
environmental and genetic factors (IEA, 2009). Within the same species, composition of
the crop differs with harvest season, for instance switchgrass harvested in October
could yield sugars as much as 110 g/kg biomass than those harvested in July under
same hydrolysis conditions (AFEX conditions ranged from 0.4 to 2 g ammonia/g dry
biomass, 0.4 to 2 g water/g dry biomass, and 5 to 30 min residence time) (Bals et al.,
2010). For enzymatic hydrolysis of wood residues, the species variation in basic
chemistry is even more significant than in agricultural residues, particularly when
comparing softwood and hardwood species. Softwood includes species of pine, spruce,
hemlock and fir that have cellulose content around 40% of total dry weight, which is
slightly higher in hardwoods up to 42%. Hemicellulose and lignin component in different
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biomass is listed in Table1-1. Hemicellulose in soft wood and hard wood are very
different, soft wood has arabinan and xylan, while hard wood and crop residue only has
xylan. The differences between the characteristics of wood, straw and vegetative
grasses can create challenges for bioconversion in multi-feedstock processing plants
(IEA, 2009).
Dedicated energy crops
Energy crops are grown specifically for production of biofuel and offer high yield
per hectare with minimum input. Energy crops include different ecotypes and species
from grasses to trees. Grasses include: big bluestem (Andropogon gerardi), wheat
grass (Thinopyrum intermedium), switchgrass (Panicum virgatum), reed canary
(Phalaris arundinacea), rye (Secale cereale) and giant reed (Arundo donax) (USDA-
NRCS, 2012; Qin et al., 2012; Feyereisen et al., 2013; Pociene, 2013; Williams, 2010).
Trees include, poplar or cottonwood (Populus tremula), willow (Salix), american
sycamore (Platanus occidentalis), sweetgum (Liquidambar styraciflua), southern beech
(Nothofagus) and ash (Fraxinus).
Advantages of switchgrass
Thirty-one sites participated in a screening trial funded by the U.S. Department of
Energy during the late 1980s to early 1990s to identify a bioenergy crop best suited for
production of next generation biofuel (Wright and Turhollow, 2010). Among thirty-four
herbaceous species tested, such as sorghums, reed canary grass, wheatgrasses, etc.
on a wide range of soil types, switchgrass was identified as having merit for further
development (Wright and Turhollow, 2010).
Switchgrass appears to be the most promising herbaceous energy crop (Wright,
2007) due to the following characteristics: (a) established from seed, (b) harvested and
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stored as hay, (c) used for biomass and forage, (d) relatively low lignin content 19-23%
(Kim et al., 2011), and (e) high genetic variability within the species providing excellent
opportunities for improvement by selection and breeding, (f) a native plant of North
America.
In the following two decades, more research was conducted on switchgrass. This
grass showed more beneficial properties to be a bioenergy crop due to its high
productivity, suitability for marginal lands, low water and nutritional requirements,
environmental benefits and flexibility for multipurpose uses (McLaughlin and Adams
Kszos, 2005). The yield can be as high as 35 t/ha⋅y (Mooney et al., 2009; Sladden et al.,
1991; Thomason et al., 2004) although the high yields are site-specific for test plots and
do not reflect realistic expectations.
Environmental benefits
Carbon sequestration is the natural uptake of atmospheric carbon dioxide by
photosynthesis. Switchgrass is an ideal crop for carbon sequestration because of its
thick, deep set root systems that can grow as much as 30 feet into the ground. The root
system can account for up to 80% of the total biomass (Liebig et al., 2005).
Researchers found that switchgrass can sequester 33 Mg/ha of carbon dioxide in one
year (Mehdi, 1998).
Pretreatment
Lignocellulosic biomass contains sugars in the form of cellulose and
hemicellulose. The monomeric sugars present in these polysaccharides are not readily
available to fermenting microorganisms due to the structural organization of the plants.
Biomass materials such as wood are composite materials with high mechanical
strength. The major components are cellulose, embedded in a matrix of lignin and
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hemicelluloses (Fengel and Wegener, 1983). Together they form tightly packed fibers
that are stacked as fiber bundles, the structural component of mature plant tissues such
as wood. Their natural function is to bear high mechanical loads, and to resist chemical
and enzymatic degradation through microorganisms. This common feature of plant
fibers is often termed as biomass recalcitrance and is a technical obstacle for bio-
refinery processes. Since fermenting microorganisms require monomeric sugars or
oligosaccharides (2-4 sugar moieties) as the feedstock for fermentation, the
polysaccharides in the plant tissue need to be converted to constituent sugars.
In general, the process for lignocellulose to bioethanol (Figure 1-1) is similar to
the steps currently employed in the sugar based bioethanol production (Figure 1-2).
With both lignocellulose and grains, the polysaccharide is released and enzymatically
hydrolyzed to sugars that are fermented by microorganisms to desired product.
However, the inherent differences in the physical characteristics and composition of the
two starting materials make it difficult to release the sugars from lignocellulose. First, the
lignocellulose is more organized to resist degradation of the polysaccharides, and
second, cellulose, as a highly crystalline glucose polymer needs to be separated from
the other polymers, hemicellulose, lignin, etc. for effective enzyme hydrolysis. The
cellulases, unlike starch hydrolyzing enzymes are not as active and as a result higher
concentration of an enzyme mixture is required. Third, to ferment all the hexose and
pentose sugars released from lignocellulose, new microorganisms need to be
discovered or developed since the yeast used by ethanol industry, Saccharomyces
cerevisiae, could not use pentose sugars, a major component of hemicellulose (IEA,
2009).
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Until now there is no native organism that can ferment lignocellulose directly and
at rates high enough to be used at commercial scale to produce ethanol. To access the
polysaccharides in biomass some form of pretreatment is essential; the only issue is
how to decrease the cost and increase the efficiency. Although many biological,
chemical, physical, thermal, and even some combination of these methods have been
tried over decades (Table 1-2), a single effective method to generate fermentable
sugars is yet to emerge.
Pretreatment is the first step to disrupt the natural structural resistance of
biomass material, to make the carbohydrates susceptible to enzymatic hydrolysis to
release sugars for fermentation. Under acidic conditions, at high temperature (160-
220oC), hemicellulose is hydrolyzed, releasing monomeric sugars and soluble oligomers
from the cell wall matrix. Even with the cellulose and lignin unaffected, increased
porosity due to the pretreatment improves enzymatic digestibility of cellulose
(Chaturvedi et al., 2013). Enzyme treatment of pretreated biomass also lowers viscosity
and improves handling of the slurry (Geddes et al., 2010). The shortcoming of dilute
acid pretreatment is inhibitors generated during the process by dehydration of both
hexose and pentose sugars. Sulfuric acid pretreatment is the most intensely
investigated dilute acid pretreatment method due to its high sugar yields and low cost.
Besides dilute sulfuric acid, low concentration of phosphoric acid has been used as well
(Fontana et al., 1984; Geddes et al., 2010).
Geddes et al. (Geddes et al., 2010) reported that sugars released after dilute
phosphoric acid pretreatment of sugar cane bagasse included glucose, xylose,
arabinose, mannose and galactose with xylose as the dominant sugar. Combined with
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commercial cellulase enzyme treatment, the phosphoric acid pretreatment process yield
almost 100% of the total sugars in biomass. Phosphoric acid pretreatment can release
up to 82.5% of the theoretical overall sugars from potato peels (Lenihan et al., 2010).
Phosphoric acid pretreatment of bamboo and corn cob also yielded high concentration
of sugars at 170°C for 45 minutes (Hong et al., 2012) and 140°C for 10 minutes
(Satimanont et al., 2012). Phosphoric acid pretreatment of corn stover at 0.5%
(v/v)/180°C/15 min and 1% (v/v)/160°C/10min achieved 85% glucose and 91.4% xylose
yields, respectively (Avci et al., 2013). These studies show that phosphoric acid
pretreatment produces relatively low amount of inhibitors compared to sulfuric acid
pretreatment while achieving comparable sugar yield as well, furthermore the
phosphoric acid remaining in the hemicellulosic hydrolysates is at adequate levels for
supplying phosphorous requirement during subsequent fermentation. The phosphorus
in the stillage can serve as a fertilizer for plant growth (Geddes et al., 2013; de
Vasconcelos et al., 2013).
Table 1-2 also lists various pretreatment methods that utilize bases, such as
lime, NaOH, etc., to pretreat cellulosic materials. Alkali work differently on the biomass
than acid to break down the crystalline matrix of the cell wall. Alkali degrade the ester
and glycosidic side chains of the materials, therefore disrupting the lignin structure,
swell the cellulose and dissolve the hemicellulose (Lindberg et al., 1984; Zhao et al.,
2010). Removal of lignin by bases favors enzymatic hydrolysis of carbohydrates by
increased porosity while decreasing lignin derived inhibitors. Generally, base
pretreatment is less severe than acid pretreatment, so it’s not suitable for woody
biomass, which is highly recalcitrant due to higher density. The drawbacks of base
23
treatment at harsh conditions include the loss of hemicellulose (Cheng et al., 2010) and
formation of inhibitors. Formation of salts during neutralization of the hydrolysates after
the pretreatment is another challenge to the disposal (Prado et al., 2012).
Base pretreatment appears to improve enzymatic biodegradability due to the
higher delignification ability of alkali, and the mild conditions produce less inhibitors
compared with acid pretreatment. Acid pretreatment of lignocellulosic biomass may
produce degradation products with an inhibitory effect on the fermentation process.
These inhibitors that have toxic effects on the fermenting organisms include furfural, 5-
hydroxymethylfurfural, acetic acid and formic acid (Figure 1-3). There are also aromatic
compounds in the hydrolysate, such as, furan aldehydes, aliphatic acids, and
extractives that also have inhibitory effect on fermenting microorganisms (Kim et at.,
2011; Hahn-Hagerdal et al., 2001; Soudham et al., 2011). In addition to the compounds
listed above, various phenolic compounds are produced during pretreatment as
additional major inhibitors of microbial growth and fermentation (Figure 1-4) (Boukari et
al., 2011; Olsen et al., 2011).
Saccharification
Cellulolytic enzymes release sugars from cellulose at high yield in short time and
in an environmentally friendly way. However, enzymatic saccharification of
lignocellulosic biomass is a complex process and the hydrolysis of all the
polysaccharides requires a repertoire of several hydrolytic enzymes. Fungi are key
microbial players in the biological conversion of plants and plant derived wastes. These
eukaryotic microorganisms secrete a vast array of carbohydrate hydrolases (collectively
termed glycosyl hydrolases) as well as a range of peptidases and lignin modifying
24
enzymes that reduce plant biomass to its simple building blocks (Ayyachamy et al.,
2013).
For decades, scientists have worked to identify the types and modes of action of
cellulases and hemicellulases produced by fungi. Cellulose hydrolysis is catalyzed by a
complex system of three enzymes that act synergistically. The three enzyme
components are, 1,4-β-D-glucan glucanohydrolase (EC3.2.1.3), 1,4-β-D-glucan
cellobiohydrolyase (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21) (Ladisch et al., 1983;
Wright, 1988). These enzymes are commonly referred to as endoglucanase,
exoglucanase and cellobiase, respectively (Keshwani and Cheng, 2009).
Hemicellulases involved with xylan hydrolysis include three main enzymes: endo-β-1-4-
xylanase, exoxylanase and β-xylosidase (Badal and Rodney, 1999). Other ancillary
enzymes responsible for cleaving side-groups of hemicellulose include, α-L-
arabinofuranosidase, α-glucuronidase, acetylxylan esterase, ferulic acid esterase, and
p-coumaric acid esterase (Badal and Rodney, 1999).
Enzymatic hydrolysis of cellulose is typically carried out by cellulases at their
optimum condition under mild conditions (pH of 5.0 and temperature of approximately
50°C). The disadvantage of commercial fungal enzyme treatment is the price of the
enzyme, which can be minimized by increasing the enzyme efficiency. The efficiency of
the enzyme is mainly affected by its own composition in addition to biomass
composition and structure. For example, one common way to improve the efficiency of
overall enzymatic hydrolysis is to supplement the enzyme formulation with β-
glucosidase to reduce inhibition by cellobiose, a reaction intermediate (Wyman et al.,
2011).
25
Although cellulases are also produced by several bacteria such as Clostridium,
Cellulomonas and Bacillus (Bisaria, 1998), fungal cellulases have the best potential for
commercial scale use.
Microorganisms
Though the biomass materials are plentiful and renewable, the recalcitrance of
these materials and the inability of native microorganisms to efficiently ferment them are
the big hurdles preventing the production of bioalcohols directly from these natural
resources. To be a sustainable bioalcohol producer, the microorganism needs to
possess properties, such as high alcohol productivities, yields, ability to convert all the
sugars in biomass, resistant to inhibitors and fermentation products, and easy to
cultivate in large fermenters. Since, there is no native microorganism with all these
properties, researchers are constructing in the laboratory microorganisms that can meet
these requirements.
Bioethanol is the most commonly produced biofuel and could potentially replace
fossil fuels in many energy applications, especially in the transportation sector
(Demirbas, 2009). Yeast, Saccharomyces cerevisiae, is the main microbial biocatalyst
at the industrial scale although several other yeasts and few bacteria produce ethanol
as the major fermentation product. However, none of the native homofermentative
ethanol producers could ferment all the sugars in the biomass, especially pentose
sugars. Several new recombinant microorganisms, ethanologenic Escherichia coli and
yeast, are currently available for fermentation of sugars derived from biomass.
Yeast
Yeast is the intensely investigated microorganism for food and beverage
production for millennia. Currently, it is also the most used microorganism in the
26
production of first generation bioethanol from sugar or starch crops. Saccharomyces
cerevisiae remains viable up to 50% (w/v) of sugar concentration (Restaino et al., 1983;
Mukherjee et al., 2014) and can survive in 2M NaCl (Gaxiola et al., 1996). Its optimum
range for fermentation is 25-37oC with an ethanol tolerance of up to 13% (v/v).
Although S. cerevisiae is the preferred microorganism for ethanol production,
there are more than 1500 yeast species (Kurtzman et al., 2011) and some of them
could have beneficial characteristics, such as the ability to ferment both pentoses and
hexoses, ability to withstand high concentration of sugar, ethanol and inhibitors
generated during pretreatment of biomass.
Escherichia coli
E. coli has certain special properties that make it an excellent organism for strain
improvement, such as rapid growth rate, high-cell density, low production cost and well-
developed physiology and genetics. Detailed knowledge of the metabolism and the
availability of genetic tools for strain improvement make E. coli a widely-used organism
of choice for molecular cloning methodologies and as a host to produce primary and
secondary metabolites (Chen et al., 2013).
Due to these beneficial traits, ethanologenic E. coli strains have been
constructed and continually improved for fermentation of all sugars in biomass
(Alterthum and Ingram, 1989; Moniruzzaman et al., 1997; Zaldivar et al., 2000; Zheng et
al., 2012; Ingram et al., 1999; Jarboe et al., 2010).
Ethanol production improvement
E. coli is naturally able to metabolize a wide variety of sugars, an important
feature in obtaining highest product yield from lignocellulose hydrolysates (Ingram et al.,
1999). Although wild type E.coli produces ethanol as a fermentation product, the yield is
27
low. E. coli KO11 that produced ethanol at close to a theoretical yield of 0.51 (g/g
glucose) was reported in 1991 by Dr. Ingram’s group by introducing Pyruvate
decarboxylase (pdc) and alcohol dehydrogenase II (adhB) from Zymomonas mobilis,
into the E. coli W chromosome at the pyruvate formate lyase (pflB) locus (Ohta et al.,
1991). Diagram of the native and introduced fermentation pathways of E. coli is
presented in Figure 1-5. Since these earlier studies, ethanologenic E. coli strains have
been evaluated for fermentation of various other sugars also (Luo et al., 2014).
Hildebrand et al. (Hildebrand et al., 2013) reported the E. coli KO11 lacking L-lactate
dehydrogenase and pyruvate formate-lyase could improve the ethanol yield from 87.5%
to 97.5% of the theoretical maximum on gluconic acid.
Inhibitor reduction
Ethanologenic E. coli with higher tolerance to inhibition by the final product and
pretreatment side products such as furfural, 5-HMF, acetate and other soluble products
have been isolated. Reduction of aldehydes to alcohol, such as furfural to furfuryl
alcohol is a key factor in this tolerance (Miller et al., 2009; Turner et al., 2011; Wang et
al., 2011).
Besides gene engineering, other methods to lower the toxicity of biomass slurry
have been researched as well. Nieves et al. (Nieves et al., 2011a) reported addition of
reduced sulfur compounds, such as, sodium metabisulfite to the pretreated biomass
slurries, could efficiently reduce the toxicity. In addition, Geddes et al. (Geddes et al.,
2015) reported a combination of treatments (vacuum evaporation, laccase, high pH,
bisulfite, small amount of O2) minimized the inhibitory effect of sugarcane acid
hydrolysates on bacterial growth and fermentation. The ability to tolerate the final
product, ethanol, has also been improved in ethanologenic E. coli (Wang et al., 2012);
28
Edwards et al., 2011). Yuan et al. (Yuan et al., 2014) reported a significantly enhanced
ethanol tolerance in both E. coli EC100 and wild type E. coli MG1655 by integrating
unique Lactobacillus plantarum genomic DNA fragments into the E. coli chromosome.
Cellulases secretion
Currently, commercial cellulases are used to hydrolyze cellulose after
pretreatment of biomass. These fungal enzyme cocktails contains a mixture of various
cellulases, xylanases, β-glucosidases and other enzymes. Despite recent efforts to
lower the cost of these enzymes, their contribution to the total cost of the resulting
biofuel remains significantly high (Klein-Marcuschamer et al., 2012; Davis et al., 2013;
Gubicza et al., 2016). No single natural microorganism, which could produce the ideal
enzyme mix for biomass hydrolysis has been described. Engineered microorganisms
offer a feasible alternative, such as a single strain of E. coli that degrade the
polysaccharides and ferment the resulting sugars into biofuels. This process is termed
as consolidated bioprocessing–CBP (Ingram et al., 1999; Lynd et al., 2005; Geddes et
al., 2011; Chanal et al., 2011). Some researchers are pursuing expression of
heterologous cellulases in fermentative ethanologenic E. coli strains (Zhou and Ingram,
2001; Kojima et al., 2012; Linger et al., 2010; Munoz-Gutierrez et al., 2014; Zheng et al.,
2012) and yeast (Kricka et al., 2014). Zhou et al. (Zhou and Ingram 2001) introduced
Erwinia cellulase into Klebsiella oxytoca and the derivative fermented amorphous
cellulose to ethanol without the addition of cellulases from other organisms. Luo et al.
(Luo and Bao, 2015) expressed bglB from Bacillus polymyxa into the ethanologenic E.
coli ZY81, to give the microorganism β-glucosidase secretion ability. E. coli ZY81/bglB
utilized cellobiose as sole carbon source for ethanol production with 34% of theoretical
yield.
29
Lignocellulose offers a good source of fermentable sugars for ethanol production,
although the conversion of biomass to ethanol faces certain challenges. The major
challenge is to select suitable microorganisms for fermentation process. Yeast has
many promising properties as a workhorse for ethanol production, but more gene
modifications need to be done to overcome the inherent limitation of fermentable sugar
use. Formation of inhibitor during pretreatment process hinders its application, hence
biocatalysts that tolerate inhibitors are necessary. The search for new ethanologenic
microorganisms as well as the improvement in the techniques of pretreatment and
fermentation may help in the advancement of cost-effective production of lignocellulosic
ethanol.
Fermentation Process Choice
Saccharification of the cellulose and fermentation of the released sugars can be
approached by two schemes depending on the sequence of hydrolysis and
fermentation; SHF vs. SSF.
Separate Hydrolysis and Fermentation (SHF)
Separate hydrolysis and fermentation is carried out in two different vessels,
which permit the enzyme hydrolysis and fermentation operate at their optimal condition
such as temperature, concentration and pH. But the SHF process has some
disadvantages related to the cost and operation. Enzyme hydrolysis end products that
will accumulate inhibit the enzyme reaction. To overcome the end-product inhibition and
increase the reaction rate higher enzyme loading may be necessary. The cost of
enzymes is expected to be higher in this process due to higher loading and in addition,
the separate process requires two large containers and associated engineering.
30
Simultaneous Saccharification and Fermentation (SSF)
The SSF process was originally developed for lignocellulosic biomass by
researchers at Gulf Oil Company in 1974. Extensive studies on SSF have since focused
on the production of ethanol from cellulosic substrates. SSF is the process in which
saccharification of the polymer and fermentation of released sugars occurred in the
same vessel, which enable the fermenting microorganism to immediately use the sugar
released by the enzyme. SSF eliminates end product inhibition of enzyme activity. SSF
can be conducted in a single vessel and is expected to improve the overall process
cost.
The down side of the SSF process is created by the combination of two steps;
both the hydrolysis and fermentation could not operate at their optimum condition using
the conventional biocatalysts (30-37oC and pH 6-7) and fungal enzymes (50oC and pH
5.0). After the process, neither enzyme nor the microorganism can be completely
separated and reused, which is also a bottleneck towards lowering the cost of biofuel
production.
To overcome the drawback of the SSF process, a SSFF, simultaneous
saccharification, filtration and fermentation has been reported by Ishola et al. (Ishola et
al., 2013). Figure 1-6 illustrates the scheme of this process. In their proposed process, a
cross-flow membrane is used to filter the hydrolysates while the retentate goes back to
the hydrolysis vessel. The sugar rich filtrate is continuously pumped to the fermenting
container. An ethanol yield of 85.0% of the theoretical value was reported from this
SSFF process and the microorganism was reused for five cultivations. Since this
process needs to pump and circulate the hydrolysates and apply a membrane, the
31
additional material and energy cost need to be taken into consideration to evaluate the
effectiveness of economy.
Comparison between SHF and SSF
Öhgren et al. (Oehgren et al., 2007) and Rana et al. (Rana et al., 2014)
specifically conducted research on comparison of SHF and SSF process with S.
cerevisae (Table 1-3).
From the results of these two studies, in SSF process, ethanol yield was
substantially higher than from the SHF process. Not displayed in the Table, the
inhibitors in the hydrolysates make the hydrolysis of carbohydrates in the SHF process
harder to be initiated than in the SSF process, since the latter exhibits lower inhibition of
the enzymes due to simultaneous fermentation.
Considering the biomass rheological properties, an improved SSF process
emerged by adding an initial short-term liquefaction step, at the optimal condition for the
enzymes followed by SScF (co-fermentation of both hexose and pentose), which is
called L+SScF (Geddes et al., 2011; Nieves et al. 2011a; Nieves et al., 2011b; Castro et
al., 2014) to overcome some additional difficulties of handling pretreated biomass.
Lignocellulosic biomass bridge among the fibers (dry solid or in an aqueous slurry) due
to its natural property, even after pretreatment, greatly affects physical handling of these
fibrous materials. Geddes, et al. (Geddes et al., 2010) concluded that low levels of
cellulase enzymes can effectively reduce viscosity and improve the flow properties of
acid-pretreated bagasse slurries. From an industry point of view, adding enzyme
liquefaction step is expected to help overcome the issues with mixing and pumping
while also generating more glucose at the optimum condition for the enzymes to support
high growth rate of the fermenting microorganism at the SScF step.
32
Proposed Method
Dilute Phosphoric Acid Catalyzed Steam Explosion Pretreatment
Steam explosion of the phosphoric acid impregnated biomass will disrupt the
crystallinity of biomass cell wall and hydrolyze the hemicellulose and increase the
surface area for down-stream enzymatic hydrolysis of cellulose (Castro et al., 2014).
This method has been applied to different biomass, such as, sugarcane bagasse
(Geddes et al., 2010), sorghum bagasse and Eucalyptus chips (Fontana et al., 1984;
Carrasco et al., 1994; Castro et al., 2014).
The proposed dilute acid step will use low concentration of phosphoric acid, for
following reasons. First, low concentration of phosphoric acid will lower the cost of the
chemical and will make the production of ethanol economically sound (Bensah and
Mensah, 2013). Second, low concentration of acid can be used in stainless steel tanks,
which reduces capital cost. Third, low concentration of this acid will reduce dehydration
of released pentoses and hexoses and minimize potential microbial growth inhibitors in
the hydrolysates (Jonsson and Martin, 2016; Larsson et al., 1999; Liu, 2009).
Saccharification
In my research, fungal enzymes from Novozymes, which is available in the lab
and has been used to other biomass before, will be used to optimize the enzymatic
hydrolysis of pretreated switchgrass.
Simultaneous Saccharification co-Fermentation Method (SScF)
Switchgrass has 31.0% of cellulose, 24.4% hemicellulose and 17.6% lignin
(Wiselogel et al., 1996) and after pretreatment the hydrolysates usually contain a
mixture of cellulose, hexoses and pentoses together with lignin. In order to make the
biofuel economy efficient, use of all sugars including hexoses and pentoses is the
33
ultimate target. A hydrolysate inhibitor-resistant mutant of E. coli LY180, designated
SL100 will be used to co-ferment hexose and pentose sugars in the pretreated slurry of
switchgrass. Before the process of SScF, liquefaction of the pretreated switchgrass will
depend on the rheology of the slurry. Since there is no literature reported about the co-
fermentation of the dilute phosphoric acid steam exploded pretreated switchgrass, all
parameters will be optimized during the research based on the work done with other
biomass (Geddes et al., 2010; Castro et al., 2014).
Research Objectives
The overall objective of this study is to develop optimal conditions for phosphoric
acid catalyzed steam pretreatment for switchgrass based on fermentability of the slurry
to cellulosic ethanol. Different pretreatment methods have been applied to switchgrass,
but not dilute phosphoric acid. This will be first phosphoric acid based process for
switchgrass and there are advantages in using this acid over sulfuric acid or ammonia
as discussed above. The specific objectives are listed below.
Pretreatment of switchgrass with various concentration (0.5%-1%) of phosphoric
acid at different temperatures (160-190oC) for various time (5-10 minutes).
Analysis of the slurry for released sugars (composition and concentration)
(cellobiose, glucose, xylose, arabinose, galactose, mannose, etc.). Determination of the
inhibitors present and their concentration (furfural, hydroxymethylfurfural, etc.) in the
slurry.
Fermentation of the pretreated slurry to ethanol using ethanologenic E. coli, in a
Liquefaction + Simultaneous Saccharification co-Fermentation (L+SScF) process, with
commercial fungal cellulases. Based on fermentation of the released sugars to ethanol,
identify the pretreatment condition with highest yield of ethanol from switchgrass.
34
Table 1-1. Composition of different biomass Cellulose Hemicellulose Lignin Hardwoods 42% 24-33% 23-30% Softwoods 40% 18-28% 27-34% Cereal straws 35-40% 26-27% 15-20% Source: OECD/IEA November2008 (IEA,2009)
35
Table 1-2. Technologies and reaction conditions for biomass pretreatment Pretreatment technology Temperature
(oC) Reaction time (min)
Pressure (atm)
Solids (wt.%) Chemical(s) Reference
DA 130-200 2-30 3-15 10-40 0.5-3.0% H2SO4 1,2,3,4 Flow through 160-220 12-24 20-24 2-4 0.0-0.1% H2SO4 5 SAA 160 60 1 12 15% NH4OH 6,2,3,8 AFEX 70-90 <5 15-20 60-90 100% anhydrous ammonia 2,3,8,9,10 LHW 200 10 1 15 Water 2,3,8 PHW 200 10 14 10 Water 11,12 LHW+K2CO3 150-190 20 33 15 Water+ 0-0.9%(w/w) K2CO3 13 Sulfur dioxide 180 40 1 10 0.05g SO2/g biomass 2 DA + SO2
140-180 1-80 1 5 0.5-2.0% H2SO4 14 180 0-60 1 10 0-0.1g SO2/ g biomass
Steam explosion +SO2 170-210 2-10 30 3% moisture the biomass 8 ARP 150-170 10-20 9-17 15-30 10-15wt.% NH3 Lime 70-130 1-6h 1-6 5-20 0.05-0.15gCa(OH)2/g biomass 2,3,7 Lime+air(oxygen) 25-60 2w-2m 1 10-20 0.05-0.15gCa(OH)2/g biomass 8 Extrusion 176 155rpm 80 Water 15 Sequential-extrusion-microwave
50-85 2.5 1 20-75 Water 16
Water/NaOH+ microwave 70-190 0.5-2 1 5-17 Water 17 0.05-0.3 g NaOH/g biomass
NaOH 21,50,121 15,30,60 1 9 0.5%-2% (w/v) Sodium hydroxide 18 NaOH + Radio-frequency based dielectric heating
90 60 1 20 0.2-0.25 g NaOH/g biomass 19
Ionic liquid+ ultra-sonication 130 2-4 1 9 1-butyl-3-C6H11CIN2 20 Formic acid 100-200 60 10 8% weight formic acid 21 SPORL 170 20 1 3(v/w) 0.2%(v/v) H2SO4
2% NaHSO3(w/w) biomass 22
Dilute phosphoric acid 140-180 10-45 1 0.5-1%(v/w) 2% W/W H3PO4 23 Electron beam irradiation 100g sample receive 250-1000 kJ/kg dosages of exposure to irradiation. 24 DA-dilute acid, SAA-soaking in aqueous ammonia, AFEX-ammonia fiber expansion, LHW-liquid hot water, PHW-pressurized hot water, ARP-ammonia recycled percolation, SPORL-sulfite pretreatment to overcome 1, Li et al., 2010; 2, Garlock et al., 2011; 3, Kim et al., 2011; 4, Zhou et al., 2012; 5, Yang and Wyman, 2004;6, Isci et al., 2009;7, Yandapalli and Mani, 2014; 8, Tao et al., 2011; 9, Alizadeh et al., 2005;10, Bals et al., 2010;11, Hu and Ragauskas, 2011;12, Papa et al., 2015;13, Kumar et al., 2011; 14, Shi et al., 2011;15, Karunanithy and Muthukumarappan, 2011;16, Karunanithy et al., 2014;17, Hu and Wen, 2008;18, Wang et al.,2012;19, Hu et al., 2008; 20, Montalbo-Lomboy and Grewell, 2015; 21, Marzialetti et al., 2011; 22, Wang et al., 2012; 23, Geddes et al., 2010; 23, Sundar et al., 2014
36
Table 1-3. SHF process vs. SSF process applied to corn stover and loblolly pine Feedstock Steam-pretreated
corn stover* Wet exploded corn stover and loblolly pine**
Water- insoluble solids (WIS) SHF 8% 5%, 10% SSF 8% 5%, 10%
Enzyme loading SHF 10FPU/g WIS 15FPU/g glucan SSF 10 FPU/g WIS 15 FPU/g glucan
Ethanol yield of theoretical SHF 59.1% 70%, 63% SSF 72.4% 76%, 67%
* Öhgren, et al.,2007 ** Rana et al., 2014
37
Figure 1-1. Ethanol production from lignocellulose (Source: IEA/OECD 2008)
38
Figure 1-2. Ethanol production from sugar or starch feedstock (Source: OECD/IEA November 2008)
39
Figure 1-3. Major types of inhibitors and their chemical structure (Source: Harmsen et al., 2010)
40
Figure 1-4. Phenolic compounds that may act as inhibitors or deactivators of cellulases (Source: Ximenes et al., 2010)
41
Figure 1-5. Fermentation pathways in E. coli with an engineered pathway for homoethanol production (Adapted from Ingram et al., 1999). LDH, Lactate dehydrogenase; PFL, Pyruvate formate-lyase; FHL, Formate hydrogenlyase; PTA, Phosphotransacetylase; ACK, Acetate kinase; ADH, Alcohol dehydrogenase; PDC, Pyruvate dehydrogenase complex.
42
Figure 1-6. Schematic representation of SSFF integrated process (Source: Ishola et al., 2013)
43
CHAPTER 2 OPTIMIZING STEAM EXPLOSION PRETREATMENT OF SWITCHGRASS WITH
DILUTE PHOSPHORIC ACID
Introduction
Cellulosic biomass is a potential feedstock for production of next generation
biofuels to replace current feedstock starch that directly competes with food supply.
Compared to starch, cellulosic biomass is more resistant to deconstruction by bacteria
and fungi due to its native components and structure. Consequently, to disassemble
and ferment the cellulosic biomass-derived carbohydrates, a complex process involving
severe physical conditions and aggressive chemicals are necessary. Before
fermentation, the carbohydrates in cellulosic biomass must be depolymerized to soluble
sugars. As the cellulosic biomass is composed primarily of crystalline cellulose (35-
50%) that is complexed with hemicellulose (20-35%) and lignin (15-25%), the process of
extracting sugars requires breaking down the structure of the biomass followed by
saccharification of the carbohydrates. This process of converting cellulosic biomass to
ethanol could involve up to 12 steps (Moniruzzaman et al., 1997; Ingram et al., 1999;
Galbe and Zacchi, 2007) (Figure 2-1).
Eliminating or combining steps to reduce process complexity and cost:
Chemical pretreatment can break the natural structure of plant cell walls and expose the
amorphous cellulose derived from crystalline cellulose fibers to enzyme hydrolysis.
There are many pretreatment methods available; base, neutral, acid, ionic and physical.
None of these pretreatments hydrolyze the cellulose and hemicellulose as selectively
and accurately as enzymes, such as glycan hydrolases. Combined steam and diluted
mineral acid pretreatment, such as 1% sulfuric acid, has the advantage of fractionation
of cellulosic biomass with less degradation of cellulose while simultaneously hydrolyzing
44
hemicellulose (Carrasco et al.,1994). But this pretreatment condition generates
unwanted side products. However, some of the side products do inhibit microbial growth
and fermentation (Palmqvist et al., 1996; Ingram et al.,1996; Larsson et al., 1999).
Pretreatment with phosphoric acid was found to produce less toxic syrups than
pretreatment with sulfuric acid, a commonly used acid (Fontana et al., 1984; Geddes et
al., 2010). In addition to minimizing corrosion of the equipment, the small amount of
phosphoric acid used during the process can serve as a nutrient for growth of the
microbial biocatalyst used for fermentation of released sugars. This contrasts with the
gypsum generated as an additional by-product of dilute sulfuric acid pretreatment that
requires disposal, a needed process step. Fontana, et al. has pioneered the use of
dilute phosphoric acid to pretreat sugarcane and sorghum bagasse (Fontana et al.,
1984). Steam explosion of dilute phosphoric acid impregnated cellulosic biomass is also
used to pretreat various biomass including olive tree pruning (Martinez et al., 2015),
sugar cane bagasse (Geddes et al., 2010), sorghum bagasse (Geddes et al., 2013), as
well as corn stover (Avci et al., 2013).
Geddes et al. have developed a simplified process that eliminated several of the
process steps listed in Figure 2-1 (L+SScF process) (Geddes et al., 2011). As a first
step in this overall process, investigation focused on finding an optimum temperature,
acid concentration, time and solids concentration for pretreatment of switchgrass to
achieve the highest ethanol yield based on the L+SScF process.
Materials and Methods
Materials
Switchgrass (Alamo) (Panicum Virgatum) grown in Virginia, USA was harvested
in Fall 2015 and kindly donated by Mr. Fred Circle (FDC enterprises, Inc.) for this study.
45
The pre-cut (average of about 1 inch) pieces with a dry weight of 89.28±0.57% was
stored in sacks at room temperature in the UF Stan Mayfield biorefinery. Enzyme Cellic
CTec3 was generously provided by Novozymes (Ames, IA). Sugarcane bagasse
standards were purchased from NIST (Gaithersburg, MD). Phosphoric acid, potassium
hydroxide and other chemicals were from Fisher Scientific (Pittsburgh, PA).
Composition Analysis
Samples of raw switchgrass, biomass after pretreatment and biomass after
fermentation were prepared and analyzed using methods from the U.S. National
Renewable Energy Laboratory (NREL method) (Hames et al., 2008; Scarlata et al.,
2011).
Pretreatment
20 kg dry weight of raw switchgrass was allocated equally into five 32-gallon
Brute heavy duty utility containers and soaked for 12 hours in 14-fold (4kg DW
switchgrass, 56kg water) excess phosphoric acid solution at various concentrations (0,
0.75%, 1.0%; wt.%). The phosphoric acid loading in wt.% was based on the amount
mixed with water prior to adding switchgrass and compensated for the moisture in the
biomass. The soaked switchgrass was dewatered using a CP-4 screw press (Vincent
Corporation, Tampa, FL) to 30-50% dry weight. The phosphoric acid impregnated
biomass was combined, mixed and separated into 500 g samples (dry weight) for
loading convenience in a steam gun before steam treatment. The Steam gun used in
this study for pretreatment of phosphoric acid soaked switchgrass was designed by Dr.
Guido Zacchi (Palmqvist et al., 1996) with a 10 L pressure vessel that was heated by
high pressure steam. Ball valves are used at both inlet and outlet, at the top and bottom
of the vessel, respectively, for rapid heating of switchgrass and quick discharge of
46
treated biomass. Temperature (160, 175, 180, and 190oC) and time (5, 7.5, and 10
minutes) of heating are controlled by a computer using software Intouch (Wonderware,
Richmond, VA, USA). The total 20kg dry weight of phosphoric acid impregnated
switchgrass was pretreated in 40 batches and then pooled and mixed before storage in
autoclave bags at 4oC.
Full Test for Pretreated Switchgrass
For determination of Water Insoluble Solid (WIS), a 5g (dry weight) sample of
pretreated biomass was washed with deionized water using a Whatman Qualitative
Filter Paper at a pore size 2.5 µ under vacuum. At least 4 L of deionized water was
used to wash the solubles, including free sugars, off the fiber. The solid residue was
collected and dried to constant weight. The fraction of WIS in the starting 5 g of
pretreated slurry was determined from the weight of the washed material.
Characterization of the Hydrolysate
Roughly 10-15 g (dry weight) of pretreated biomass slurry was pressed with RA
Chand J210 Manual citrus juicer. The pass-through was collected and, centrifuged for
12 minutes at 2,000 rpm (600 x g) using Thermo scientific Sorvall evolution RC
Superspeed centrifuge with Fiberlite F14-6X250y carbon fiber fixed-angle Rotor. The
supernatant was used for determination of pH, density, conductivity, dry weight and
composition of the hydrolysate. pH and conductivity were determined using a pH-
Conductivity Meter (Model 220; Denver Instruments, Bohemia, NY). Dry weight was
determined using a moisture analyzer (Kern model MLB 50–3; Balingen, Germany). The
hydrolysate composition was determined by HPLC using an Agilent Technologies 1260
HPLC equipped with a model G1314B refractive index detector. Sugars were separated
using a BioRad (Hercules, CA) Aminex HPX-87P ion exclusion column (300x7.8 mm)
47
fitted with a Phenomenex (Torrance, CA) Carbo-Ca 4 guard column (4x3 mm) at 80 °C
using nano-pure water as the mobile phase (0.6 ml/min). Organic acids and furans were
determined by HPLC using an Agilent Technologies 1260 HPLC equipped with dual
detectors (UV and refractive index, in series) and a BioRad Aminex HPX- 87H column
(45 °C; 4 mM H2SO4 as the mobile phase, 0.4 ml/ min flow rate).
Results and Discussion
The main purpose of pretreatment is to condition the biomass for release of
sugars for fermentation by microorganisms to desired products. As discussed above,
there are several steps in the process of generating fermentable sugars and L+SScF
process has reduced these steps to two (Geddes et al., 2011). These are considered
one at a time and presented in subsequent chapters; acid and steam treatment of
biomass and liquefaction of the slurry by enzyme hydrolysis of carbohydrates. Each
pretreatment condition is described as temperature-acid concentration-time, for
example switchgrass pretreated at 180oC with phosphoric acid at a concentration of 1%
for 10 minutes will be listed as 180-1-10. The switchgrass (Alamo) used in this study
contained 38% cellulose, 28% hemicellulose, 27% lignin and 0.5% ash. The total
fermentable carbohydrate in the biomass was 64%. This composition of the switchgrass
is comparable to the values reported by Hu et al. for Alamo (Hu et al., 2010).
Effect of Phosphoric Acid as a Catalyst on Hemicellulose Hydrolysis
Various biomass pretreatment methods have been described to increase sugar
yield, minimize side products and maximize ethanol production (Wang et al., 2012; Avci
et al., 2013; Bensah and Mensah, 2013). Dilute acid pretreatment does not significantly
remove lignin from biomass compared to pretreatment with alkali that in addition to
solubilizing lignin also improves porosity of the biomass. However, dilute acid
48
pretreatment does lead to hydrolysis of hemicellulose to soluble sugars, such as xylose,
arabinose and galactose. Dilute sulfuric acid pretreatment of switchgrass has been
investigated extensively (Yat et al., 2008; Shi et al., 2011; Xu et al., 2011). Detailed
phosphoric acid pretreatment of switchgrass is yet to be conducted, although as
indicated previously, phosphoric acid pretreatment of sugarcane and sorghum bagasse
has been reported (Fontana et al., 1984; Geddes et al., 2013). This research is focused
on steam explosion of phosphoric acid impregnated switchgrass. The pretreatment
conditions selected for evaluation in this study, temperature, acid concentration and
steam treatment time, were based on published values from sugarcane and sorghum
bagasse pretreatment (Geddes et al., 2010; Geddes et al., 2013). As a baseline,
switchgrass was treated with steam at 190˚C for 10 min without added acid. This
treatment released 1.3 g of xylose /kg of biomass. The major sugar released during this
pretreatment condition was glucose (2.12 g/kg biomass), cellobiose (7.20g/kg biomass)
and arabinose (2.62g/kg biomass) (Table 2-1). The furfural concentration in this
hydrolysate was only 0.2 g/kg biomass and formic acid was the dominant inhibitor (4.24
g/kg biomass). With 1.3 g acetic acid /kg biomass, formic acid and acetic acid (total of
5.5 g /kg biomass) released during pretreatment apparently catalyzed biomass
degradation.
When switchgrass was treated with phosphoric acid at 0.75% and at 190˚C for 5
min, the amount of xylose and glucose released was 38.5 g and 7.0 g per kg of biomass
respectively (Table 2-1). As seen from the results, acid also increased the concentration
of all the inhibitors over the control without acid (furfural, hydroxymethyl furfural, acetic
acid and formic acid). Due to the increase in the concentration of inhibitors in the slurry
49
from acid and steam treated biomass, various conditions were attempted towards the
goal of identifying a pretreatment condition that yielded a slurry that can be fermented to
high yield of ethanol.
Effect of Temperature on Switchgrass Pretreatment
In order to evaluate the effect of temperature on pretreatment of switchgrass,
biomass was treated with 0.75 % phosphoric acid followed by steam explosion for 5
minutes at three different temperatures. Composition of the hydrolysate after the
pretreatment is presented in Figure 2-2. Increasing the temperature of pretreatment
increased the amount of xylose released into the hydrolysate although the amount of
glucose and arabinose did not vary significantly (Figure 2-2 and 2-3). Xylose released at
160oC was 5.3±0.6 g/kg of biomass. This value increased to 23.6 ±1.6 g/kg biomass at
175˚C and then to 38.5±0.89 g/kg biomass at 190˚C. Xylose release from the three
pretreatment temperatures was linear with temperature (y= 16.6x-10.7) (R2=0.9966)
This differential release of xylose is also reflected in the total sugars released by the
dilute acid steam pretreatment. Total sugars in the hydrolysates from the three
pretreatment conditions was also linear with temperature, and it can be described with a
linear regression, (R2=0.999) y=19.9x-4.5 (Figure 2-3). This positive correlation between
pretreatment temperature and hemicellulose hydrolysis observed with phosphoric acid
pretreatment of switchgrass is similar to the effect of pretreatment temperature on
hydrolysis of hemicellulose from other biomass (Nguyen et al., 1998; Castro et al.,
2014; Geddes et al., 2010). The fraction of hemicellulose that was hydrolyzed at 160°C
was only about 2% of the total hemicellulose. At 190°C, this value increased to 15%. A
slight drop in xylose concentration at 190oC could be dehydration to furfural.
50
Although sugar yield increased with increasing temperature, there was also an
increase in the amount of inhibitors, acetic acid, formic acid, furfural and
hydroxymethylfurfural, as the temperature of the pretreatment increased to 190oC. Total
inhibitor concentration in the hydrolysate after 190oC pretreatment was 12.6±1.3 g/kg
biomass with furfural contributing 3.31±0.3 g/kg of biomass.
Effect of Acid Concentration on Switchgrass Pretreatment
To evaluate the effect of acid concentration, an intermediate temperature of
175˚C was used with two different acid concentrations (0.75 and 1 %, w/w). The
temperature and time (5 min) were kept constant (Figure 2-4). There was no significant
difference in the amount of xylose released or total sugar yield after pretreatment with
either concentration of phosphoric acid. Similarly, inhibitor concentration of the
hydrolysate was also not significantly different between the two acid concentrations.
Effect of Pretreatment Time on Switchgrass Hydrolysis
A temperature of 175oC was also selected to evaluate the pretreatment time on
switchgrass. Phosphoric acid concentration in this experiment was 1% (w/w). Highest
xylose and total sugar yield was obtained after 10 minutes of steam pretreatment of
acid-impregnated switchgrass (Figure 2-5). However, the differences between 5, 7.5
min and 10 min pretreatment are not significant. Similar results were also obtained with
the inhibitors.
The effect of all the nine pretreatment conditions used in this study on the
amount of sugars and inhibitors released is summarized in Table 2-2 and Table 2-3 and
Figure 2-6. Based on these results, the highest xylose yield was 20% of theoretical with
a pretreatment condition of 190-1-7.5 but this pretreatment condition also released the
highest level of furfural and hydroxymethylfurfural.
51
Summary
Steam explosion of dilute phosphoric acid impregnated switchgrass is an
effective method to destabilize switchgrass biomass structure and hydrolyze
hemicellulose with low level of sugar converted inhibitory side products.
The amount of sugar monomers released by pretreatment increased mostly with
temperature and minimally with acid concentration and time. Total sugar (glucose,
xylose and arabinose) released varied from 15 to 72 g/kg dry switchgrass depending on
the pretreatment condition (Table 2-2; Figure 2-6). Among the sugars, xylose
contributed the most to the total sugar released due to the effectiveness of acid
catalyzed hydrolysis of hemicellulose.
The highest amount of sugar released was at a pretreatment condition of 190oC,
with 1% phosphoric acid (w/w) and steam treated for 7.5 min. At this condition, about
6% of the cellulose was hydrolyzed and released as cellobiose and glucose while about
21% of the hemicellulose in the biomass was also hydrolyzed.
At pretreatment 190o, with 1% phosphoric acid (w/w) and steam treated 7.5 min.,
the total inhibitors released was highest at 15.75g/kg dry switchgrass, which was
contributed mainly by formate at 6.97g/kg dry switchgrass and furfural at 3.89 g/kg dry
switchgrass (Figure 2-7). At temperature from 160oC to 175oC, the main inhibitor furfural
increased not apparently until the temperature reached 190oC. HMF remained low until
temperature at 190oC and 1% acid concentration. Even in the presence of these
inhibitors, the sugars in the pretreated slurry from 190-1-7.5 was fermented successfully
by ethanologenic E. coli SL 100, at 10% solids loading, as presented in subsequent
chapters.
52
With these pretreatment conditions, water insoluble solids correspondingly
decreased as more sugars are released from the biomass (Figure 2-8). This is primarily
due to the hydrolysis of hemicellulose with xylose dominating as the free sugar (Samuel
et al., 2011). This is also seen as the change in the composition of biomass before and
after pretreatment (Figure 2-9). In addition to a decrease in hemicellulose, soluble lignin
also decreased (160-0.75-5 to 190-1-7.5). Even without acid (190-0-10), steam
pretreated biomass had more insoluble lignin and less xylan compared to untreated
biomass which was also observed by Kumar et al., suggesting that at even moderate
severity of pretreatment the hemicellulose could form pseudo-lignin (Kumar et al.,
2013).
53
Table 2-1. Comparison of pretreatment of switchgrass with and without phosphoric acid Soluble sugars (g/kg dry switchgrass)
Pretreatment conditiona
190-0-10 190-0.75-5 Cellobiose 7.20±0.25 8.32±0.32 Glucose 2.12±1.00 6.95±0.45 Xylose 1.33±1.83 38.52±0.89 Arabinose 2.62±1.20 9.27±0.40 Furfural 0.23±0.30 3.31±0.30 HMF 0.09±0.02 0.42±0.05 Acetic acid 1.30±0.20 2.49±0.20 Formic acid 4.24±0.83 5.55±0.65 a Switchgrass was pretreated at the indicated temperature (˚C), acid concentration (% by weight) and time (min). The pretreatment conditions are listed in that order.
54
Table 2-2. Effect of pretreatment conditions on the release of sugars from switchgrass
Pretreatment conditionb [Sugar] (g/kg pretreated switchgrass) Glucose Xylose Arabinose Total sugara
190-0-10 2.12±1.00 1.33±1.80 2.62±1.20 6.07±4.00 160-0.75-5 3.43±0.50 5.31±0.60 6.28±0.30 15.02±2.50 160-1-7.5 4.31±0.30 23.17±0.30 6.52±0.70 34.00±2.20 175-0.75-5 4.80±0.50 23.60±1.60 7.39±0.40 35.80±3.00 175-1-5 4.98±2.00 23.91±3.20 7.67±1.20 36.56±5.60 175-1-7.5 5.92±2.30 28.38±3.50 8.68±1.50 42.98±6.40 175-1-10 6.31±3.00 34.96±2.90 8.69±1.00 49.96±5.50 190-0.75-5 6.95±0.50 38.52±0.90 9.27±0.40 54.74±2.60 190-1-7.5 9.35±3.50 52.80±2.00 10.22±2.00 72.36±5.60 a Total sugar includes all three sugars. b Switchgrass was pretreated at the indicated temperature (˚C), acid concentration (% by weight) and time (min). The pretreatment conditions are listed in that order.
55
Table 2-3. Inhibitors in the hydrolysate of pretreated biomass
Pretreatment conditiona [Inhibitor] (g/kg dry pretreated switchgrass) HMF Furfural Acetate Formate Total Inhibitor
190-0-10 0.09±0.02 0.23±0.30 1.30±0.20 4.24±0.83 5.86±1.35 160-0.75-5 0.12±0.03 0.22±0.20 0.74±0.10 3.62±0.50 4.70±1.00 160-1-7.5 0.22±0.03 1.18±0.30 1.37±0.20 3.67±0.50 6.45±2.20 175-0.75-5 0.25±0.05 1.45±0.20 1.52±0.15 3.87±0.54 7.09±1.20 175-1-5 0.26±0.08 1.48±0.50 1.69±0.21 4.28±0.34 7.71±1.50 175-1-7.5 0.30±0.04 1.69±0.45 1.92±0.34 4.83±0.20 8.73±2.00 175-1-10 0.30±0.03 1.70±0.48 2.01±0.46 5.42±0.32 9.44±2.60 190-0.75-5 0.42±0.05 3.31±0.30 2.49±0.20 5.55±0.65 11.77±1.20 190-1-7.5 1.35±0.05 3.89±0.50 2.89±0.50 6.97±0.70 15.10±1.75 aSwitchgrass was pretreated at the indicated temperature (˚C), acid concentration (% by weight) and time (min). The pretreatment conditions are listed in that order.
56
Figure 2-1. Process for ethanol production from cellulosic biomass
57
Figure 2-2. Effect of pretreatment temperature on switchgrass hydrolysis
(Total sugar includes, glucose, xylose and arabinose)
0
10
20
30
40
50
60
70
Glucose Xylose ArabinoseTotal sugar HMF Furfural Acetate Formate Lactate Total Inhibitor
[Sug
ars]
and
[Inh
ibito
rs] (
g/kg
DW
sw
itchg
rass
)160-0.75-5175-0.75-5190-0.75-5
58
Figure 2-3. Effect of pretreatment temperature on switchgrass hydrolysis
y = 16.605x - 10.733R² = 0.9966
y = 19.856x - 4.5263R² = 0.9993
y = 1.5474x - 1.4366R² = 0.9862
0
10
20
30
40
50
60
70
160 175 190
[Sug
ar]&
[Fur
fura
l] (g
/kg
DW sw
itchg
rass
)
Temperature (oC)
XyloseTotal sugarFurfuralLinear (Xylose)Linear (Total sugar )Linear (Furfural)
59
Figure 2-4. Effect of acid concentration during pretreatment on switchgrass hydrolysis
0
5
10
15
20
25
30
35
40
45
Glucose Xylose Arabinose Total sugar HMF Furfural Acetate Formate Lactate TotalInhibitor
[Sug
ars]
and
[Inh
ibito
rs] (
g/kg
DW
switc
hgra
ss
175-0.75-5175-1-5
60
Figure 2-5. Effect of residence time during pretreatment on switchgrass hydrolysis
0
10
20
30
40
50
60
Glucose Xylose ArabinoseTotal sugar HMF Furfural Acetate Formate Lactate TotalInhibitor
[Sug
ars]
and
[Inh
ibito
rs] (
g/kg
DW
switc
hgra
ss
175-1-5175-1-7.5175-1-10
61
Figure 2-6. Sugars in the hydrolysate of slurries from all pretreatments
0
10
20
30
40
50
60
70
80
190-0-10 160-0.75-5 160-1-7.5 175-0.75-5 175-1-5 175-1-7.5 175-1-10 190-0.75-5 190-1-7.5
[Sug
ar] (
g/kg
dry
switc
hgra
ss)
Pretreatment Conditions (temperature-acid concentration-time)
Glucose
Xylose
Arabinose
Total sugar (include glucose, xylose, arabinose)
62
Figure 2-7. Furans and organic acids in the hydrolysate from all pretreatment conditions
0
5
10
15
20
190-0-10 160-0.75-5 160-1-7.5 175-0.75-5 175-1-5 175-1-7.5 175-1-10 190-0.75-5 190-1-7.5
[Fur
an] a
nd[ O
rgan
ic A
cid]
(g/k
g dr
y sw
itchg
rass
)
Pretreatment Conditions (temperature-acid concentration-time)
HMF Furfural Acetate Formate Lactate Total Inhibitor
63
Figure 2-8. Water insoluble solids (WIS) from all pretreatment conditions
0
100
200
300
400
500
600
700
800
900
190-0-10 160-0.75-5 160-1-7.5 175-0.75-5 175-1-5 175-1-7.5 175-1-10 190-0.75-5 190-1-7.5
WIS
(g/k
g dr
y sw
itchg
rass
)
Pretreament Conditions (temperature-acid concentration-time)
64
Figure 2-9. Composition of switchgrass solids before and after pretreatment
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
100.00%
Raw
190-
0-10
160-
0.75
-5
160-
1-7.
5
175-
0.75
-5
175-
1-5
175-
1-7.
5
175-
1-10
190-
0.75
-5
190-
1-7.
5
Com
posit
ion
(%)
Pretreatment Conditions ( temperature-acid concentration-time)
Glucan Xylan Arabinan Acetate soluble Lignin Insoluble Lignin Ash
65
CHAPTER 3 OPTIMIZING ENZYMETIC LIQUEFACTION OF ACID PRETREATED SWITCHGRASS
SLURRY
Introduction
Cellulosic biomass is built to resist microbial deconstruction and is difficult to
break down the complex structure to release the fermentable sugars. Even with efficient
pretreatment methods, the carbohydrates in the biomass tend to bridge together in the
pretreatment slurry. With the dilute acid steam pretreatment discussed in the previous
chapter, hemicellulose is partially hydrolyzed and removed from the biomass. However,
the remaining biomass solids bridge across the particles, apparently through the
cellulose fibers. This structure makes handling of the slurry difficult in subsequent steps.
Geddes et al. (Geddes et al., 2010) reported that small amount of cellulases can lower
the viscosity of the slurry by hydrolyzing the cellulose and increase the flow properties
of the slurry for the following fermentation process (L+SScF) (Geddes et al., 2013). This
partial hydrolysis (liquefaction) step also released additional sugars into the slurry.
In this chapter, the effect of pretreatment conditions described in Chapter 2 on
the release of sugars during liquefaction of the slurry by a commercial enzyme
preparation (Cellic CTec3) is presented and discussed.
Materials and Methods
Materials
Switchgrass was size reduced to 1 inch and stored at room temperature in sack.
Enzyme Cellic CTec3 was provided by Novozymes (Ames, IA). Potassium hydroxide
and other salts were purchased from Thermo-Fisher Scientific (Waltham, MA).
Phosphoric acid hydrolysates of switchgrass hemicellulose were prepared at IFAS Stan
Mayfield Biorefinery Pilot Plant of University of Florida.
66
Liquefaction of Pretreated Slurry
The pH of the slurry obtained after pretreatment with phosphoric acid and steam
was adjusted to 5.0 with ammonium hydroxide (5N). A well-mixed sample of the slurry
(10% solids, w/v) was transferred to a one gallon zipper bag and commercial cellulase
(Cellic CTec3, Novozymes) at 5% (v/dry weight of the biomass) was added to the slurry.
Upon assay, the enzyme CTec3 was found to have about 230 FPU/ml. The bag with its
contents was incubated at 50oC, in a water bath for 6 hours. The samples were mixed
thoroughly every hour by gently kneading the bag contents for 5 minutes.
Composition of the Slurry before Liquefaction
A sample of the slurry obtained after pretreatment was dried in a 60o oven to
constant weight. The dried sample was cut and passed through a sieve with 0.841 mm
opening. Composition of the sample was determined using NREL method (Scarlata et
al., 2011). Moisture content of the slurry was obtained using a moisture analyzer (Kern
model MLB 50-3 Balingen, Germany). Sugars, organic acids and furans were
determined by HPLC, as described in Chapter 2.
Detoxification of Pretreated Slurry for Liquefaction and Downstream Fermentation
The slurry obtained after 190-1-0.75 pretreatment condition was detoxified before
L+SScF process at 15% solids loading to remove some of the inhibitory compounds
generated during pretreatment. For detoxification, 300g dry weight of pretreated
switchgrass slurry was mixed with 360.32 mL water and 50.25 mL of ammonium
hydroxide (5N) to bring the pH to 9.0 in a volume of 2 L. With these additions, the solids
loading during L+SScF, after addition of enzyme, nutrients and inoculum, will be 15%
(w/v) in a final volume of 2L. The mixture was incubated for 12 hours at 4oC. During this
67
incubation period, pH of the sample decreased to about 7.0 and the pH was further
lowered to 5.0 by phosphoric acid before adding enzymes for liquefaction.
Results and Discussion
Composition of the Slurry after Pretreatment
In this study, the slurry (solids and hydrolysate) obtained after pretreatment was
used directly for liquefaction and simultaneous saccharification and co-fermentation.
The composition of the slurry, determined using the NREL method, is presented in
Table 3-1 and Figure 2-9. The values listed in Table 3-1 for glucan, xylan and arabinan
also include the free sugars released during pretreatment. The hemicellulose (xylan and
arabinan) content of the slurries decreased while the glucan and insoluble lignin content
increased upon pretreatment compared to raw switchgrass. This is unexpected since
the sugars released during pretreatment, especially from hemicellulose, are expected to
remain in the slurry. This may reflect the conversion of some of the sugars to furan and
insoluble degradation products, collectively termed as chars and/or pseudo-lignin during
the harsh sulfuric acid treatment used by the NREL method to determine the
composition of the slurries. Kumar et al. have reported that hemicellulose (xylan)-
derived-pseudo-lignin is formed at even moderate severities of pretreatment and these
insoluble degradation products can significantly retard cellulose hydrolysis at moderate
to low enzyme loadings (Kumar et al., 2013).
Effect of Pretreatment Temperature on Enzyme Liquefaction
Switchgrass pretreated with 0.75% (w/w) of phosphoric acid for 5 minutes but at
different temperatures (160oC, 175oC, 190oC) was treated with enzymes (5% (v/w);
about 11.5 FPU/g dry switchgrass) at a solid loading of 10% (w/v) for 6 hours. At the
end of liquefaction, the amount of glucose released by the enzymes increased with
68
increasing pretreatment temperature (Figure 3-1) from 96 g/kg of biomass at 160°C to
154 g/kg of biomass at 190°C. The amount of xylose released by the enzymes from the
slurries pretreated at 160 and 175 °C was comparable (about 90 g/kg biomass).
However, the amount of xylose released from the 190°C slurry (73 g/kg biomass) was
lower than the level of xylose obtained from the other two pretreatment temperatures,
reflecting a reduced level of xylan in the slurry from the 190°C pretreatment. The higher
temperature condition hydrolyzed a comparatively higher amount of hemicellulose than
the pretreatment at the lower temperatures (Figure 2-2). As expected, this difference is
not seen when the total amount of xylose in the slurries from the three pretreatment
samples at the end of liquefaction (about 110-115 g/kg of biomass) (Figure 3-1). These
results show that of the three temperatures evaluated in this study, the 190°C
pretreatment provided a better cellulose substrate for the enzymes (Figure 3-1C). This
can be due to removal of a higher percent of hemicellulose and/or alteration of the
cellulose crystal structure at the 190°C pretreatment with steam and phosphoric acid.
The temperature of pretreatment appears to have a minimal effect on the
concentration of arabinose in the slurries post-liquefaction. The low concentration of
arabinan in the biomass could account for this. Alternatively, this can be a reflection of
the enzyme composition of the commercial enzymes that may lack appropriate glycan
hydrolases for arabinan.
Effect of Acid Concentration during Pretreatment on Enzyme Liquefaction
To evaluate the effect of acids on liquefaction process, switchgrass was
pretreated for 5 minutes at 175°C at two phosphoric acid concentrations (0.75 and 1 %;
w/w). In this study, enzyme hydrolysis is used as an indicator of the degree of
destabilization of the biomass structure. This temperature and time were chosen to
69
minimize loss of xylan due to acid hydrolysis and release of sugars from such a
pretreated slurry would depend on enzyme action and structure of the carbohydrates in
the biomass. For comparison, slurry from switchgrass pretreated at 190°C for 10 min
without added acid was used. This pretreatment condition is the highest time and
temperature used in this study.
The results from these experiments show that increasing the acid concentration
had minimal effect, if any, on glucose release during liquefaction (Figure 3-2). The
amount of glucose released (about 110 g/kg of biomass) is about the same as that from
the slurry obtained after pretreatment without acid. This amounted to about 25% of the
theoretical level of glucose of the biomass. The higher acid concentration of 1% did
support release of more xylose compared to 0.75% acid during pretreatment (Table 2-2)
and this reduced the amount of xylose produced during liquefaction. However, the total
xylose at the end of pretreatment and liquefaction is comparable at both acid
concentrations.
Enzymes released about 9.4% of the theoretical arabinose level from the slurry
of the no-acid sample which is higher than other two conditions with acid. Due to the
release of arabinose during acid pretreatment, the total arabinose in the slurry after
liquefaction was higher in the acid-treated samples
These results show that an acid concentration of 0.75% phosphoric acid is
sufficient for pretreatment of switchgrass at this temperature. As seen above, increasing
the temperature to 190°C at 0.75% acid concentration did support higher glucose
release along with a higher level of inhibitors.
70
Effect of Pretreatment Time on Enzyme Liquefaction
In addition to the temperature and acid concentration, residence time of the
biomass at the chosen temperature contributes to destabilization of the biomass to
enhance the rate of enzyme hydrolysis. To evaluate the effect of time of pretreatment
on enzyme-hydrolyzed sugar release, phosphoric acid (1%, w/w) impregnated
switchgrass was pretreated at 175oC for different times. The steam gun used in this
study allows more precise control of time by rapid injection and release of steam. The
slurry obtained from such a pretreatment was submitted to enzyme liquefaction with 5%
(v/w) enzyme loading at 10% (w/v) solids. The amount of glucose released was about
20 g/kg biomass higher when the residence time was 10 min compared to 7.5 min
(Figure 3-3). The difference in the amount of glucose released from the slurry obtained
after 5 or 7.5 min of pretreatment was not significantly different. This trend of enzyme
hydrolysis of cellulose to pretreatment time also match with the ratio of solubilized sugar
to its theoretical value in pretreated biomass.
The pretreatment time did not influence the amount of xylose or arabinose
released from the glycans (Figure 3-3). The amount of xylose released was about 80
g/kg of biomass under all three residence times. The amount of arabinose released by
the enzymes was about 1 g/kg of biomass. It is interesting to note that 10 min
pretreatment condition that helped release more glucose from the cellulose did not
result in a decrease in the amount of xylose released as seen with the highest amount
of acid or temperature of pretreatment (Figure 3-1, Figure 3-2). This suggests that even
after 10 min of pretreatment at this temperature and acid concentration, significant
amount of hemicellulose remained in the solids.
71
The influence of various pretreatment conditions on enzyme hydrolysis of
residual solids is summarized in Tables 3-2 and 3-3. A pretreatment temperature of
190˚C appears to be helpful for cellulose hydrolysis providing about 40 g additional
glucose per kg biomass compared to a pretreatment at 175˚C. The highest yield of total
sugars from the pretreatment and liquefaction was 328 g/kg of biomass at a
pretreatment condition of 190-1-7.5 (Table 3-3). This is about 46% of the total sugars
recoverable from a kg of switchgrass (716 g/kg biomass).
Concentration of all the inhibitors increased post-liquefaction compared to the
values before liquefaction (Tables 2-3 and 3-4). The reason for this unexpected
increase in furfural and hydroxymethylfurfural during liquefaction is not clear. It is
possible that some of the furans are trapped in the solids matrix that were not pressed
out of the slurry due to the high viscosity. Lowering the viscosity during the liquefaction
step apparently improved the flow characteristics of the slurry resulting in the higher
concentration of the furans.
Effect of Enzyme Level on Enzyme Liquefaction
Acid and steam based pretreatment of biomass although releases significant
amount of pentose sugars, leaves behind almost all the cellulose. Hydrolysis of
cellulose to fermentable sugars requires enzymes, and the commercial enzymes are a
significant cost component of the overall process. To determine the minimum usage of
enzymes that will yield the highest amount of fermentable sugars, the pretreated slurry
was liquefied with various concentrations of Cellic CTec3 and the amount of sugars
released during this step was determined (Figure 3-4). In this experiment, switchgrass
pretreated at 190oC with 1% (w/w) phosphoric acid for 7.5 minutes was used.
72
The amount of glucose released by the enzymes was exponential with increasing
enzyme concentration until 5% (v/w) was reached (Figure 3-4 C). Increasing enzyme
concentration beyond 5% had no significant effect on the amount of glucose released
during the 6 hours of liquefaction. The highest amount of glucose released by hydrolysis
of cellulose was about 160 g per kg of biomass. This ceiling in glucose concentration is
a result of product inhibition of the enzymes cannot be ruled out.
Although average xylose concentration in the samples increased, this increase
appears not be significant above 3% enzyme level. The highest amount of xylose
released by the enzymes was 59 g/kg biomass (Figure 3-4;Table 3-5). The amount of
arabinose released by the enzymes was only about 1-2 g/kg biomass in the enzyme
concentration range of 1 to 5 %. Increasing the enzyme concentration beyond this level
had a significant positive effect on arabinan hydrolysis with arabinose concentration
reaching 12 g/kg biomass at 7.5% enzyme loading. As discussed above, higher amount
of total enzyme may be needed to compensate for the low arabinan hydrolysis activity
of the commercial enzyme.
These results show that under this experimental condition (190-1-7.5 at 10%
solids loading), 5% enzyme loading is optimal and after liquefaction, the samples that
are ready for SScF contained glucose, xylose and arabinose at a concentration of about
165 g, 147 g and 17 g/kg biomass respectively (Table 3-5; Figure 3-4 B).
Increase in Solids Concentration on Hydrolysis
The highest expected ethanol titer based on the composition of switchgrass is
about 365 g/kg of dry biomass. At a 10% solids loading, the theoretical ethanol titer is
about 36.5 g/L of fermentation broth. To minimize the energy required for ethanol
distillation, higher than 10% solids loading during L+SScF is needed (Stampe et al.,
73
1983). Due to the difficulty in mixing slurries with higher solids content, liquefaction of a
15% solids sample was attempted to lower the viscosity.
At 15% solids loading, the amount of glucose released by the enzymes (about
109 g/kg biomass) was lower than the amount from the 10% solids loading (about 150
g/kg biomass) (Figure 3-5; Table 3-6). To minimize the potential inhibitory effect of
furfural and other compounds in the slurry at 15% solids loading, detoxified slurry (as
described above to support fermentation) was also liquefied with enzymes at this solids
loading. The amount of glucose released from cellulose was about the same
irrespective of the detoxification. The lower sugar yield at the 15% solids suggests that
the enzymes are not uniformly mixed at 15% solids loading. An alternate possibility that
the inhibitors present in the slurry at a higher concentration in the 15% slurry (Figure 3-
5C) is inhibiting the enzymes cannot be ruled out (Ximenes et al., 2010; Kumar et al.,
2013).
For xylose, 15% solid loading released more xylose than detoxified 15% solid
loading and 10% solid loading. Detoxification process also affected enzyme action,
which make the liquefaction of detoxified 15% solid loading release less glucose, xylose
and arabinose. This phenomenon was reported by Martinez, et al. that overliming
hydrolysate with base Ca(OH)2 could reduce total furan, phenolic compounds and sugar
as well (Martinez et al., 2001).
74
Table 3-1. Composition of switchgrass and slurry from various pretreatment conditions
Pretreatment conditiona
Component (%)
Glucan Xylan Arabinan Acetate Soluble Lignin Insoluble Lignin Ash
Raw 38.10±0.2 22.20±0.13 3.59±0.38 2.89±0.52 4.75±0.12 22.69±0.34 0.16±0.08 190-0-10 39.50±1.28 20.34±1.21 2.20±0.11 2.46±0.48 4.57±0.17 23.99±0.06 1.38±0.14 160-0.75-5 38.67±1.35 20.40±0.95 2.32±0.16 2.64±0.63 6.97±0.59 23.89±0.45 1.03±0.04 160-1-7.5 39.45±1.04 20.09±0.14 2.36±0.17 2.52±0.35 7.29±0.10 23.99±0.49 0.76±0.10 175-0.75-5 39.89±1.16 19.03±1.15 2.79±0.60 2.40±0.12 5.01±0.22 25.31±2.13 0.69±0.09 175-1-5 40.16±0.34 18.89±0.78 2.58±0.11 2.30±0.12 5.21±0.10 26.09±0.36 0.50±0.05 175-1-7.5 40.23±1.14 18.85±0.17 2.45±0.34 2.25±0.16 4.91±0.32 26.41±0.54 0.16±0.02 175-1-10 40.36±0.48 18.54±0.26 2.10±0.07 2.10±0.25 4.79±0.34 26.28±0.13 0.22±0.20 190-0.75-5 42.54±1.21 17.56±0.54 2.20±0.17 2.05±0.11 3.65±0.16 27.83±1.26 1.45±0.14 190-1-7.5 43.49±0.31 17.65±0.31 1.87±0.05 2.00±0.19 3.54±0.14 28.44±0.34 0.19±0.04 The slurries also include the soluble sugars released during pretreatment. aSwitchgrass was pretreated at the indicated temperature (˚C), acid concentration (% by weight) and time (min). The pretreatment conditions are listed in that order.
75
Table 3-2. Inhibitors in the slurry after liquefaction of various pretreatment condition
Pretreatment conditiona
Total inhibitor content of the slurry after liquefaction (g/kg dry switchgrass)
HMF Furfural Acetate Formate 190-0-10 0.06±0.10 0.54±0.47 18.44±1.61 4.59±1.23 160-0.75-5 0.26±0.16 1.95±0.23 12.38±1.55 0.81±1.14 160-1-7.5 0.54±0.07 2.54±0.48 20.96±1.53 1.27±1.04 175-0.75-5 1.43±0.11 2.88±0.34 15.01±0.65 2.73±0.48 175-1-5 1.48±0.33 3.68±0.35 18.77±1.88 1.99±0.15 175-1-7.5 1.28±0.30 3.01±0.19 16.68±1.18 0.29±0.22 175-1-10 1.32±0.12 4.94±0.14 23.53±3.16 2.11±0.44 190-0.75-5 1.12±0.10 3.27±0.20 18.36±1.53 2.59±0.96 190-1-7.5 1.77±0.58 4.9±0.23 28.23±1.18 2.83±0.36 a Switchgrass was pretreated at the indicated temperature (˚C), acid concentration (% by weight) and time (min). The pretreatment conditions are listed in that order.
76
Table 3-3. Effect of various pretreatment conditions on enzyme hydrolysis of the slurry during liquefaction
Pretreatment conditiona Amount of sugar released by enzyme hydrolysis (g/kg dry switchgrass) Glucose Xylose Arabinose 190-0-10 112.05±2.24 94.46±6.42 3.83±1.91 160-0.75-5 96.01±8.48 88.99±7.43 2.93±1.00 160-1-7.5 110.33±1.97 114.32±3.53 3.28±0.97 175-0.75-5 109.49±1.04 93.26±2.35 2.43±0.93 175-1-5 110.87±2.59 80.07±5.70 1.15±0.73 175-1-7.5 116.60±4.40 77.51±0.96 1.15±1.65 175-1-10 134.52±3.07 81.53±2.05 1.24±0.82 190-0.75-5 154.30±3.70 73.24±2.51 2.01±1.15 190-1-7.5 147.46±5.28 50.52±5.21 1.11±0.99 The listed sugar concentrations represent the amount released during liquefaction step and does not include the amount released during pretreatment. a Switchgrass was pretreated at the indicated temperature (˚C), acid concentration (% by weight) and time (min). The pretreatment conditions are listed in that order.
77
Table 3-4. Total amount of sugars released by the combined pretreatment and
liquefaction steps Pretreatment conditiona Total sugar content of the slurry after liquefaction (g/kg dry switchgrass) Glucose Xylose Arabinose Total sugars 190-0-10 114.78±2.36 94.46±6.42 7.76±3.77 217.00±14.01 160-0.75-5 99.68±9.00 96.46±7.88 13.71±0.74 209.85±11.21 160-1-7.5 117.01±1.58 140.27±3.49 16.23±0.84 273.51±10.02 175-0.75-5 115.03±1.30 114.98±2.86 11.37±0.97 241.38±5.23 175-1-5 116.05±2.59 115.99±5.23 11.02±0.37 243.06±9.00 175-1-7.5 122.16±4.27 113.00±1.29 10.48±1.11 245.64±11.28 175-1-10 143.36±2.96 139.20±5.28 18.10±1.42 300.66±10.05 190-0.75-5 162.51±3.37 110.69±3.38 12.45±0.38 285.65±8.92 190-1-7.5 164.15±5.51 146.52±1.40 17.49±1.17 328.16±7.02 a Switchgrass was pretreated at the indicated temperature (˚C), acid concentration (% by weight) and time (min). The pretreatment conditions are listed in that order.
78
Table 3-5. Effect of enzyme concentration on the amount of sugars released from pretreated switchgrass slurry
Enzyme concentration (% v/w) Sugar concentration (g/kg of dry switchgrass) Glucose Xylose Arabinose Sugars released during liquefaction 1.5 55.17±1.92 35.74±5.98 1.76±1.56 3.0 83.61±1.58 45.47±4.17 1.24±1.21 4.0 106.58±3.01 48.58±1.91 1.89±1.28 5.0 147.46±5.28 50.52±5.21 1.11±0.99 6.0 151.70±11.33 56.53±6.07 8.51±0.27 7.5 161.88±3.32 59.06±3.14 12.04±1.44 Total sugars after liquefaction 1.5 57.57±1.38 107.20±1.60 5.91±0.17 3.0 100.01±1.48 138.43±2.26 11.64±0.72 4.0 123.35±2.80 143.32±0.82 17.40±0.64 5.0 164.15±5.51 146.52±1.40 17.49±1.17 6.0 168.02±11.10 148.31±2.74 17.20±0.46 7.5 178.17±2.66 153.28±0.85 17.96±0.85 The slurry used for this experiment was pretreated at 190-1-7.5 and the solids concentration was 10%. The enzyme CTec3 was used and the samples were incubated for 6 hours at pH 5.0 and 50oC.
79
Table 3-6. Enzyme-catalyzed release of sugars from pretreated switchgrass slurry at 15% solids loading
Solids content during liquefaction (%) [Sugars] (g/kg of dry biomass) Glucose Xylose Arabinose Sugars released during liquefaction 10 147.46±5.28 50.52±5.21 1.11±0.99 15 109.36±3.26 88.23±4.78 2.01±1.01 15 (detoxified slurry) 103.95±3.74 37.51±3.54 1.12±0.31 Total sugars after liquefaction
10 164.15±5.51 146.52±1.40 17.49±1.17 15 117.78±2.95 152.43±3.55 11.84±1.10 15 (detoxified slurry) 106.29±3.54 96.29±4.00 6.54±0.34 The slurry used in this experiment was pretreated at 190-1-7.5. Solids loading was either 10 or 15 %. In addition, a 15% solids containing slurry was also detoxified by base treatment before liquefaction by enzymes. The enzyme CTec3 was used at 5% (v/w) and the samples were incubated for 6 hours at pH 5.0 and 50°C.
80
Figure 3-1. Effect of pretreatment temperature on hydrolysis of carbohydrates during
enzyme liquefaction. A) Sugars released during liquefaction, B) The ratio of released sugar during liquefaction to the theoretical sugar yield from switchgrass, C) Total sugar concentration in the slurry at the end of enzyme liquefaction (pretreatment + liquefaction).
0
20
40
60
80
100
120
140
160
180
Glucose Xylose Arabinose
[Sug
ar] (
g/kg
dry
switc
hgra
ss)
C
160-0.75-5175-0.75-5190-0.75-5
0%
10%
20%
30%
40%
50%
Glucose Xylose Arabinose
Suga
r (%
of t
heor
etic
al su
gar
in p
retr
eate
d sw
itchg
rass
)
B 160-0.75-5 175-0.75-5 190-0.75-5
0
50
100
150
Glucose Xylose Arabinose[Sug
ar] (
g/kg
dry
switc
hgra
ss
A 160-0.75-5 175-0.75-5 190-0.75-5
81
Figure 3-2. Effect of phosphoric acid concentration during pretreatment on enzyme
liquefaction. A) Sugar released during liquefaction, B) Ratio of released sugar during liquefaction to the theoretical level of sugars from switchgrass, C) Total concentration of sugars in the slurry after liquefaction.
0
50
100
150
Glucose Xylose Arabinose
[Sug
ar] (
g/kg
dry
switc
hgra
ss)
A 190-0-10 175-0.75-5 175-1-5
0%
10%
20%
30%
40%
50%
Glucose Xylose Arabinose
Suga
r (%
of t
heor
etic
al su
gar i
n pr
etre
ated
switc
hgra
ss)
B190-0-10 175-0.75-5 175-1-5
0
20
40
60
80
100
120
140
Glucose Xylose Arabinose
[Sug
ar] (
g/kg
dry
switc
hgra
ss)
C190-0-10175-0.75-5175-1-5
82
Figure 3-3. Effect of pretreatment time on enzyme liquefaction. A) Sugar released
during liquefaction, B) Ratio of released sugar during liquefaction process to the theoretical sugar from switchgrass, C) Total sugar concentration in the slurry at the end of enzyme liquefaction.
0
50
100
150
Glucose Xylose Arabinose
[Sug
ar] (
g/kg
dry
switc
hgra
ss)
A 175-1-5175-1-7.5175-1-10
0%
10%
20%
30%
40%
50%
Glucose Xylose Arabinose
Suga
r (%
of t
heor
etic
al su
gar i
n pr
etre
ated
switc
hgra
ss)
B 175-1-5175-1-7.5175-1-10
0
20
40
60
80
100
120
140
160
Glucose Xylose Arabinose
[Sug
ar] (
g/kg
dry
switc
hgra
ss)
C
175-1-5175-1-7.5175-1-10
83
Figure 3-4. Effect of enzyme concentration during liquefaction on the amount of sugars
released from a slurry from 190-1-7.5 pretreatment. A) Sugar released during liquefaction, B) Total concentration of sugars in the samples after liquefaction, C) Glucose and xylose release from different Cellic Ctec3 concentration.
0
50
100
150
200
Glucose Xylose Arabinose[Sug
ar] (
g/kg
dry
switc
hgra
ss)
A 1.5%3%4%5%6%7.5%
020406080
100120140160180200
Glucose Xylose Arabinose
[Sug
ar] (
g/kg
dry
switc
hgra
ss)B 1.5%
3%4%5%6%7.50%
1
2
3
1.5%% 3% 4% 5% 6% 7.5%
Log[
Suga
r]
[Enzyme] %
CGlucose
Xylose
84
Figure 3-5. Effect of solids loading on enzyme liquefaction. A) Sugars released during
liquefaction, B) Ratio of released sugar during liquefaction to the theoretical sugar from switchgrass, C) Total sugar concentration at the end of enzyme liquefaction.
0
20
40
60
80
100
120
140
160
Glucose Xylose Arabinose
[Sug
ar] (
g/kg
dry
switc
hgra
ss)A
10% 15% 15% Detoxified
0%
10%
20%
30%
40%
Glucose Xylose Arabinose
Suga
r (%
of t
he th
eore
tical
su
gar i
n pr
etre
ated
sw
itchg
rass
)
B 10% 15% 15% Detoxified
020406080
100120140160
Glucose Xylose Galactose Arabinose HMF Furfural Acetate Formate Lactate[Sug
ar] (
g/kg
dry
switc
hgra
ss)
C10% 15% 15% Detoxified
85
CHAPTER 4 ETHANOL PRODUCTION FROM PHOSPHORIC ACID PRETREATED
SWITCHGRASS USING INHIBITORY PRODUCTS TOLERANT ESCHERICHIA COLI STRAIN SL100 THROUGH LIQUEFACTION PLUS SIMULTANEOUS
SACCHARIFICATION AND CO-FERMENTATION PROCESS
Introduction
Switchgrass as a primary source of cellulosic material for biofuel production has
been investigated from various aspects (Bai et al., 2010; Garlock et al., 2011; Dien et
al., 2013). Pretreatment is essential to break the structure of cellulosic biomass to
release cell wall sugars for making the downstream fermentation more efficient.
Although various pretreatment and fermentation conditions were tested with switchgrass
(Marzialetti et al., 2011; Wyman et al., 2011), a detailed investigation of fermentation of
phosphoric acid pretreated switchgrass is not available.
Geddes et al. demonstrated that dilute phosphoric acid pretreatment effectively
hydrolyzed hemicellulose from sugarcane bagasse with low level of side products
(Geddes et al., 2010a). Commercial cellulolytic enzymes reduced the viscosity of the
pretreated slurry (liquefaction). The added enzymes continued to hydrolyze the
carbohydrates in the water-insoluble solids to fermentable sugars during fermentation of
the released sugars to ethanol, although the fermentation condition is not optimal for the
enzyme activity. Using an inhibitor-tolerant ethanologenic E. coli, the pentoses and
hexoses derived from the sugarcane bagasse were co-fermented to ethanol. This
process named Liquefaction + Simultaneous Saccharification and co-Fermentation
(L+SScF), also has been applied to sorghum bagasse (Geddes et al., 2013) and
eucalyptus (Castro et al., 2014). These studies show that the optimum condition of
pretreatment to achieve the highest ethanol titer is slightly different with each tested
biomass. In this study, switchgrass was pretreated with phosphoric acid and steam
86
under 8 different conditions and the slurry was liquefied with cellulolytic enzymes (Cellic
CTec3) as described in the previous chapters. In this Chapter, the fermentation and
liquefaction profiles of E. coli strain SL100 using the slurries obtained from these
pretreatment conditions are presented to identify the phosphoric acid based
pretreatment condition that can yield the highest ethanol titer from switchgrass.
Materials and Methods
Materials
Switch grass, provided by FDC Enterprises, Inc. from Virginia was size reduced
to 1 inch and stored at room temperature in sack. Enzyme CTec3 was provided by
Novozymes (Ames, IA). Ammonium hydroxide and other salts were purchased from
Thermo-Fisher Scientific (Waltham, MA). Steam exploded switchgrass with phosphoric
acid impregnated were prepared at IFAS Stan Mayfield Biorefinery Pilot Plant of
University of Florida. Bioflo 110 was purchased from New Brunswick Scientific Co.
(Edison, NJ)
Ethanologenic E. coli
Ethanologenic E. coli strain SL100 was used in this study. E. coli SL100 is a
derivative of E. coli ATCC 9637 that carries the genes encoding pyruvate decarboxylase
(pdc) and alcohol dehydrogenase (adhAB) from Zymomonas mobilis (Miller et al.,2009).
Strain SL 100 was isolated after metabolic adaptation of ethanologenic strain LY180 in
AM1 medium containing sugarcane bagasse hydrolysate obtained after pretreatment
with phosphoric acid and steam (Shi et al., 2016). This strain is tolerant to furfural, the
most abundant inhibitor in the acid pretreated biomass (Larsson et al., 1999). Strain
SL100 also carries deletions of the following genes encoding the enzymes that catalyze
competing reactions; fumarate reductase (frdBC), lactate dehydrogenase (ldhA), native
87
alcohol dehydrogenase (adhE), acetate kinase (ackA) and methylglyoxal synthase
(mgsA). Strain SL100 also carries a mutation in nemR that enhanced nemA expression
(N-ethylmaleimide reductase) and a mutation in an uncharacterized gene, yafC*. In
addition to removing the competing reactions at the pyruvate node, these mutations
conferred tolerance to some of the inhibitors in the hydrolysate other than furfural (Shi et
al., 2016). Due to the higher tolerance of strain SL100 to inhibitors in a biomass
hydrolysate, this E. coli strain was used as the microbial biocatalyst in this study for
fermentation of pretreated slurries of switchgrass.
Media, Seed Strain Propagation and Growth Conditions
Medium for cultivation of seed cultures
Switchgrass slurry obtained after pretreatment at 180oC for 10 minutes with 1%
phosphoric acid was pressed with model CP-4 screw press (Vincent Corporation,
Tampa FL) to obtain the hydrolysate that is used for propagation of E. coli strain SL100.
This hydrolysate was stored at 4˚C without separating the liquid from the particles and
fibers. The hydrolysate had the following characteristics: pH 2.4; conductivity, 3.57
µs/cm; density, 1.03 g/ml; dry weight, 0.39 %. The hydrolysate had cellobiose (4.86
g/L), glucose (3.4 g/L), xylose (19.34 g/L), galactose (2.52 g/L), arabinose (4.07 g/L),
furfural (0.74 g/L) and HMF (0.27 g/L). Prior to use, pH of the hydrolysate was adjusted
to 9.0 with NH4OH and let stand overnight with mixing by a magnetic stirrer at room
temperature (23oC). This empirical method helps lower the toxicity of the hydrolysate to
fermenting microorganism. During the overnight incubation, the pH of the hydrolysate
decreased to about 7.0. The pH-adjusted hydrolysate was filtered through a GF/D glass
microfiber filter to remove fibers and particles. The filtrate was sterilized by filtering
88
through a 0.2 μm Nalgene nylon membrane filter and used for growing E. coli strain
SL100 as inoculum for fermenters.
Preparation of seed cultures
The modified AM1 mineral salts medium used for growing the seed culture
contained the following : (NH4)2HPO4, 19.92 mM; NH4H2PO4, 7.56 mM; MgSO4·7H2O,
1.50 mM; 1 mL of trace metals solution prepared in 120 mM HCl per liter of medium
(composition of trace metal stock; FeCl3 ·6H2O, 8.88mM; CoCl2· 6H2O, 1.26 mM;
CuCl2· 2H2O, 0.88 mM; ZnCl2, 2.20 mM; Na2MoO4 ·2H2O, 1.24 mM; H3BO3, 1.21 mM;
MnCl2· 4H2O, 2.50 mM) (Martinez et al., 2007); switchgrass hydrolysate at the indicated
concentration. The final volume of 1L was achieved by a mixture of hydrolysate and
water. E. coli cultures were grown in 50, 60 or 70 % switchgrass hydrolysate (v/v)
containing medium to evaluate the tolerance of strain SL100 in this hydrolysate. The
lowest hydrolysate concentration that supported highest growth rate and cell yield was
used for preparation of seed cultures for the fermentations.
E. coli strain SL100 grown in sugarcane bagasse hydrolysate was stored at -
80oC as frozen stocks in 40% glycerol (about 8 ml in a 15ml polypropylene tube). This
culture was thawed by immersing in water at 25oC before inoculation into 1L of AM1-
hydrolysate medium in a 2L Erlenmeyer flask. Cultures were grown at 37 oC on a
shaking incubator at 100 RPM for 24 hours. Fifty mL of this culture was transferred to
1L of AM1-hydrolysate medium in a 3L vessel with a working volume of 2L (New
Brunswick BioFlo 110 fermenter; New Brunswick Scientific Co. Inc., Edison, NJ) and
grown at 37oC for 20 hours. The agitation speed was 200 RPM. Culture pH was
automatically controlled at pH 6.3 by addition of 5N ammonium hydroxide and aerated
89
with air at 0.01 vvm (20mL/min). This culture served as the seed culture for
fermentations of switchgrass slurry.
Simultaneous Saccharification and co-Fermentation of Liquefied Switchgrass Slurry
Fermentations were conducted in New Brunswick BioFlo 110 fermenters using a
3L vessel with a 2L working volume (37oC, 200 rpm). Agitation and pH were
automatically controlled by computer through the Primary Control Unit (PCU). Cooled
liquefied slurry obtained after pretreatment was added to the fermentation vessel to
obtain a 10% solids content of switchgrass (in 2L final volume) and the pH was adjusted
to 6.3 with NH4OH (5N). Appropriate amount of AM1 medium and sodium meta-bisulfite
(SMB, 2M) were added to the slurry before inoculation. SMB when added to aqueous
solution converts to two bisulfite molecules that decrease hydrolysate toxicity (Nieves et
al., 2011a; Soudham et al., 2011). The fermentation process was initiated by
transferring 200 mL of the seed culture grown in AM1-hydrolysate medium into the
fermenter to bring the final volume to 2L. The culture was aerated at 0.01 vvm (20
ml/min) that increased the growth rate by rapid metabolic removal of inhibitors in the
slurry. Fermentation pH was maintained at 6.3 by automatic addition of 5N NH4OH.
Temperature of the fermentations were controlled at 37˚C using a water bath. Growth of
the culture and fermentation profile were monitored for 96 hours. Concentration of
sugars, ethanol and co-products during fermentation was determined and normalized to
the starting culture volume of 2L after adjustment for volume change due to sample
withdrawal and base addition. Fermentations were conducted in triplicate and the mean
and standard deviation were calculated from the fermentations profiles.
90
Chemical Analysis
Concentration of sugars, furans and organic acids in the fermentations were
determined using an Agilent Technologies 1260 HPLC equipped with a model G1314B
refractive index detector. Sugars were separated using a BioRad (Hercules, CA)
Aminex HPX-87P ion exclusion column (300x7.8 mm) fitted with a Phenomenex
(Torrance, CA) Carbo-Ca guard column (4x3 mm) at 80°C using nano-pure water as the
mobile phase (0.6 ml/min). Organic acids and furans were determined by HPLC using
an Agilent Technologies 1260 HPLC equipped with dual detectors (UV and refractive
index, in series) and a BioRad Aminex HPX- 87H column (45°C; 4 mM H2SO4 as the
mobile phase, 0.4 ml/ min). Ethanol was measured using an Agilent Technologies 6890
N Network gas chromatograph equipped with a wide bore HP-PLOT Q column (0.5 mm
diameter 30 m; J&W Scientific, Folsom, CA).
Results and Discussion
Acid and steam pretreated switchgrass slurry that contained both solids and
liquid was used for L+SScF to ethanol using E. coli strain SL100. Solids content of the
slurry during the fermentations was 10% (w/w). The viscosity of the slurry was reduced
by the enzyme Cellic CTec3 during the liquefaction stage, as described in Chapter 3.
This slurry still contained all the inhibitors generated during the pretreatment process.
Fermentations were at 37°C and pH 6.3. Although the optimum pH and temperature for
Cellic CTec3 is about 5.0 and 50°C, respectively, certain level of enzyme activity is
expected, but at a lower rate, during the fermentation conditions in support of SScF.
The ethanol titer reflects fermentation of the sugars that are released at various stages
of the overall process, pretreatment, liquefaction and saccharification during
fermentation.
91
Preparation of Seed Cultures of E. coli Strain SL100
Strain SL100 was pre-grown in AM1 medium with switchgrass hydrolysate to
minimize lag time before growth in experimental fermentations. However, the behavior
of this strain on switchgrass hydrolysate is not known. Towards this objective, the
growth and fermentation properties of strain SL100 was evaluated using the hydrolysate
prepared from 180-1-10 pretreatment regime at three different concentrations. A frozen
stock culture that was activated in modified AM1 medium with 30% switchgrass
hydrolysate was inoculated into AM1-hydrolysate medium containing 50, 60 or 70% of
hydrolysate. Growth of all three cultures started after about 3 h lag and the initial growth
rate depended on the hydrolysate concentration of the medium (Figure 4-1 A). Higher
the hydrolysate concentration, the lower the growth rate. This sensitivity to higher
concentration of hydrolysate is not uncommon among fermenting microorganisms (Lau
et al., 2008; Miller et al., 2009; Kothari and Lee, 2011). Although the growth rate was
higher in the medium with 50% hydrolysate, sugar was limiting the final cell density. The
cell density of the cultures with 60 and 70% hydrolysate at 15h were comparable
although at 21h, the culture in a medium with 70% hydrolysate was slightly higher.
Irrespective of the hydrolysate concentration, all the sugar in the medium was
fermented to ethanol in about 20 h. Ethanol concentration increased with increasing
sugar (hydrolysate) concentration with an ethanol titer of 6.24g/L at 24 hours in 70 %
hydrolysate medium (Figure 4-1B). Since the main objective of this experiment was to
define a hydrolysate concentration for growing seed cultures for fermentation, 60%
switchgrass hydrolysate medium was chosen in this study. The sugar concentration in
this medium was almost saturating for growth while the inhibitor concentration was not
92
high to prolong the lag period. A 20 hours old culture was chosen as inoculum for
pretreated switchgrass slurry fermentations.
Fermentation of Pretreated and Liquefied Switchgrass Slurry
Two pretreatment conditions, one with acid and one without acid (190-0-10 and
190-1-7.5), were chosen to initially evaluate the SScF of released sugars to ethanol by
strain SL100. The slurry from the pretreatment condition 190-0-10 after liquefaction
contained 217 g/kg biomass of total sugars and 0.32 g/kg biomass of inhibitors (furfural
+ HMF). On the other hand, the 190-1-7.5 pretreatment condition yielded a slurry with
328 g/kg of total sugars after liquefaction and 5.24 g/kg dry switchgrass of inhibitory
aldehydes (furfural + HMF).
When these slurries were inoculated with strain SL100, ethanol production was
completed by 48 h when the slurry from 190-0-10 was used (Figure 4-2 A). An ethanol
yield of 132.59±6.1 g/kg of biomass is 36% of the theoretical yield of ethanol. On the
other hand, with the slurry from the 190-1-0.75 pretreatment condition, a lag of almost
24 h in ethanol production was observed. Furfural disappeared linearly from the medium
during the first 48 h. Apparently, the observed lag in fermentation of glucose and
ethanol production is related to inhibition of growth of strain SL100 until the furfural
concentration reached an acceptable level. Due to the presence of solids, growth of the
culture was not directly monitored in these fermentations. At about 24 h, glucose
fermentation started and was completely fermented during the next 48 h. The slight
increase in glucose concentration at 24h is due to continued hydrolysis of cellulose
during fermentation. In this fermentation, glucose was preferentially fermented and
xylose fermentation started at about 48 h and continued during the next 48 h when the
93
experiment was terminated. The ethanol yield on fermented sugars was
190.39±2.93g/kg dry switchgrass and this yield is 52.11% of the theoretical yield.
These results show that the inhibitors produced during pretreatment with
phosphoric acid exert a significant negative effect on growth and fermentation of sugars
to ethanol and strain SL100 was able to overcome this inhibitory effect after a lag of about
24 hours.
Effect of Pretreatment Temperature on Ethanol Production
Cellulose is a highly organized crystalline structure in the plant cell wall. The
pretreatment condition is designed to disrupt the cellulose structure for enzyme
hydrolysis. Whether it is SHF or SSF, pretreatment condition plays a significant role in
enabling the substrate polysaccharides amenable to enzyme hydrolysis. Since the
enzymes are inhibited by the products SSF is preferred over SHF and the ethanol titer
and yield serve as an indication of the effectiveness of pretreatment. To evaluate the
effect of temperature at the time of pretreatment, slurries from three different
temperatures of pretreatment were used in this experiment; 160, 175 and 190 ˚C. The
phosphoric acid concentration was 0.75% and the time of pretreatment was 5 min.
Glucose in the slurries from all three pretreatment conditions was rapidly fermented and
a major fraction of ethanol titer was obtained during the first 24 h (Figure 4-3).
Fermentation of glucose in all three slurries was almost complete in 48 h. Fermentation
of xylose in the 190-0.75-5 slurry was slower than the other two fermentations and even
after 96 h, small amount of xylose remained in the medium (Figure 4-3 E). With the
other two slurries, xylose fermentation was complete by 48 h. The highest ethanol titer
from the three slurries was 13.57±0.58 g/L for 160oC, 15.77±0.04 g/L for 175oC,
94
16.78±0.77 for 190oC. Pretreatment at 160°C yielded about 80% of the ethanol as
compared to the pretreatment temperature of 190°C.The ethanol titer increased almost
linearly with the rise in temperature during pretreatment (160 to 190 °C), an indication of
the availability of sugars (Figure 4-3 B). As noted in Chapter 3, higher temperature of
pretreatment apparently supported increased access of the carbohydrates to enzyme
hydrolysis.
Although pretreatment at 190oC increased ethanol titer and yield (16.78± 0.77g/L,
150.99± 6.89g/kg dry switchgrass), it also increased the inhibitor concentration in the
slurry (Table 2-3) that introduces a lag before fermentation starts, as presented in later
part of this chapter.
Effect of Acid Concentration in Pretreatment on Ethanol Titer and Yield
To evaluate the role of phosphoric acid concentration on L+SScF, 0.75 and 1%
were used to pretreat switchgrass at 175°C for 5 min. This pretreatment temperature
and time were chosen to minimize the inhibitor concentration in the slurry. The results
show no significant difference between the two pretreatment conditions on the
fermentation profile or ethanol titer (Figure 4-4) except that with 1% acid condition,
fermentation of xylose required slightly longer time than 48 h, attributable to slightly
higher furfural concentration (Table 3-4). The titer of ethanol was 15.77±0.04 g/L from
0.75%, 16.03±0.01 g/L from 1% acid concentration. The yield of ethanol was
141.94±0.37 g/kg dry switchgrass from 0.75%, 144.30±0.05 g/kg switchgrass from 1%
acid. These values corresponded to 38.85% and 39.49% of theoretical ethanol yield
from switch grass.
95
Effect of Pretreatment Time on Ethanol Titer and Yield
To evaluate the effect of time of pretreatment on overall ethanol titer and yield, a
pretreatment temperature of 175oC and phosphoric acid concentration of 1% were
selected. The three different times of pretreatment were 5, 7.5 and 10 minutes. Under
these conditions, no significant difference in the fermentation profiles of the 5 and 7.5
min pretreatment samples was observed, although the ethanol titer of the 7.5 min
sample (17.20±0.01 g/L) was slightly higher than the 5min sample (16.03±0.01 g/L)
(Figure 4-5). The rate of ethanol production with the 10min pretreatment slurry was
slightly lower than the other two samples and at 72 h the ethanol titer was comparable
to the slurry from the 7.5 min pretreatment (17.65±0.87 g/L). This difference can be
related to the slightly higher furfural concentration of the liquefied slurry from the 10
minutes pretreatment compared to the 7.5 minutes pretreatment (4.94±0.14 vs
3.01±0.19 g/kg dry switchgrass for the 10 and 7.5 min pretreatment, respectively)
(Table 3-4). Due to the combination of higher concentration of sugars (Table 3-3) and
lower ethanol production rate, about 4 g/L xylose remained in the beer at the end of 96
h while all the glucose and xylose were completely fermented in the other two slurries
by 48 h. In spite of the lower rate of ethanol production, pretreatment with longer
retention time supported release of more sugars (Table 3-3) resulting in higher ethanol
yield. It is likely that the higher glucose release from the 10 min pretreatment condition
combined with the inhibitors retarded xylose fermentation resulting in a lower
fermentation rate of this sugar observed with this slurry (Figure 4-5 E).
Based on the results on various pretreatments, the pretreatment condition that
yielded the highest ethanol was 190-1-7.5 (Table 4-1). However, this pretreatment
96
condition also gave the highest inhibitor concentration that resulted in a lag of about 24h
before fermentation of the sugars in the slurry to ethanol started (Figure 4-2 B).
Effect of Enzyme Concentration on Ethanol Production and Yield
As stated earlier, enzymes are critical components of cellulose hydrolysis and
thus, cellulosic ethanol production. The cost of enzymes is a significant component of
the overall cost of products produced from biomass, including ethanol. To determine the
optimum concentration of enzymes needed for ethanol production in the L+SScF
process, pretreated slurries obtained from 190-1-7.5 was treated with varying
concentrations of Cellic CTec3. The enzymes were added to the liquefaction step at
these concentrations and this resulted in an increase in glucose that is proportional to
the enzyme concentration up to 5% enzyme concentration (Table 3-5). Ethanol
production increased with time at all enzyme concentrations (Figure 4-6) but with
different rates during the process. Fermentation profiles of the pretreated slurries with
the enzyme concentrations tested were comparable except for the 1.5% enzyme
loading. The reason for the lack of lag in ethanol production at 1.5% enzyme is not
clear. Ethanol production reached the maximum at 72 h of SScF with an enzyme
concentration of 1.5, 3 and 4 % (w/w). With an enzyme loading of 5% and higher,
ethanol production continued to 96 h when the experiment was terminated. A positive
correlation between the enzyme concentration and ethanol titer was observed up to an
enzyme concentration of 5% (Figure 4-6 B). Increasing the enzyme concentration above
5% had minimal effect on the 96h ethanol titer (22.57±0.04 g/L ethanol at 7.5% enzyme
loading compared to 21.15±0.32 g/L ethanol at 5.0 % enzyme loading). This
corresponds to the increase in sugars released by different concentrations of the
enzymes at the liquefaction step (Table 3-5; Figure 3-5) These results show that about
97
5% (v/w) Celli CTec 3 (11.5 FPU/g switchgrass) is the optimum for ethanol production in
the L+SScF process of phosphoric acid pretreated switchgrass (Table 4-2).
Effect of Solid Loading Level on Ethanol Production and Yield
At 10% solids loading the theoretical ethanol titer is about 350 g/L based on the
glycan composition of switchgrass. This theoretical yield may require higher enzyme
concentration and longer fermentation time; both are not optimal for an efficient
Biorefinery. The highest ethanol concentration achieved with 10% solid loading for 96 h
fermentation with 5% enzyme loading was about 21.15 g/L (about 52% yield) (Table 4-
1). The energy required for distillation of ethanol from this beer is more than 4-times
higher than a beer with about 10% ethanol (Huang and Percival Zhang, 2011). The only
way to achieve this higher ethanol concentration is to increase solids content of the
L+SScF process. Increasing solids loading is inherently plagued with mixing the slurry.
To evaluate higher solids loading switchgrass slurry with 15% solids was attempted.
The switchgrass slurry from the 190-1-7.5 pretreatment was liquefied with 5% enzymes
and the liquefied slurry was fermented in a Bioflo at a volume of 2L. The results are
presented in Figure 4-7.
The slurry at 15% solids loading was not fermented by strain SL100 apparently
due to the presence of various inhibitors in the slurry (Figure 4-7). Although strain
SL100 was isolated as a furfural tolerant strain, the concentration of furfural and/or other
inhibitors in the 15% solids loading are too high for this organism (Shi et al., 2016). The
enzymes continued to hydrolyze the polysaccharides in the solids and the concentration
of glucose increased to 31.95 g/L at the end of 96 h. The xylose concentration did not
change during the first 48 h and then slightly decreased. It is interesting to note that the
furfural concentration increased during the 24 and 48 h suggesting that some of the
98
furfural is released from a solid fraction. After 48 h, the furfural started to decline
indicating metabolic activity of the culture. At 96 h, the furfural concentration was 0.72
g/L.
Holding the slurry at pH 9.0 overnight lowered the toxicity of the slurry. The
detoxified slurry had a lower furfural concentration (0.75g/L) at the beginning of the
fermentation and this slurry was readily fermented by E. coli strain SL100. The ethanol
titer of detoxified slurry at 15% solids loading was 22.57 g/L. Unexpectedly, this titer is
only 1.4 g/L higher than the 21.15 g/L of ethanol from the L+SScF of 10% solids
loading. Carbohydrate content of the solids after 96h fermentation of the detoxified
slurry was much higher than the solids obtained after fermentation of slurry at 10%
solids (Table 4-3). These results suggest that under the experimental conditions used
the enzymes are not mixed uniformly with the solids at high solids loading. An alternate
possibility that the high lignin content of the 15% solids is binding the enzymes
irreversibly and thus lowering the overall activity of the added enzymes cannot be ruled
out.
An ethanologenic microorganism that can ferment the slurries at high solids
loading even in the presence of associated inhibitors is needed for fermentation of the
sugars at high solids loading. Although detoxification of the slurry can support
fermentation of the slurry at high solids loading, this is an additional step in the overall
process of the Biorefinery. Design of impeller that can mix the slurries at high solids
loading is another needed improvement for high solids fermentation.
99
Table 4-1. Ethanol production from the slurry of various pretreatment conditions
Pretreatment conditiona Ethanol titer Ethanol yield (g/L) (g/kg dry biomass)
190-0-10 14.73±0.68 132.59±6.10 160-0.75-5 13.57±0.58 122.15±5.18 160-1-7.5 16.07±0.01 144.63±0.05 175-0.75-5 15.77±0.06 141.94±0.37 175-1-5 16.03±0.01 144.29±0.05 175-1-7.5 17.20±0.01 154.76±0.03 175-1-10 17.65±0.89 158.88±7.98 190-0.75-5 16.78±0.77 150.99±6.89 190-1-7.5 21.15±0.32 190.39±2.92 The reported ethanol concentrations are the highest values observed during the 96h fermentations. Fermentation of the slurries were at 10% solids with an enzyme concentration of 5% (v/w). a Switchgrass was pretreated at the indicated temperature (˚C), acid concentration (% by weight) and time (min). The pretreatment conditions are listed in that order.
100
Table 4-2. Ethanol production from the slurry at various enzyme loading [Enzyme] (%) Ethanol titer (g/L) Ethanol yield (g/kg dry biomass) 1.5 14.27±0.50 128.54±4.51 3.0 16.74±0.56 150.83±5.05 4.0 17.63±0.39 158.83±3.51 5.0 21.15±0.32 190.39±2.92 6.0 21.45±0.20 193.01±1.88 7.5 22.57±0.04 203.12±0.40 Pretreatment condition was 190-1-7.5 and liquefaction of the slurry was at 10% solids loading for 6 hours at pH 5.0 before SScF. The reported values are the highest observed during the 96 h fermentations.
101
Table 4-3. Comparison of residue after fermentation of 10% and 15% solids loading
Solids content Remaining free sugar *
(g/kg dry biomass)
Solid residue composition (g/kg dry biomass)**
Glucose Xylose Arabinose 10% 53.46±8.88 160.62±2.02 54.27±3.91 9.71±1.84 15% 316.93±6.04 210.46±1.60 77.81±1.99 11.22±1.92 15%, Detoxified 15.43±3.67 210.32±5.56 113.07±1.71 13.29±1.03 **Unfermented sugars remaining at the end of 96 and is a sum of all the sugars, glucose, xylose and arabinose. **The amount of carbohydrates remaining in the solids represented as sugar equivalents.
102
Figure 4-1. Effect of switchgrass hydrolysate concentration on the growth and fermentation of E. coli strain SL100. A) Growth, B) Ethanol production.
0.00
1.00
2.00
3.00
4.00
5.00
0 3 6 9 12 15 18 21 24
OD
(560
nm)
Time (h)
A50%
60%
70%
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
0 3 6 9 12 15 18 21 24
[Eth
anol
] (g/
L)
Time (h)
B50%
60%
70%
103
A
0 24 48 72 960
5
10
15
20
0.00
0.01
0.01
0.02 CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfuralEthanolLactic acid
Formic acidAcetic acid
Time (h)
[Sug
ar] a
nd [E
than
ol] (
g/L)
[Fur
fura
l] (g
/L)
B
0 24 48 72 960
10
20
30
0.00
0.25
0.50
0.75 CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfuralEthanolLactic acid
Formic acidAcetic acid
Time (h)
[Sug
ars]
and
[Eth
anol
] (g/
L)
[Fur
fura
l] (g
/L)
Figure 4-2. Fermentation of pretreated and liquefied switchgrass slurry to ethanol by E.
coli strain SL100. A) Pretreatment condition of 190-0-10, B) Pretreatment condition of 190-1-7.5.
104
A
0 24 48 72 960
5
10
15
20
25
160-0.75-5175-0.75-5190-0.75-5
Time (h)
[Eth
nol]
(g/L
)B
160 170 180 19012
14
16
18
100
120
140
160
Ethanol titerEthanol yield
Temperature of pretreatment( oC)
Etha
nol t
iter (
g/L)
Etha
nol y
ield
(g/k
g dr
y sw
itchg
rass
)
C
0 24 48 72 960
10
20
30
0.0
0.5
1.0
1.5
2.0
CellubiosGlucoseXyloseGalactoseArabinoseHMFFurfuralLactic acidFormic acidAcetic acid
Time(h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
0 24 48 72 960
10
20
30
0.0
0.5
1.0
1.5
2.0CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfuralLactic acidFormic acidAcetate
D
Time (h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
0 24 48 72 960
10
20
30
0.0
0.5
1.0
1.5
2.0
CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfuralLactic acidFormic acidAcetic acid
E
Time (h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
Figure 4-3. Effect of pretreatment temperature on fermentation. A) Ethanol production
from various pretreatment temperature, B) Ethanol titer and yield vs. pretreatment temperature, C) Fermentation of 160-0.75-5, D) Fermentation of 175-0.75-5, E) Fermentation of 190-0.75-5.
105
0 24 48 72 960
10
20
30
0.0
0.5
1.0
1.5
2.0CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfuralLactic acidFormic acidAcetate
B
Time (h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
C
0 24 48 72 960
10
20
30
0.0
0.5
1.0
1.5
2.0
CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfural
AcetateFormic acidLactic acid
Time (h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
A
0 24 48 72 960
5
10
15
20
25
175-0.75-5
175-1-5
Time (h)
[Eth
anol]
(g/L
)
Figure 4-4. Effect of pretreatment acid concentration on ethanol production. A) Ethanol
production from different acid concentration of pretreatment, B) Fermentation of 175-0.75-5, C) Fermentation of 175-1-5.
106
A
0 24 48 72 960
5
10
15
20
25
0.0
0.2
0.4
0.6
0.8
1.0175-1-5175-1-7.5175-1-10
Time (h)
[Eth
anol
] (g/
L)
Noth
ing
B
5 6 7 8 9 1015
16
17
18
140
145
150
155
160
Ethanol titer
Ethanol yield
Time (minute)
Etha
nol t
iter (
g/L)
Etha
nol y
ield
(g/k
g dr
y sw
itchg
rass
)
C
0 24 48 72 960
10
20
30
0.0
0.5
1.0
1.5
2.0
CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfural
AcetateFormic acidLactic acid
Time (h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
0 24 48 72 960
10
20
30
0.0
0.5
1.0
1.5
2.0
CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfural
AcetateFormic acidLactic acid
D
Time(h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
0 24 48 72 960
10
20
30
0.0
0.5
1.0
1.5
2.0
CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfuralLactic acidFormic acidAcetic acid
E
Time(h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
Figure 4-5. Effect of pretreatment time on ethanol production. A) Ethanol production as
a function of pretreatment time, B) Ethanol titer and yield from various pretreatment condition, C) Fermentation of 175-1-5, D) Fermentation of 175-1-7.5, E) Fermentation of 175-1-10.
107
A
0 24 48 72 960
5
10
15
20
25
1.5%3%4%5%6%7.5%
Time (h)
[Eth
anol
] (g/
L)
C
0 24 48 72 960
10
20
30
0.0
0.5
1.0
1.5
2.0
CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfuralLactic acidFormic acidAcetate
Time(h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
D
0 24 48 72 960
10
20
30
0.0
0.5
1.0
1.5
2.0
CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfural
AcetateFormic acidLactic acid
Time (h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
B
1 2 3 4 5 6 7 8
15
20
150
200
Ethanol titerEthanol yield
Enzyme loading level % (v/w) of dry switchgrass
Etha
nol t
iter (
g/L)
Etha
nol y
ield
(g/k
g dr
y sw
itchg
rass
)
F
0 24 48 72 960
10
20
30
0.0
0.5
1.0
1.5
2.0CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfuralLactic acidFormic acidAcetic acid
Time(h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
G
0 24 48 72 96 1200
10
20
30
0.0
0.5
1.0
1.5
2.0
CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfuralLactic AcidFormic AcidAcetic Acid
Time(h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
H
0 24 48 72 96 1200
10
20
30
0.0
0.5
1.0
1.5
2.0
CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfuralLactic AcidFormic AcidAcetic Acid
Time(h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
E
0 24 48 72 960
10
20
30
0.0
0.5
1.0
1.5
2.0
CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfural
AcetateFormic acidLactic acid
Time(h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
Figure 4-6. Effect of enzyme concentration on ethanol production from the liquefied
slurry from pretreatment condition of 190-1-7.5. A) Ethanol production from various enzyme concentration, B) Ethanol titer and yield from various enzyme levels, C) Fermentation of 1.5% enzyme loading, D) Fermentation of 3% enzyme loading, E) Fermentation of 4% enzyme loading, F) Fermentation of 5% enzyme loading, G) Fermentation of 6% enzyme loading, H) Fermentation of 7.5% enzyme loading.
108
A
0 24 48 72 960
5
10
15
20
25
10% Solid15% solidDetoxified 15% solid
Time (h)
[Eth
anol
] (g/
L)B
0 24 48 72 960
10
20
30
0.0
0.5
1.0
1.5
2.0CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfuralLactic acidFormic acidAcetic acid
Time(h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
C
0 24 48 72 960
10
20
30
0.0
0.5
1.0
1.5
2.0
CellobioseGlucoseXyloseGalactoseArabinose
HMFFurfuralLactic acidFormic acidAcetate
Time(h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
D
0 24 48 72 960
10
20
30
0.0
0.5
1.0
1.5
2.0
CellobioseGlucoseXyloseGalactoseArabinoseHMFFurfuralLactic AcidFormic AcidAcetic Acid
Time(h)
[Sug
ars]
and
[Inh
ibito
rs] (
g/L)
[Fur
fura
l] (g
/L)
Figure 4-7. Effect of solid loading during fermentation on ethanol production and yield
from pretreatment condition 190-1-7.5. A) Ethanol production from different solids loading, B) Fermentation of 10% solids loading, C) Fermentation of 15% solids loading, D) Fermentation of detoxified 15% solids loading. Liquefied slurry from the pretreatment condition of 190-1-7.5 was used in this experiment.
109
CHAPTER 5 MASS BALANCE OF ETHANOL PRODUCTION FROM ACID PRETREATED
SWITCHGRASS WITH E. COLI SL100 THROUGH L+SScF PROCESS
Introduction
In this study, the switchgrass was pretreated with phosphoric acid and steam.
The slurry from the pretreatment was fermented to ethanol using the L+SScF process
(Geddes et al., 2011). In this process, the solids and liquid are not separated and both
glucose and pentose sugars were co-fermented. The inhibitors generated by the acid-
based pretreatment were not physically removed and the inhibitors in the slurry were
detoxified by the fermenting microorganism. The ethanol titer, yield and mass balance
of various pretreatment conditions are discussed in this chapter.
Result and Discussion
Switchgrass (Alamo) was pretreated with phosphoric acid and steam.
Pretreatment temperature, time and acid concentration were evaluated using ethanol
yield as the benchmark to identify a pretreatment condition for this biomass. In addition,
the amount of enzyme minimally required to produce highest ethanol titer and yield was
also determined.
The composition of the solids remaining after L+SScF indirectly reveals the
effectiveness of pretreatment in destabilizing the biomass components and especially,
cellulose, for enzyme hydrolysis (Table 5-1). It can be inferred based on the
composition of the solids, as the severity of pretreatment increased higher amount of
carbohydrates in the switchgrass was hydrolyzed to sugars. This is more pronounced
with cellulose than with hemicellulose that is partly hydrolyzed by the acid (catalyst)
during the pretreatment. The amount of glucose remaining as cellulose in the solids
after L+SScF of the slurry from a pretreatment condition of 160-0.75-5 was 286.14g/kg
110
biomass compared to the glucose equivalent of 160.62 g/kg biomass for the
pretreatment condition of 190-1-7.5. The theoretical yield of total sugars from a kg of
raw switchgrass is 716.44g calculated based on the composition. This difference in
pretreatment condition in sugar release due to altered structure of the cellulose is also
reflected in the ethanol titer (Table 4-1). Increasing the enzyme concentration, as
expected, hydrolyzed more sugars for fermentation.
At 15% solids loading, the amount of total sugars remaining in the solids after
detoxification and L+SScF was about 350 g/kg of biomass, about 49% of the theoretical
yield of total sugars (716.44 g/kg of switchgrass). This is significantly lower (11%) than
the amount of total sugars remaining (about 430 g/kg biomass) in the solids after a 10%
solids loading (Table 5-2). The amount of sugars fermented combined with the amount
remaining (free sugars and in the solids) represent a mass balance of 97% of the
theoretical yield of sugars. These results are in agreement with the poor mixing of the
enzymes with the solids under these experimental conditions.
Except for three pretreatment conditions (175-1-10 and pretreatment at 190oC),
all other pretreatment conditions yielded a slurry in which almost all the released sugars
were fermented by E. coli strain SL100 (Table 5-2). Small amount of free xylose (about
25-40 g/kg of biomass) remained at the end of 96 h of SScF of the slurries from the
three listed pretreatment conditions. This is apparently due to the higher inhibitor
concentration in these slurries that increased the lag period before fermentation started
and the termination of the experiment at 96h.
The soluble and insoluble lignin content of raw switchgrass is 47.53g/kg and
226.85g/kg (dry weight), respectively. After pretreatment at 190-1-7.5 and 5% enzyme
111
loading, almost all of the insoluble lignin was recovered (222.72 g/kg) while only about
55% (26.7 g/kg) of soluble lignin was detected in the solids (Table 5-1). The soluble
lignin content of the solids after fermentation varied with the pretreatment condition and
with the enzyme concentration. It is likely, a fraction of the soluble lignin was released
into the beer during the process and was not accounted for.
The yield of ethanol also depended on the enzyme level, with 190-1-7.5 at 7.5 %
enzyme loading about 200 g/kg of biomass ethanol was yield (Table 5-3). This converts
to about 60 gallons of ethanol per ton of switchgrass. At 5% enzyme loading, the
ethanol yield was 190 g/kg of biomass and 58 gallons/ton of biomass.
Values that are similar to this yield has been reported by others for other biomass
including sugarcane bagasse, sorghum bagasse, etc. (Geddes et al., 2013). The mass
balance of the overall process for all pretreatment conditions and enzyme loadings
averages to 96.5±2.5 % (Table 5-4).
Further increase in ethanol yield in a fixed time fermentation would depend on
increasing the sugar yield from the biomass, reducing the concentration of inhibitors and
improving the volumetric productivity of the fermenting microbial biocatalyst, especially
for the pentose sugars.
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Table 5-1. Composition of residue after fermentation
Pretreatment condition a
[Enzyme] (%)
Composition of solids after fermentation (g/kg DW biomass)
Glucose Xylose Arabinose Acetate Soluble lignin
Insoluble Lignin
Ash
190-0-10 5.0 270.16 112.18 7.89 14.83 30.61 232.71 9.95 160-0.75-5 5.0 286.14 108.43 10.26 14.97 34.06 232.56 6.29 160-1-7.5 5.0 228.46 95.76 12.39 19.58 30.62 226.47 3.63 175-0.75-5 5.0 222.88 94.82 16.51 18.79 30.06 222.38 8.33 175-1-5 5.0 228.20 95.58 14.21 19.24 33.42 227.44 10.52 175-1-7.5 5.0 221.40 89.71 9.16 19.57 35.03 243.29 9.26 175-1-10 5.0 178.96 69.73 6.09 8.49 30.36 214.41 3.13 190-0.75-5 5.0 184.58 80.12 8.27 12.25 30.51 211.19 6.21 190-1-7.5 1.5 194.52 82.09 13.17 13.98 32.74 238.88 3.58 190-1-7.5 3.0 185.11 77.44 12.06 12.65 31.07 236.17 3.24 190-1-7.5 4.0 177.43 76.31 9.93 15.79 30.26 232.88 2.81 190-1-7.5 5.0 160.62 54.27 9.71 11.17 26.74 222.72 3.05 190-1-7.5 6.0 135.07 65.64 4.88 1.78 18.11 205.06 3.23 190-1-7.5 7.5 113.56 57.13 8.37 9.62 21.53 197.16 2.61 190-1-7.5* 5.0 210.46 77.81 11.22 14.01 45.21 228.18 5.44 190-1-7.5** 5.0 210.32 113.07 13.29 4.04 28.94 232.1 5.54 The sugars listed represent the sugar equivalents of the carbohydrates remaining in the solids. *15% solids, **15% solids, detoxified a Switchgrass was pretreated at the indicated temperature (˚C), acid concentration (% by weight) and time (min). The pretreatment conditions are listed in that order.
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Table 5-2. Effect of pretreatment condition on the amount of sugars fermented and remaining after fermentation‡
Pretreatment conditiona
[Enzyme] (%)
Sugar consumed based on the product* (g/kg dry switchgrass)
Unfermented sugars** (g/kg dry switchgrass)
Ethanol Lactate Acetate Total Glucose Xylose Arabinose Total
190-0-10 5.0 259.98 1.31 54.64 315.94 0.00 0.00 0.00 0.00
160-0.75-5 5.0 239.52 17.94 43.91 301.37 0.31 1.89 3.07 5.27
160-1-7.5 5.0 283.59 28.32 51.48 363.39 0.12 4.87 7.06 12.05
175-0.75-5 5.0 278.31 37.62 50.93 366.85 0.00 4.86 2.09 6.94
175-1-5 5.0 282.93 39.64 43.13 365.70 0.18 0.00 5.00 5.18
175-1-7.5 5.0 303.46 49.81 45.01 398.28 0.10 0.62 2.92 3.63
175-1-10 5.0 311.54 56.17 46.91 414.61 4.11 30.44 9.12 43.66
190-0.75-5 5.0 296.07 36.99 42.99 376.06 2.46 35.55 8.73 46.74
190-1-7.5 1.5 252.04 3.48 93.36 348.88 0.00 5.93 0.00 5.93
190-1-7.5 3.0 295.75 50.00 48.03 393.78 0.00 23.78 7.46 31.24
190-1-7.5 4.0 311.42 53.67 48.23 413.32 0.10 24.32 8.17 32.58
190-1-7.5 5.0 373.31 9.52 50.34 433.16 6.42 39.74 7.30 53.46
190-1-7.5 6.0 378.46 8.69 48.60 435.75 7.99 52.31 8.65 68.95
195-1-7.5 7.5 398.28 8.71 49.52 456.51 8.69 59.75 8.26 76.70
190-1-7.5b 5.0 10.42 32.48 33.21 76.12 181.03 122.25 13.65 316.93
190-1-7.5c 5.0 264.32 25.70 59.73 349.76 0.00 13.89 1.55 15.43 ‡Carbohydrate content of raw switchgrass corresponds to 423.31g/kg glucose, 252.32g/kg xylose, 40.81 g/kg arabinose. *Total sugar consumed was calculated from the product profile. Following theoretical values for sugar conversion to product were used in this calculation; 0.51 g ethanol / g of sugar, 0.67 g acetate / g sugar and 1 g lactate / g sugar. **Free sugars remaining in the liquid fraction after fermentation for 96 hours. a Switchgrass was pretreated at the indicated temperature (˚C), acid concentration (% by weight) and time (min). The pretreatment conditions are listed in that order. b15% solids c15% solids, detoxified
114
Table 5-3. Effect of pretreatment condition on the yield of ethanol from switchgrass
Pretreatment conditiona
Enzyme (%)
Total sugars fermented Yield of ethanol
g/kg dry switchgrass g/kg dry switchgrass
gallons/tonne dry switchgrass
gallons/ton dry switchgrass
190-0-10 5.0 315.94 132.59 44.39 40.27
160-0.75-5 5.0 301.37 122.15 40.90 37.10
160-1-7.5 5.0 363.39 144.63 48.42 43.93
175-0.75-5 5.0 366.85 141.94 47.52 43.11
175-1-5 5.0 365.70 144.29 48.31 43.83
175-1-7.5 5.0 398.28 154.76 51.82 47.01
175-1-10 5.0 414.61 158.88 53.20 48.26
190-0.75-5 5.0 376.06 150.99 50.56 45.86
190-1-7.5 1.5 348.88 128.54 43.04 39.04
190-1-7.5 3.0 393.78 150.83 50.50 45.81
190-1-7.5 4.0 413.32 158.83 53.18 48.24
190-1-7.5 5.0 433.16 190.39 63.74 57.83
190-1-7.5 6.0 435.75 193.01 64.62 58.63
190-1-7.5 7.5 456.51 203.12 68.01 61.70
190-1-7.5* 5.0 76.120 5.32 1.78 1.61
190-1-7.5** 5.0 349.760 134.80 45.13 40.94 a Switchgrass was pretreated at the indicated temperature (˚C), acid concentration (% by weight) and time (min). The pretreatment conditions are listed in that order. *15% solids, **15% solids, detoxified
115
Table 5-4. Mass balance from fermentation of switchgrass Pretreatment
conditiona [Enzyme] Total sugar fermented
Remaining unfermented free
sugars
Remaining sugars in the solids*
Total sugars**
Total lignin Ash Total mass
recovered
Mass Balance
(%) (g/kg dry switchgrass) (%)
190-0-10 5.0 315.94 0.00 390.23 706.17 263.32 9.96 979.46 97.95
160-0.75-5 5.0 301.37 5.27 404.83 711.47 266.62 6.29 984.39 98.44
160-1-7.5 5.0 363.39 12.05 336.61 712.05 257.09 3.64 972.78 97.28
175-0.75-5 5.0 366.85 6.94 334.21 708.00 252.45 8.33 968.79 96.88
175-1-5 5.0 365.70 5.18 337.99 708.87 260.87 10.52 980.26 98.03
175-1-7.5 5.0 398.28 3.63 320.27 722.19 278.33 9.26 1009.79 100.98
175-1-10 5.0 414.61 43.66 254.78 713.06 244.77 3.13 960.96 96.10
190-0.75-5 5.0 376.06 46.74 272.97 695.76 241.71 6.21 943.68 94.37
190-1-7.5 1.5 348.88 5.93 289.78 644.60 271.62 3.59 919.80 91.98
190-1-7.5 3.0 393.78 31.24 274.61 699.63 267.24 3.25 970.12 97.01
190-1-7.5 4.0 413.32 32.58 263.67 709.57 263.14 2.81 975.53 97.55
190-1-7.5 5.0 433.16 53.46 224.60 711.22 249.47 3.06 963.75 96.37
190-1-7.5 6.0 435.75 68.95 205.59 710.29 223.18 3.23 936.70 93.67
195-1-7.5 7.5 456.51 76.70 179.06 712.27 218.70 2.62 933.59 93.36
190-1-7.5b 5.0 76.12 316.93 299.49 692.54 273.39 5.44 971.38 97.14
190-1-7.5c 5.0 349.76 15.43 336.68 701.87 261.05 5.55 968.48 96.85 Theoretical 716.44 274.38 1.60 1000.00 *Remaining sugars in the solids is expressed as sugar equivalents (hexoses and pentoses). **Total sugars is the sum of the amount fermented, remaining as free sugars (in the liquid fraction) and the remaining carbohydrates in the solids, as sugar equivalents. The calculated amount of total sugar equivalent in the biomass is 716.44 g/kg of raw switchgrass. a Switchgrass was pretreated at the indicated temperature (˚C), acid concentration (% by weight) and time (min). The pretreatment conditions are listed in that order. b 15% solids, c 15% solids detoxified
116
CHAPTER 6 GENERAL CONCLUSION AND FUTURE DIRECTIONS
Switchgrass is a potential energy crop native to north America and can be used
for multiple functions, such as forage and soil conservation due to its drought tolerance
and high yield. The average yield for Alamo, the lowland cultivar used in this study was
reported to be 12.9 metric tons per hectare (Wullschleger et al., 2010). Like other
herbaceous biomass, it is naturally resistant to chemical and biological degradation.
Pretreatment is necessary to break the structure and make the biomass more porous
for enzymes to access the carbohydrates. A simplified process utilizing dilute
phosphoric acid based pretreatment developed by Geddes et al. (Geddes et al.,2011)
has been adapted for switchgrass and this is presented in Figure 6-1 and summarized
below.
Dilute phosphoric acid impregnated into switchgrass served as a catalyst in
steam explosion pretreatment. This pretreatment although did not solubilize lignin, it did
hydrolyze hemicellulose. Switchgrass pretreated at 190oC with 1% (W/W) phosphoric
acid for 7.5 minutes released 52.80 g xylose /kg dry biomass and this is 20.93% of
theoretical xylose in the switchgrass. This slurry was fermented by ethanologenic E. coli
strain SL100 to ethanol after liquefaction by simultaneous saccharification and co-
fermentation with 5% (v/w) (11.5 FPU/g biomass) commercial fungal enzyme
preparation Cellic CTec3. This enzyme concentration was determined to be sufficient in
this process condition. Fermentation of this slurry yielded 190.4g ethanol /kg dry
switchgrass or 63.75 gal ethanol /tonne. This translates to an average yield of 886
gallons of ethanol per hectare.
117
Phosphoric acid pretreatment, a milder condition than the sulfuric acid based
pretreatment of biomass, does not require expensive alloys for equipment material;
normally stainless steel could be used. This process (Figure 6-1) uses the entire
pretreated slurry without separating the solids from the liquid that still contains the
inhibitors generated during the pretreatment step, which lowered the capital and
operating cost of the overall process while also minimizing contamination. The microbial
biocatalyst used in this study has been developed to tolerate the inhibitors in the slurry
and ferments the sugars to ethanol. In addition, the phosphoric acid used during the
pretreatment serves as a nutrient for the microorganism during fermentation and later
can be recycled as a fertilizer for growing plants.
Although pretreatment parameters, including temperature, acid concentration
and time have been optimized for ethanol yield and productivity, further investigation is
needed to minimize inhibitor production. Future studies need to focus on increasing the
ratio of total sugars released from the biomass and fermented to over 90%, compared
to the present 60%. This could be accomplished by using a thermotolerant ethanologen
that can perform SScF at the optimum temperature for both the enzyme and the
microbial biocatalyst. To improve the efficiency of distillation, higher ethanol titer needs
to be a goal and this can be achieved by higher than 10% solids loading. However,
higher solids loading requires fermenter designs that would allow efficient mixing of the
enzymes with the solids to achieve the needed high sugar yield to support high ethanol
titer.
118
Figure 6-1. Ethanol production from switchgrass through L+SScF process
119
LIST OF REFERENCES
Alizadeh, H., Teymouri, F., Gilbert, T.I., Dale, B.E., 2005. Pretreatment of switchgrass by ammonia fiber explosion (AFEX), Appl. Biochem. Biotechnol. 124, 1133-1141.
Alterthum, F., Ingram, L.O., 1989. Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli, Appl. Environ. Microbiol. 55, 1943-1948.
Avci, A., Saha, B.C., Dien, B.S., Kennedy, G.J., Cotta, M.A., 2013. Response surface optimization of corn stover pretreatment using dilute phosphoric acid for enzymatic hydrolysis and ethanol production, Bioresour. Technol. 130, 603-612.
Ayyachamy, M., Gupta, V.K., Cliffe, F.E., Tuohy, M.G., 2013. Enzymatic saccharification of lignocellulosic biomass, Anonymous Laboratory Protocols in Fungal Biology. Springer, pp. 475-481.
Bai, Y., Luo, L., van der Voet, E., 2010. Life cycle assessment of switchgrass-derived ethanol as transport fuel, The International Journal of Life Cycle Assessment. 15, 468-477.
Bals, B., Rogers, C., Jin, M., Balan, V., Dale, B., 2010. Evaluation of ammonia fibre expansion (AFEX) pretreatment for enzymatic hydrolysis of switchgrass harvested in different seasons and locations, Biotechnology for biofuels. 3, 1.
Bensah, E.C., Mensah, M., 2013. Chemical pretreatment methods for the production of cellulosic ethanol: technologies and innovations, International Journal of Chemical Engineering. 2013.
Bioenergy, I., 2008. From 1st-to 2nd-Generation biofuel technologies, An overview of current industry and RD&D activities. IEA-OECD.
Bisaria, V., 1998. Bioprocessing of agro-residues to value added products, Anonymous Bioconversion of waste materials to industrial products. Springer, pp. 197-246.
Boukari, I., O’Donohue, M., Rémond, C., Chabbert, B., 2011. Probing a family GH11 endo-β-1, 4-xylanase inhibition mechanism by phenolic compounds: Role of functional phenolic groups, J Molec Catal B. 72, 130-138.
Carrasco, J., Sáiz, M.C., Navarro, A., Soriano, P., Saez, F., Martinez, J., 1994. Effects of dilute acid and steam explosion pretreatments on the cellulose structure and kinetics of cellulosic fraction hydrolysis by dilute acids in lignocellulosic materials, Appl. Biochem. Biotechnol. 45, 23-34.
Castro, E., Nieves, I.U., Mullinnix, M.T., Sagues, W.J., Hoffman, R.W., Fernández-Sandoval, M.T., Tian, Z., Rockwood, D.L., Tamang, B., Ingram, L.O., 2014. Optimization of dilute-phosphoric-acid steam pretreatment of Eucalyptus benthamii for biofuel production, Appl. Energy. 125, 76-83.
120
Chanal, A., Mingardon, F., Bauzan, M., Tardif, C., Fierobe, H.P., 2011. Scaffoldin modules serving as "cargo" domains to promote the secretion of heterologous cellulosomal cellulases by Clostridium acetobutylicum, Appl. Environ. Microbiol. 77, 6277-6280.
Chaturvedi, V., Verma, P., 2013. An overview of key pretreatment processes employed for bioconversion of lignocellulosic biomass into biofuels and value added products, 3 Biotech. 3, 415-431.
Chen, X., Zhou, L., Tian, K., Kumar, A., Singh, S., Prior, B.A., Wang, Z., 2013. Metabolic engineering of Escherichia coli: a sustainable industrial platform for bio-based chemical production, Biotechnol. Adv. 31, 1200-1223.
Cheng, Y., Zheng, Y., Yu, C.W., Dooley, T.M., Jenkins, B.M., VanderGheynst, J.S., 2010. Evaluation of high solids alkaline pretreatment of rice straw, Appl. Biochem. Biotechnol. 162, 1768-1784.
Davis, R., Tao, L., Tan, E., Biddy, M., Beckham, G., Scarlata, C., Jacobson, J., Cafferty, K., Ross, J., Lukas, J., 2013. Process design and economics for the conversion of lignocellulosic biomass to hydrocarbons: dilute-acid and enzymatic deconstruction of biomass to sugars and biological conversion of sugars to hydrocarbons.
Davis, S.C., Diegel, S.W., Boundy, R.G., Transportation Energy Data Book: Edition 32, from the Center for Transportation Analysis (CTA).
de Vasconcelos, S.M., Santos, A.M.P., Rocha, G.J.M., Souto-Maior, A.M., 2013. Diluted phosphoric acid pretreatment for production of fermentable sugars in a sugarcane-based biorefinery, Bioresour. Technol. 135, 46-52.
Demirbas, A., 2009. Political, economic and environmental impacts of biofuels: a review, Appl. Energy. 86, S108-S117.
Dien, B.S., O'Bryan, P.J., Hector, R.E., Iten, L.B., Mitchell, R.B., Qureshi, N., Sarath, G., Vogel, K.P., Cotta, M.A., 2013. Conversion of switchgrass to ethanol using dilute ammonium hydroxide pretreatment: influence of ecotype and harvest maturity, Environ. Technol. 34, 1837-1848.
Edwards, M.C., Henriksen, E.D., Yomano, L.P., Gardner, B.C., Sharma, L.N., Ingram, L.O., Doran Peterson, J., 2011. Addition of genes for cellobiase and pectinolytic activity in Escherichia coli for fuel ethanol production from pectin-rich lignocellulosic biomass, Appl. Environ. Microbiol. 77, 5184-5191.
Evans, M., 1997. The Economic Impact of the Demand for Ethanol, prepared for Midwestern Governor's Conference, Lombard, Illinois, February.
Fengel, D., Wegener, G., 1983. Wood: chemistry, ultrastructure, reactions. Walter de Gruyter.
121
Feyereisen, G.W., Camargo, G.G., Baxter, R.E., Baker, J.M., Richard, T.L., 2013. Cellulosic biofuel potential of a winter rye double crop across the US corn–soybean belt, Agron. J. 105, 631-642.
Fontana, J., Correa, J., Duarte, J., Barbosa, A., Blumel, M., 1984. Aqueous phosphoric acid hydrolysis of hemicelluloses from sugarcane and sorghum bagasses.
Galbe, M., Zacchi, G., 2007. Pretreatment of lignocellulosic materials for efficient bioethanol production, Anonymous Biofuels. Springer, pp. 41-65.
Garlock, R.J., Balan, V., Dale, B.E., Pallapolu, V.R., Lee, Y., Kim, Y., Mosier, N.S., Ladisch, M.R., Holtzapple, M.T., Falls, M., 2011. Comparative material balances around pretreatment technologies for the conversion of switchgrass to soluble sugars, Bioresour. Technol. 102, 11063-11071.
Gaxiola, R., Corona, M., Zinker, S., 1996. A halotolerant mutant of Saccharomyces cerevisiae, J. Bacteriol. 178, 2978-2981.
Geddes, C.C., Peterson, J., Roslander, C., Zacchi, G., Mullinnix, M., Shanmugam, K., Ingram, L., 2010. Optimizing the saccharification of sugar cane bagasse using dilute phosphoric acid followed by fungal cellulases, Bioresour. Technol. 101, 1851-1857.
Geddes, C., Mullinnix, M., Nieves, I., Hoffman, R., Sagues, W., York, S., Shanmugam, K., Erickson, J., Vermerris, W., Ingram, L., 2013. Seed train development for the fermentation of bagasse from sweet sorghum and sugarcane using a simplified fermentation process, Bioresour. Technol. 128, 716-724.
Geddes, C.C., Mullinnix, M., Nieves, I., Peterson, J., Hoffman, R., York, S., Yomano, L., Miller, E., Shanmugam, K., Ingram, L., 2011. Simplified process for ethanol production from sugarcane bagasse using hydrolysate-resistant Escherichia coli strain MM160, Bioresour. Technol. 102, 2702-2711.
Geddes, R., Shanmugam, K.T., Ingram, L.O., 2015. Combining treatments to improve the fermentation of sugarcane bagasse hydrolysates by ethanologenic Escherichia coli LY180, Bioresour. Technol. 189, 15-22.
Gubicza, K., Nieves, I.U., Sagues, W.J., Barta, Z., Shanmugam, K., Ingram, L.O., 2016. Techno-economic analysis of ethanol production from sugarcane bagasse using a liquefaction plus simultaneous saccharification and co-fermentation process, Bioresour. Technol. 208, 42-48.
Hahn-Hägerdal, B., Galbe, M., Gorwa-Grauslund, M., Lidén, G., Zacchi, G., 2006. Bio-ethanol–the fuel of tomorrow from the residues of today, Trends Biotechnol. 24, 549-556.
122
Hahn-Hägerdal, B., Wahlbom, C.F., Gárdonyi, M., van Zyl, W.H., Otero, R.R.C., Jönsson, L.J., 2001. Metabolic engineering of Saccharomyces cerevisiae for xylose utilization, Anonymous Metabolic Engineering. Springer, pp. 53-84.
Hashimoto, K., Kumagai, N., Izumiya, K., Takano, H., Kato, Z., 2014. The production of renewable energy in the form of methane using electrolytic hydrogen generation, Energy, Sustainability and Society. 4, 17.
Hildebrand, A., Schlacta, T., Warmack, R., Kasuga, T., Fan, Z., 2013. Engineering Escherichia coli for improved ethanol production from gluconate, J. Biotechnol. 168, 101-106.
Hillring, B., 2002. Rural development and bioenergy—experiences from 20years of development in Sweden, Biomass Bioenergy. 23, 443-451.
Hong, B., Xue, G., Weng, L., Guo, X., 2012. Pretreatment of moso bamboo with dilute phosphoric acid, BioResources. 7, 4902-4913.
Hu, Z., Wen, Z., 2008. Enhancing enzymatic digestibility of switchgrass by microwave-assisted alkali pretreatment, Biochem. Eng. J. 38, 369-378.
Hu, Z., Ragauskas, A.J., 2011. Hydrothermal pretreatment of switchgrass, Ind Eng Chem Res. 50, 4225-4230.
Hu, Z., Sykes, R., Davis, M.F., Brummer, E.C., Ragauskas, A.J., 2010. Chemical profiles of switchgrass, Bioresour. Technol. 101, 3253-3257.
Huang, W., Zhang, Y.P., 2011. Analysis of biofuels production from sugar based on three criteria: thermodynamics, bioenergetics, and product separation, Energy & Environmental Science. 4, 784-792.
Huntington, G.B., 1997. Starch utilization by ruminants: from basics to the bunk. J. Anim. Sci. 75, 852-867.
Independence, E., 2007. Security Act (EISA), 110-140.2007.
Ingram, L., Aldrich, H., Borges, A., Causey, T., Martinez, A., Morales, F., Saleh, A., Underwood, S., Yomano, L., York, S., 1999. Enteric bacterial catalysts for fuel ethanol production, Biotechnol. Prog. 15, 855-866.
Isci, A., Himmelsbach, J.N., Strohl, J., Pometto, A.L., Raman, D.R., Anex, R.P., 2009. Pilot-scale fermentation of aqueous-ammonia-soaked switchgrass, Appl. Biochem. Biotechnol. 157, 453.
Ishola, M.M., Jahandideh, A., Haidarian, B., Brandberg, T., Taherzadeh, M.J., 2013. Simultaneous saccharification, filtration and fermentation (SSFF): A novel method for bioethanol production from lignocellulosic biomass, Bioresour. Technol. 133, 68-73.
123
Jarboe, L.R., Zhang, X., Wang, X., Moore, J.C., Shanmugam, K.T., Ingram, L.O., 2010. Metabolic engineering for production of biorenewable fuels and chemicals: contributions of synthetic biology, J. Biomed. Biotechnol. 2010, 761042.
Jönsson, L.J., Martín, C., 2016. Pretreatment of lignocellulose: formation of inhibitory by-products and strategies for minimizing their effects, Bioresour. Technol. 199, 103-112.
Karunanithy, C., Muthukumarappan, K., 2011. Optimization of switchgrass and extruder parameters for enzymatic hydrolysis using response surface methodology, Industrial Crops and Products. 33, 188-199.
Karunanithy, C., Muthukumarappan, K., Gibbons, W., 2014. Sequential extrusion-microwave pretreatment of switchgrass and big bluestem, Bioresour. Technol. 153, 393-398.
Keshwani, D.R., Cheng, J.J., 2009. Switchgrass for bioethanol and other value-added applications: a review, Bioresour. Technol. 100, 1515-1523.
Kim, Y., Mosier, N.S., Ladisch, M.R., Pallapolu, V.R., Lee, Y., Garlock, R., Balan, V., Dale, B.E., Donohoe, B.S., Vinzant, T.B., 2011. Comparative study on enzymatic digestibility of switchgrass varieties and harvests processed by leading pretreatment technologies, Bioresour. Technol. 102, 11089-11096.
Kim, Y., Ximenes, E., Mosier, N.S., Ladisch, M.R., 2011. Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass, Enzyme Microb. Technol. 48, 408-415.
Klein-Marcuschamer, D., Oleskowicz-Popiel, P., Simmons, B.A., Blanch, H.W., 2012. The challenge of enzyme cost in the production of lignocellulosic biofuels, Biotechnol. Bioeng. 109, 1083-1087.
Kline, K.L., Oladosu, G.A., Wolfe, A.K., Perlack, R.D., Dale, V.H., McMahon, M., 2008. Biofuel feedstock assessment for selected countries, ORNL/TM-2007/224.
Kojima, M., Akahoshi, T., Okamoto, K., Yanase, H., 2012. Expression and surface display of Cellulomonas endoglucanase in the ethanologenic bacterium Zymobacter palmae, Appl. Microbiol. Biotechnol. 96, 1093-1104.
Kothari, U.D., Lee, Y.Y., 2011. Inhibition effects of dilute-acid prehydrolysate of corn stover on enzymatic hydrolysis of solka floc, Appl. Biochem. Biotechnol. 165, 1391-1405.
Kricka, W., Fitzpatrick, J., Bond, U., 2014. Metabolic engineering of yeasts by heterologous enzyme production for degradation of cellulose and hemicellulose from biomass: a perspective, Frontiers in microbiology. 5, 174.
124
Kumar, S., Kothari, U., Kong, L., Lee, Y., Gupta, R.B., 2011. Hydrothermal pretreatment of switchgrass and corn stover for production of ethanol and carbon microspheres, Biomass Bioenergy. 35, 956-968.
Kurtzman, C., Fell, J., Boekhout, T., 2011. Definition, classification and nomenclature of the yeasts, The yeasts, a taxonomic study. 1, 3-5.
Ladisch, M., Lin, K., Voloch, M., Tsao, G.T., 1983. Process considerations in the enzymatic hydrolysis of biomass, Enzyme Microb. Technol. 5, 82-102.
Lange, J., 2007. Lignocellulose conversion: an introduction to chemistry, process and economics, Biofuels, bioproducts and biorefining. 1, 39-48.
Larsson, S., Palmqvist, E., Hahn-Hägerdal, B., Tengborg, C., Stenberg, K., Zacchi, G., Nilvebrant, N., 1999. The generation of fermentation inhibitors during dilute acid hydrolysis of softwood, Enzyme Microb. Technol. 24, 151-159.
Lau, M.W., Dale, B.E., Balan, V., 2008. Ethanolic fermentation of hydrolysates from ammonia fiber expansion (AFEX) treated corn stover and distillers grain without detoxification and external nutrient supplementation, Biotechnol. Bioeng. 99, 529-539.
Lenihan, P., Orozco, A., O’neill, E., Ahmad, M., Rooney, D., Walker, G., 2010. Dilute acid hydrolysis of lignocellulosic biomass, Chem. Eng. J. 156, 395-403.
Li, C., Knierim, B., Manisseri, C., Arora, R., Scheller, H.V., Auer, M., Vogel, K.P., Simmons, B.A., Singh, S., 2010. Comparison of dilute acid and ionic liquid pretreatment of switchgrass: biomass recalcitrance, delignification and enzymatic saccharification, Bioresour. Technol. 101, 4900-4906.
Liebig, M., Johnson, H., Hanson, J., Frank, A., 2005. Soil carbon under switchgrass stands and cultivated cropland, Biomass Bioenergy. 28, 347-354.
Lindberg, J.E., Ternrud, I.E., Theander, O., 1984. Degradation rate and chemical composition of different types of alkali-treated straws during rumen digestion, J. Sci. Food Agric. 35, 500-506.
Linger, J.G., Adney, W.S., Darzins, A., 2010. Heterologous expression and extracellular secretion of cellulolytic enzymes by Zymomonas mobilis, Appl. Environ. Microbiol. 76, 6360-6369.
Liu, Z., Song, M., 2009. Genomic adaptation of Saccharomyces cerevisiae to inhibitors for lignocellulosic biomass conversion to ethanol, Appl Mycol. 136,.
Luo, Z., Bao, J., 2015. Secretive expression of heterologous β-glucosidase in Zymomonas mobilis, Bioresources and Bioprocessing. 2, 1-6.
125
Luo, Z., Zhang, Y., Bao, J., 2014. Extracellular secretion of β-glucosidase in ethanologenic E. coli enhances ethanol fermentation of cellobiose, Appl. Biochem. Biotechnol. 174, 772-783.
Lynd, L.R., Van Zyl, W.H., McBride, J.E., Laser, M., 2005. Consolidated bioprocessing of cellulosic biomass: an update, Curr. Opin. Biotechnol. 16, 577-583.
Mabee, W., McFarlane, P., Saddler, J., 2011. Biomass availability for lignocellulosic ethanol production, Biomass Bioenergy. 35, 4519-4529.
Martínez-Patiño, J.C., Romero-García, J.M., Ruiz, E., Oliva, J.M., Álvarez, C., Romero, I., Negro, M.J., Castro, E., 2015. High solids loading pretreatment of olive tree pruning with dilute phosphoric acid for bioethanol production by Escherichia coli, Energy Fuels. 29, 1735-1742.
Marzialetti, T., Miller, S.J., Jones, C.W., Agrawal, P.K., 2011. Switchgrass pretreatment and hydrolysis using low concentrations of formic acid, Journal of Chemical Technology and Biotechnology. 86, 706-713.
McAloon, A., Taylor, F., Yee, W., Ibsen, K., Wooley, R., 2000. Determining the cost of producing ethanol from corn starch and lignocellulosic feedstocks, National Renewable Energy Laboratory Report.
McLaughlin, S.B., Kszos, L.A., 2005. Development of switchgrass (Panicum virgatum) as a bioenergy feedstock in the United States, Biomass Bioenergy. 28, 515-535.
Mehdi, B., Zan, C., Girouard, P., Samson, R., 1999. Soil organic carbon sequestration under two dedicated perennial bioenergy crops, 17-23.
Miller, E.N., Jarboe, L.R., Yomano, L.P., York, S.W., Shanmugam, K.T., Ingram, L.O., 2009. Silencing of NADPH-dependent oxidoreductase genes (yqhD and dkgA) in furfural-resistant ethanologenic Escherichia coli, Appl. Environ. Microbiol. 75, 4315-4323.
Misra, D., 1993. Cereal straw, Pulp and paper manufacture. Hamilton, F., Leopold, B.(ed.). 3, 82-93.
Moniruzzaman, M., Lai, X., York, S.W., Ingram, L.O., 1997. Isolation and molecular characterization of high-performance cellobiose-fermenting spontaneous mutants of ethanologenic Escherichia coli KO11 containing the Klebsiella oxytoca casAB operon, Appl. Environ. Microbiol. 63, 4633-4637.
Montalbo-Lomboy, M., Grewell, D., 2015. Rapid dissolution of switchgrass in 1-butyl-3-methylimidazolium chloride by ultrasonication, Ultrason. Sonochem. 22, 588-599.
Mooney, D.F., Roberts, R.K., English, B.C., Tyler, D.D., Larson, J.A., 2009. Yield and breakeven price of ‘Alamo’switchgrass for biofuels in Tennessee, Agron. J. 101, 1234-1242.
126
Mukherjee, V., Steensels, J., Lievens, B., Van de Voorde, I., Verplaetse, A., Aerts, G., Willems, K.A., Thevelein, J.M., Verstrepen, K.J., Ruyters, S., 2014. Phenotypic evaluation of natural and industrial Saccharomyces yeasts for different traits desirable in industrial bioethanol production, Appl. Microbiol. Biotechnol. 98, 9483-9498.
Muñoz-Gutiérrez, I., Moss-Acosta, C., Trujillo-Martinez, B., Gosset, G., Martinez, A., 2014. Ag43-mediated display of a thermostable β-glucosidase in Escherichia coli and its use for simultaneous saccharification and fermentation at high temperatures, Microbial cell factories. 13, 106.
Nguyen, Q., Tucker, M., Boynton, B., Keller, F., Schell, D., 1998. Dilute acid pretreatment of softwoods scientific note, anonymous biotechnology for fuels and chemicals. Springer, pp. 77-87.
Nieves, I., Geddes, C., Miller, E., Mullinnix, M., Hoffman, R., Fu, Z., Tong, Z., Ingram, L., 2011a. Effect of reduced sulfur compounds on the fermentation of phosphoric acid pretreated sugarcane bagasse by ethanologenic Escherichia coli, Bioresour. Technol. 102, 5145-5152.
Nieves, I., Geddes, C., Mullinnix, M., Hoffman, R., Tong, Z., Castro, E., Shanmugam, K., Ingram, L., 2011b. Injection of air into the headspace improves fermentation of phosphoric acid pretreated sugarcane bagasse by Escherichia coli MM170, Bioresour. Technol. 102, 6959-6965.
Öhgren, K., Bura, R., Lesnicki, G., Saddler, J., Zacchi, G., 2007. A comparison between simultaneous saccharification and fermentation and separate hydrolysis and fermentation using steam-pretreated corn stover, Process Biochemistry. 42, 834-839.
Ohta, K., Beall, D.S., Mejia, J.P., Shanmugam, K.T., Ingram, L.O., 1991. Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II, Appl. Environ. Microbiol. 57, 893-900.
Olsen, S.N., Bohlin, C., Murphy, L., Borch, K., McFarland, K., Sweeny, M., Westh, P., 2011. Effects of non-ionic surfactants on the interactions between cellulases and tannic acid: A model system for cellulase–poly-phenol interactions, Enzyme Microb. Technol. 49, 353-359.
Palmqvist, E., Hahn-Hägerdal, B., Galbe, M., Larsson, M., Stenberg, K., Szengyel, Z., Tengborg, C., Zacchi, G., 1996. Design and operation of a bench-scale process development unit for the production of ethanol from lignocellulosics, Bioresour. Technol. 58, 171-179.
127
Papa, G., Rodriguez, S., George, A., Schievano, A., Orzi, V., Sale, K., Singh, S., Adani, F., Simmons, B., 2015. Comparison of different pretreatments for the production of bioethanol and biomethane from corn stover and switchgrass, Bioresour. Technol. 183, 101-110.
Pocienė, L., Šarūnaitė, L., Tilvikienė, V., Šlepetys, J., Kadžiulienė, Ž., 2013. The yield and composition of reed canary grass biomass as raw material for combustion, Biologija. 59.
Prado, R., Erdocia, X., Serrano, L., Labidi, J., 2012. Lignin purification with green solvents, Cellulose Chemistry and Technology. 46, 221.
Qin, Z., Zhuang, Q., Chen, M., 2012. Impacts of land use change due to biofuel crops on carbon balance, bioenergy production, and agricultural yield, in the conterminous United States, Gcb Bioenergy. 4, 277-288.
Rana, V., Eckard, A.D., Ahring, B.K., 2014. Comparison of SHF and SSF of wet exploded corn stover and loblolly pine using in-house enzymes produced from T. reesei RUT C30 and A. saccharolyticus, SpringerPlus. 3, 516.
Restaino, L., Bills, S., Tscherneff, K., Lenovich, L.M., 1983. Growth characteristics of Saccharomyces rouxii isolated from chocolate syrup, Appl. Environ. Microbiol. 45, 1614-1621.
Saha, B.C., Bothast, R.J., 1999. Enzymology of xylan degradation, Anonymous ACS Publications.
Samuel, R., Foston, M., Jiang, N., Allison, L., Ragauskas, A.J., 2011. Structural changes in switchgrass lignin and hemicelluloses during pretreatments by NMR analysis, Polym. Degrad. Stab. 96, 2002-2009.
Satimanont, S., Luengnaruemitchai, A., Wongkasemjit, S., 2012. Effect of temperature and time on dilute acid pretreatment of corn cobs, International Journal Chemical Biological Engineering. 6, 333-337.
Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2011. Determination of structural carbohydrates and lignin in biomass, National Renewable Energy Laboratory-NREL/TP-510-42618.Laboratory Analytical Procedure (LAP). Golden, CO.
Shi, J., Ebrik, M.A., Wyman, C.E., 2011. Sugar yields from dilute sulfuric acid and sulfur dioxide pretreatments and subsequent enzymatic hydrolysis of switchgrass, Bioresour. Technol. 102, 8930-8938.
Shi, A., Zheng, H., Yomano, L.P., York, S.W., Shanmugam, K.T., Ingram, L.O., 2016. Plasmidic expression of nemA and yafC* Increased resistance of ethanologenic Escherichia coli LY180 to nonvolatile side products from dilute acid treatment of sugarcane bagasse and artificial hydrolysate, Appl. Environ. Microbiol. 82, 2137-2145.
128
Sladden, S., Bransby, D., Aiken, G., 1991. Biomass yield, composition and production costs for eight switchgrass varieties in Alabama, Biomass Bioenergy. 1, 119-122.
Soudham, V.P., Alriksson, B., Jönsson, L.J., 2011. Reducing agents improve enzymatic hydrolysis of cellulosic substrates in the presence of pretreatment liquid, J. Biotechnol. 155, 244-250.
Soudham, V.P., Alriksson, B., Jönsson, L.J., 2011. Reducing agents improve enzymatic hydrolysis of cellulosic substrates in the presence of pretreatment liquid, J. Biotechnol. 155, 244-250.
Stampe, S., Alcock, R., Westby, C., Chisholm, T., 1983. Energy consumption of a farm-scale ethanol distillation system, Energy Agric. 2, 355-368.
Sundar, S., Bergey, N.S., Salamanca-Cardona, L., Stipanovic, A., Driscoll, M., 2014. Electron beam pretreatment of switchgrass to enhance enzymatic hydrolysis to produce sugars for biofuels, Carbohydr. Polym. 100, 195-201.
Tao, L., Aden, A., Elander, R.T., Pallapolu, V.R., Lee, Y., Garlock, R.J., Balan, V., Dale, B.E., Kim, Y., Mosier, N.S., 2011. Process and technoeconomic analysis of leading pretreatment technologies for lignocellulosic ethanol production using switchgrass, Bioresour. Technol. 102, 11105-11114.
Thomason, W., Raun, W., Johnson, G., Taliaferro, C., Freeman, K., Wynn, K., Mullen, R., 2005. Switchgrass response to harvest frequency and time and rate of applied nitrogen, J. Plant Nutr. 27, 1199-1226.
Turner, P.C., Miller, E.N., Jarboe, L.R., Baggett, C.L., Shanmugam, K., Ingram, L.O., 2011. YqhC regulates transcription of the adjacent Escherichia coli genes yqhD and dkgA that are involved in furfural tolerance, J. Ind. Microbiol. Biotechnol. 38, 431-439.
USDA-NRCS Plant Materials Program, Cape May Plant Materials Center, 2012. Suther Germplasm Big Bluestem Andropogon gerardii Vitman.
Wang, Z., Zhu, J., Zalesny, R.S., Chen, K., 2012. Ethanol production from poplar wood through enzymatic saccharification and fermentation by dilute acid and SPORL pretreatments, Fuel. 95, 606-614.
Wang, X., Miller, E.N., Yomano, L.P., Shanmugam, K.T., Ingram, L.O., 2012. Increased furan tolerance in Escherichia coli due to a cryptic ucpA gene, Appl. Environ. Microbiol. 78, 2452-2455.
Wang, X., Miller, E.N., Yomano, L.P., Zhang, X., Shanmugam, K.T., Ingram, L.O., 2011. Increased furfural tolerance due to overexpression of NADH-dependent oxidoreductase FucO in Escherichia coli strains engineered for the production of ethanol and lactate, Appl. Environ. Microbiol. 77, 5132-5140.
129
Williams, C.M., Biswas, T., 2010. Commercial potential of giant reed for pulp, paper and biofuel production. Rural Industries Research and Development Corporation.
Wiselogel, A., Agblevor, F., Johnson, D., Deutch, S., Fennell, J., Sanderson, M., 1996. Compositional changes during storage of large round switchgrass bales, Bioresour. Technol. 56, 103-109.
World Agricultural Outlook Board WAOB, 2015. World Agricultural Supply and Demand Estimates, 546.
Wright, J.D., 1987. Ethanol from lignocellulose; An overview.
Wright, L., 2007. Historical perspective on how and why switchgrass was selected as a “model” high-potential energy crop, ORNL/TM-2007/109 Oak Ridge, TN: Bioenergy Resources and Engineering Systems.
Wright, L., Turhollow, A., 2010. Switchgrass selection as a “model” bioenergy crop: a history of the process, Biomass Bioenergy. 34, 851-868.
Wullschleger, S.D., Davis, E.B., Borsuk, M.E., Gunderson, C.A., Lynd, L., 2010. Biomass production in switchgrass across the United States: database description and determinants of yield, Agron. J. 102, 1158-1168.
Wyman, C.E., Balan, V., Dale, B.E., Elander, R.T., Falls, M., Hames, B., Holtzapple, M.T., Ladisch, M.R., Lee, Y., Mosier, N., 2011. Comparative data on effects of leading pretreatments and enzyme loadings and formulations on sugar yields from different switchgrass sources, Bioresour. Technol. 102, 11052-11062.
Xu, J., Cheng, J.J., 2011. Pretreatment of switchgrass for sugar production with the combination of sodium hydroxide and lime, Bioresour. Technol. 102, 3861-3868.
Yandapalli, V., Mani, S., 2014. Effect of lime pretreatment on granulation of switchgrass, BioEnergy Research. 7, 833-844.
Yang, B., Wyman, C.E., 2004. Effect of xylan and lignin removal by batch and flowthrough pretreatment on the enzymatic digestibility of corn stover cellulose, Biotechnol. Bioeng. 86, 88-98.
Yat, S.C., Berger, A., Shonnard, D.R., 2008. Kinetic characterization for dilute sulfuric acid hydrolysis of timber varieties and switchgrass, Bioresour. Technol. 99, 3855-3863.
Yuan, Y., Bi, C., Nicolaou, S.A., Zingaro, K.A., Ralston, M., Papoutsakis, E.T., 2014. Overexpression of the Lactobacillus plantarum peptidoglycan biosynthesis murA2 gene increases the tolerance of Escherichia coli to alcohols and enhances ethanol production, Appl. Microbiol. Biotechnol. 98, 8399-8411.
130
Zaldivar, J., Martinez, A., Ingram, L.O., 2000. Effect of alcohol compounds found in hemicellulose hydrolysate on the growth and fermentation of ethanologenic Escherichia coli, Biotechnol. Bioeng. 68, 524-530.
Zhao, X., Zhang, L., Liu, D., 2010. Pretreatment of Siam weed stem by several chemical methods for increasing the enzymatic digestibility, Biotechnology journal. 5, 493-504.
Zheng, H., Wang, X., Yomano, L.P., Shanmugam, K.T., Ingram, L.O., 2012. Increase in furfural tolerance in ethanologenic Escherichia coli LY180 by plasmid-based expression of thyA, Appl. Environ. Microbiol. 78, 4346-4352.
Zheng, J., Liu, L., Liu, C., Jin, Q., 2012. Molecular cloning and heterologous expression of a true lipase in Pichia pastoris isolated via a metagenomic approach, J. Mol. Microbiol. Biotechnol. 22, 300-311.
Zhou, S., Ingram, L., 2001. Simultaneous saccharification and fermentation of amorphous cellulose to ethanol by recombinant Klebsiella oxytoca SZ21 without supplemental cellulase, Biotechnol. Lett. 23, 1455-1462.
Zhou, X., Xu, J., Wang, Z., Cheng, J.J., Li, R., Qu, R., 2012. Dilute sulfuric acid pretreatment of transgenic switchgrass for sugar production, Bioresour. Technol. 104, 823-827.
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BIOGRAPHICAL SKETCH
Wei Wu was born in Jilin, China in 1975. She attended schools in Jilin, China and
later in Tianjin, China where she joined the Chemical Engineering Department at Tianjin
University of Science and Technology in 1993 and graduated with a Master of
Engineering degree in April 2000. After graduation, she worked in Tianjin University of
Science and Technology as a teacher and office manager. In 2008, along with her
family she moved to Gainesville, Florida. In 2014, she decided to pursue a graduate
degree and joined Dr. Pratap Pullammanappallil’s lab at the University of Florida’s
Department of Agricultural and Biological Engineering. She received her Ph.D. from the
University of Florida in the summer of 2017.