fermentation of carbohydrates in ......1-2 ethanol production from sugar or starch feedstock..... 38...

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

To my husband, my parents and my two sweet girls who supported me make every progress on my way

4

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.

5

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

6

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


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


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

7

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

8

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

9

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

10

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

11

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

12

(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.

13

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.

14

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

15

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

16

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.

17

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

18

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

19

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

20

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).

21

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

22

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

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.


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).

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Table 3-1. Composition of switchgrass and slurry from various pretreatment conditions


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


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.

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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

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120

140

160

180

Glucose Xylose Arabinose

[Sug

ar] (

g/kg

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switc

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C

160-0.75-5175-0.75-5190-0.75-5

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Glucose Xylose Arabinose[Sug

ar] (

g/kg

dry

switc

hgra

ss

A 160-0.75-5 175-0.75-5 190-0.75-5

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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

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[Sug

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A 190-0-10 175-0.75-5 175-1-5

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140


[Sug

ar] (

g/kg

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switc

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C190-0-10175-0.75-5175-1-5

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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

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[Sug

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A 175-1-5175-1-7.5175-1-10

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[Sug

ar] (

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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

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switc

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A 1.5%3%4%5%6%7.5%

020406080

100120140160180200


[Sug

ar] (

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switc

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3%4%5%6%7.50%

1

2

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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

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[Sug

ar] (

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10% 15% 15% Detoxified

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020406080

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Glucose Xylose Galactose Arabinose HMF Furfural Acetate Formate Lactate[Sug

ar] (

g/kg

dry

switc

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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

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

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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).


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

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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



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



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



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


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



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


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.

113

Table 5-2. Effect of pretreatment condition on the amount of sugars fermented and remaining after fermentation‡


[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


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

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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.

fermentation of carbohydrates in ......1-2 ethanol production from sugar or starch feedstock..... 38...

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