impact of pretreatment methods on enzymatic … · 2.1.2 hardwood ... figure 2-8 dry pine...

Impact of Pretreatment Methods on Enzymatic Hydrolysis of Softwood

by

Tim Tze Wei Sun

A thesis submitted in conformity with the requirements for the degree of Masters of Applied Science

Graduate Department of Chemical Engineering and Applied Chemistry

University of Toronto

Copyright © 2013 by Tim Tze Wei Sun

II

Abstract

Impact of Pretreatment Methods on Enzymatic Hydrolysis of Softwood

Tim Tze Wei Sun

Masters of Applied Science

Graduate Department of Chemical Engineering and Applied Chemistry University of Toronto

2013

Bioethanol is an appealing alternative to petroleum-based liquid fuel due to drivers such as

environmental regulations and government mandates. Second generation lignocellulosic

feedstocks are abundant, but their resistance to hydrolysis continues to be problematic. Different

pretreatments have been proposed to increase cellulose reactivity.

Softwood pine autohydrolyzed at different severities was subjected to further treatment to

increase fibre reactivity. Liquid hot water is most effective at removing barriers, with the highest

increase in sugar yield after enzymatic hydrolysis. Alkaline (NaOH) is found to be the worst

option compared to dilute acid and organosolv. In addition, higher chemical concentrations and

longer treatment times do not guarantee higher enzymatic hydrolysis yield.

Process modifications such as fiber washing and multistage enzymatic hydrolysis are observed to

be effective at increasing yield. However, more research is required to bring the enzymatic

hydrolysis yield to a level where commercialization is feasible.

III

Acknowledgement

My sincere gratitude goes towards Professor Bradley Saville for providing this opportunity to

tackle a global problem that has many implications in terms of applications. His guidance,

knowledge, and support were invaluable throughout this project, especially during times when

nothing seemed to work. In addition, I would like to thank NSERC Bioconversion Network for

providing the funding for this project and the opportunities to meet and exchange ideas with

other researchers working in the field. To Mascoma Canada for providing the necessary

autohydrolysis equipment.

I would also like to thank Professor Emma Master and Professor Krishna Mahadevan for serving

on the defense committee and taking the time to give valuable insight into the project.

Special thanks go out to everyone in Biozone for making the past 2 years fun and worthwhile.

Thanks to all the friends that have kept me in check and helped me balance research and social

life.

Lastly but most importantly, to my parents for their continual support in everything I do. Their

constant encouragement kept me focused and brought me back to my feet when I was at my

lowest. Without their support, I would not be where I am today.

IV

Table of Contents

Abstract .................................................................................................................................................... II Acknowledgement .............................................................................................................................. III Table of Contents .................................................................................................................................. IV List of Figures ........................................................................................................................................ VI List of Tables .......................................................................................................................................... IX Nomenclature ......................................................................................................................................... X 1 Introduction ................................................................................................................................... 1 2 Background .................................................................................................................................... 3

2.1 Second Generation Feedstock: Lignocellulosic Material ................................................ 3 2.1.1 Agricultural Residues................................................................................................ 6 2.1.2 Hardwood .................................................................................................................. 7 2.1.3 Softwood ................................................................................................................... 8

2.2 Bioconversion Processes ................................................................................................ 10 2.3 Pretreatment ................................................................................................................... 11

2.3.1 Physical Treatment.................................................................................................. 13 2.3.2 Chemical Treatment ................................................................................................ 13 2.3.3 Physicochemical Treatment .................................................................................... 15 2.3.4 Potential Adverse Effects of Pretreatment .............................................................. 16

2.4 Enzymatic Hydrolysis - Kinetics .................................................................................... 17 2.4.1 Particle Size Reduction ........................................................................................... 19 2.4.2 Inhibitors ................................................................................................................. 19 2.4.3 Non-productive Binding ......................................................................................... 19

2.5 Enzymatic Hydrolysis - Scale-up.................................................................................... 20 2.6 Current Status - Softwood as a Second-Generation Biofuel Feedstock ......................... 21

3 Research Focus and Aim .......................................................................................................... 23 4 Experimental Protocol and Design ...................................................................................... 26

4.1 Materials ........................................................................................................................ 26 4.1.1 Substrate .................................................................................................................. 26 4.1.2 Enzymes .................................................................................................................. 26 4.1.3 Pretreatment Chemicals .......................................................................................... 27

4.2 Apparatus/Equipment ..................................................................................................... 27 4.2.1 Pretreatment Reactor ............................................................................................... 27 4.2.2 Incubator Hydrolysis Reactions .............................................................................. 28 4.2.3 Jacketed Reactor ..................................................................................................... 28 4.2.4 Milling..................................................................................................................... 30

4.3 Experimental Protocol ................................................................................................... 30 4.3.1 Pretreatment ............................................................................................................ 30 4.3.2 Enzymatic Hydrolysis ............................................................................................. 31 4.3.3 Multistage Enzymatic Hydrolysis ........................................................................... 31 4.3.4 Solids Characterization ........................................................................................... 32

4.4 Sample Analysis.............................................................................................................. 32 4.4.1 High Performance Liquid Chromatography (HPLC) ............................................. 32

V

4.4.2 Brix Refractometer.................................................................................................. 32 4.5 Methods to Evaluate Enzyme Performance ................................................................... 33

4.5.1 Enzyme Hydrolysis Yield ....................................................................................... 33 4.5.2 Graphical Analysis of Rates.................................................................................... 33 4.5.3 Enzyme Performance Index .................................................................................... 34

5 Observation and Results .......................................................................................................... 36 5.1 Pretreatment Conditions ................................................................................................ 36

5.1.1 Autohydrolysis ........................................................................................................ 36 5.1.2 Chemical Treatment ................................................................................................ 38 5.1.3 Particle Size Reduction ........................................................................................... 40

5.2 Effect of Enzyme Loading and Cocktail ......................................................................... 40 5.2.1 Enzyme Inhibition ................................................................................................... 43

5.3 Effect of Substrate Loading and Reaction Scale ............................................................ 45 5.3.1 Reaction Scale-up ................................................................................................... 45 5.3.2 Higher Solids Loading ............................................................................................ 46

6 Discussion and Analysis ........................................................................................................... 48 6.1 Autohydrolysis Pretreatment .......................................................................................... 49 6.2 Post-autohydrolysis Treatment ...................................................................................... 52

6.2.1 Physical Treatment.................................................................................................. 52 6.2.2 Chemical Treatment ................................................................................................ 55

6.3 Enzymatic Hydrolysis – Reaction Rate and Yield .......................................................... 62 6.4 Enzymatic Hydrolysis - Inhibitors .................................................................................. 66

6.4.1 Liquid Removal ...................................................................................................... 66 6.4.2 Staggered Enzyme Addition ................................................................................... 67 6.4.3 Higher Solids Loading ............................................................................................ 67 6.4.4 Multistage Enzymatic Hydrolysis/Washing ........................................................... 69

7 Conclusions and Recommendations .................................................................................... 71 7.1 Recommendations........................................................................................................... 72

References ............................................................................................................................................. 75 Appendix A - Calculating Yield Through Total Mass Balance For Multistage Enzymatic Hydrolysis ............................................................................................................................................... 80

VI

List of Figures

Figure 2-1 Lignocellulosic Biomass Structure and Component (Adapted from Rubin 2008) ....... 3

Figure 2-2 Cellulose Chain (Adapted from Gardner and Blackwell 1974) .................................... 4

Figure 2-3 Structure of Crystalline Cellulose - Red Circles Illustrate the Alternating β-1,4 glycosidic Bonds (Adapted from Greer, Pemberton and Tan, 2006) ............................................. 5

Figure 2-4 The Building Blocks of Lignin (Adapted from Rubin 2008) ........................................ 6

Figure 2-5 Hardwood Roadside Harvest Residue Distribution Across Canada ............................. 7

Figure 2-6 Canada's Total Forested Area (Hardwood and Softwood) ............................................ 8

Figure 2-7 Softwood Roadside Harvest Residue Distribution Across Canada ............................... 9

Figure 2-8 Dry Pine Composition by Mass (w/w%) (Galbe and Zacchi 2007) ............................ 10

Figure 2-9 Bioconversion Process ................................................................................................ 10

Figure 2-10 Schematic Representation of Effect of Pretreatment on Biomass (Adapted from Mosier et al. 2005) ........................................................................................................................ 12

Figure 2-11 Enzymes and substrates used in biomass hydrolysis (Adapted from Binod et al. 2011). ............................................................................................................................................ 18

Figure 4-1 Agitator Blade Arrangement for SBI Reactor ............................................................. 29

Figure 4-2 Typical Sugar Generation Curves ............................................................................... 34

Figure 5-1 Pine Autohydrolysis Product - 200oC/8min (left) and 205oC/10min (right)............... 36

Figure 5-2 Pine Autohydrolysis Product - 215oC/8min ................................................................ 37

Figure 5-3 Douglas Fir Autohydrolysis Product - 215oC/8min, batch ......................................... 37

Figure 5-4 Yield Profile in Terms of Total Glucan Available for Different Enzyme Cocktails Using Autohydrolyzed Fibres from 200oC/8min Condition (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10% Solids Loading, n = 1) ................................................................ 41

Figure 5-5 Yield Profile in Terms of Total Glucan Available for Different Enzyme Cocktails Using Autohydrolyzed Fibres from 205oC/10min Condition (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10% Solids Loading, n = 1) ................................................................ 42

VII

Figure 5-6 Impact of Enzyme Load with No Buffer on Enzymatic Hydrolysis Yield in Terms of Total Glucan Available (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10% Solids Loading, 5% Enzyme Loading, n = 1) ............................................................................... 42

Figure 5-7 Impact of Liquid Removal on Enzymatic Hydrolysis Yield in Terms of Total Glucan Available (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10% Solids Loading, n = 1) ................................................................................................................................................ 44

Figure 5-8 Impact of Staggered Enzyme Addition on Enzymatic Hydrolysis Glucose Generation (Enzymatic Hydrolysis Conditions – 1L Reaction Volume, 10% Solids Loading, 5% Enzyme Loading, n = 1) .............................................................................................................................. 44

Figure 5-9 Impact of Interstage Wash on Enzymatic Hydrolysis Yield in Terms of Total Glucan Available (Enzymatic Hydrolysis Conditions – 1L Reaction Volume, 20% Solids Loading, 5% Enzyme Loading with Addition of Half the Initial Amount after Solid/Liquid Separation, n = 2 averaged value) ............................................................................................................................. 45

Figure 5-10 Impact of Solids Loading on Enzymatic Hydrolysis Yield in Terms of Total Glucan Available (Enzymatic Hydrolysis Conditions – 1L Reaction Volume, 5% Enzyme Loading, n = 1)....................................................................................................................................................... 47

Figure 6-1 Effect of Autohydrolysis Severity on Glucose Enzymatic Hydrolysis Yield in Terms of Total Glucan Available (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10% Solids Loading, 5% Enzyme Loading) ......................................................................................... 50

Figure 6-2 Impact of Particle Size Reduction on Enzymatic Hydrolysis Yield in Terms of Total Glucan Available (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10% Solids Loading, 5% Enzyme Loading, n = 1) .......................................................................................... 53

Figure 6-3 Impact of Particle Size on Initial Amount of Sugar Generated During Enzymatic Hydrolysis (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10% Solids Loading, 5% Enzyme Loading, n = 1) ......................................................................................................... 54

Figure 6-4 Impact of Duration of NaOH Treatment at 65oC on Enzymatic Hydrolysis Yield in Terms of Total Glucan Available (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10% Solids Loading, 5% Enzyme Loading, n = 1) ...................................................................... 56

Figure 6-5 Comparing the Impact of Alkaline Treatment and Organosolv Treatment on Enzymatic Hydrolysis Yield in Terms of Total Glucan Available (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10% Solids Loading, 5% Enzyme Loading, n = 1) ...... 58

Figure 6-6 Impact of IPA Concentration Used in Chemical Treatment on Enzymatic Hydrolysis Yield in Terms of Total Glucan Available (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10% Solids Loading, 5% Enzyme Loading, n = 1) ........................................................ 59

VIII

Figure 6-7 Impact of Using Different Chemical Treatment on Enzymatic Hydrolysis Yield in Terms of Total Glucan Available (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10% Solids Loading, 5% Enzyme Loading, n = 1) ...................................................................... 60

Figure 6-8 Impact of Cocktail Blend on Enzymatic Hydrolysis Yield Within First 24hr (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10% Solids Loading, 5% Enzyme Loading, n = 1) ............................................................................................................... 64

Figure 6-9 Glucose Generation Profile with Second Enzyme Addition after First 72hr of Hydrolysis (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10% Solids Loading, 5% Enzyme Loading, n = 1) ......................................................................................................... 65

Figure A-1 Block Flow Diagram for Multistage Enzymatic Hydrolysis ...................................... 82

IX

List of Tables

Table 2-1 Typical Woody Biomass Composition (Mass Basis) ..................................................... 6

Table 2-2 Select Results from Past Investigations Using Different Softwood as a Biofuel Feedstock (Mosier et al; 2005; Galbe and Zacchi 2007; Zhu and Pan 2010) ............................... 21

Table 5-1 Impact of Autohydrolysis Condition on Enzymatic Hydrolysis Yield ........................ 38

Table 5-2 Impact of Length of Alkaline Treatment Time (at 65oC) on Enzymatic Hydrolysis Yield .............................................................................................................................................. 38

Table 5-3 Impact of Alkaline Concentration and Autohydrolysis Severity (at 125oC) on Enzymatic Hydrolysis Yield ......................................................................................................... 39

Table 5-4 Impact of Organosolv Treatment (at 65oC) on Enzymatic Hydrolysis ........................ 39

Table 5-5 Impact of Different Chemical Treatment (at 125oC) on Enzymatic Hydrolysis Yield 39

Table 5-6 Impact of Particle Size Reduction on Enzymatic Hydrolysis Yield............................. 40

Table 5-7 Different Enzyme Cocktails Used ................................................................................ 41

Table 5-8 Impact of Different Enzyme Cocktails on Enzymatic Hydrolysis Yield ..................... 41

Table 5-9 Impact of Citrate Buffer on Enzymatic Hydrolysis Yield ............................................ 43

Table 5-10 Impact of Reactor Volume on Enzymatic Hydrolysis Yield ...................................... 46

Table 5-11 Impact of Solids Loading on Enzymatic Hydrolysis Yield ........................................ 46

Table 6-1 Summary of Impact of Pretreatment on Enzymatic Hydrolyis of Softwood Pine ....... 48

Table 6-2 Enzymatic Hydrolysis Yield and Enzyme Performance Index of Different Solid's Loading ......................................................................................................................................... 68

Table 6-3 Enzymatic Hydrolysis Yield for Multistage Hydrolysis Using Different Yield Calculations................................................................................................................................... 70

X

Nomenclature

AF100 AlternaFuel100 (Xylanase Cocktail)

AH Autohydrolysis

H2SO4 Sulphuric Acid

HMF Hydroxymethylfurfural

HPLC High Performance Liquid Chromatography

IPA Isopropyl Alcohol

NaOH Sodium Hydroxide

NREL National Renewable Energy Labotory

O.D. Outer Diameter

PEG Polyethylene Glycol (Surfactant)

SBI SunOpta BioProcess Inc

Chapter 1 Introduction

1

1 Introduction

Renewable energy has been the focus of the past decade in light of the stricter environmental

regulations placed on the use of petroleum-based fuel. Bioprocessing and bioconversion have

both been considered as a method of engineering for renewability and sustainability. Currently,

the world consumes 88 millions of barrels of oil per day (accounting for 33% of the world's

energy consumption), with Canada using roughly 2 thousand barrels per day (BP Statistical

Review, 2012). The world consumption is predicted to increase by 2 to 3 percent per annum as

developing countries such as China and India become more productive and competitive at the

global-scale (Mabro 2003). Though the energy consumed may not come from petroleum fuel

itself, an increase in demand is inevitable and will need to be addressed. It should be noted that

the demand for energy used per capita is still very low when compared to that of industrialized

countries, and the time will be long before it reaches the same level; however, during this time,

energy demand will only rise. It is estimated that the world's reserve of 1600 billion barrels is

only sufficient to last 54 years (BP Statistical Review, 2012).

The current debate on the issue of peak oil raises the question of where society needs to focus:

earth’s fossil reserve or earth’s capacity to handle products from fossil fuel use. Carbon that has

been sequestered within fossil fuel will ultimately be released back into the atmosphere if there is

no adequate way of re-capturing it in the global carbon cycle. From the growing environmental

awareness and government mandates for change (either reduction in the emission of greenhouse

gas (GHG) or specific replacement of gasoline with renewable liquid fuel), it is apparent that

petroleum-based fossil fuel must be first displaced by renewable energy and then ultimately

replaced.

Chapter 1 Introduction

2

Some renewable sources include solar, wind, geothermal, and hydrothermal. However, these are

currently not feasible to replace the liquid fuel consumption, especially in the transportation

sector. This puts strong favour on biomass derived bioethanol. Ethanol has always been of

interest as a source of energy due to its octane rating (107-111) and higher combustion efficiency

when compared to gasoline. The world oil crisis in 1973 and the ban on tetraethyl-lead increased

the attention to ethanol. Rise in production has been observed in many parts of the world; 13

billion barrels of ethanol was produced in 2007 and about 80% of that was used as a fuel source

(Scheffran, 2010). Currently, sugar crop serves as the main feedstock source for the biomass

conversion process. First generation bioethanol has been in production for over a decade and is

well-established. One of the biggest producers of bioethanol, Brazil, is self-sustaining in terms of

the amount produced. However, the use of starch-based feedstock has prompted many debates

over the years, including the food versus fuel debate. Thus, focus has been placed on second

generation feedstock, namely lignocellulosic biomass.

It is expected that second generation biofuel will mature within the next decade. In actuality, it

must mature by 2030 in order to meet the ethanol demand. Thus, more research is required to

bring lignocellulosic biomass conversion technology to a level such that it can be

commercialized. In addition, the economics of the process and feasibility will need to be

evaluated before full implementation can occur.

Chapter 2 Background

3

2 Background

2.1 Second Generation Feedstock: Lignocellulosic Material

Woody biomass is rich in lignocellulosic material. Forests cover about 9.5% of the Earth's

surface (Soccol et al., 2011), and thus, using woody biomass to make biofuel is a favourable

option for replacement of conventional petroleum-based liquid fuel, because of the mass of

biomass available. A US feasibility study aimed at replacing at least 30% of the petroleum usage

by biomass-based fuel was conducted by Perlack et al., and they found that forest and

agricultural resources provide 1.4 billion tons (dry) per year, where only roughly 1 billion tons is

required to reach the replacement goal (Perlack et al. 2005).

Lignocellulosic biomass contains three major components: cellulose, hemicellulose, and lignin,

as illustrated in Figure 2-1. Each component can be utilized for the production of fuel or value-

added products.

Figure 2-1 Lignocellulosic Biomass Structure and Component (Adapted from Rubin 2008)


4

Cellulose is a linear long chain polymer with anhydrous glucose (C6H12O6) building blocks

joined by β-1,4 glycosidic bonds, as illustrated by Figure 2-2. The actual repeating unit is

cellobiose, which is noted by the blue brackets. Cellulose contains a reducing end and a non-

reducing end. In biomass, cellulose is present both in a crystalline and in an amorphous form.

Crystalline cellulose, shown in Figure 2-3, contains multiple hydroxyl groups which allow

hydrogen bonding between adjacent strands and microfibril packing, resulting in cellulose's

insolubility in water and resistance to degradation. Cellulose has been a focal point of

biocomposites due to its high structural integrity and its biocompatibility with most biological

organisms. On the other hand, amorphous cellulose, which is present in different mass

proportion with crystalline cellulose depending on specie and is located along the cellulose chain

every 100 to 150nm, lengthwise, is less structured and is much easier to digest (Ruel, Nishiyama,

and Joseleau 2012). Accordingly, cellulose is a suitable candidate for biofuel production as long

as enzymes are efficient and cost is minimal.

Figure 2-2 Cellulose Chain (Adapted from Gardner and Blackwell 1974)


5

Figure 2-3 Structure of Crystalline Cellulose - Red Circles Illustrate the Alternating β-1,4

glycosidic Bonds (Adapted from Greer, Pemberton and Tan, 2006)

Hemicellulose is a collective term that describes any sugar that is not cellulose within a biomass,

including hexoses such as glucose, mannose, and galactose; pentoses such as xylose and

arabinose; and may contain acids such as uronic and acetic acids. It is often shorter and branched

due to the presence of the substituents, and thus has a lower degree of polymerization. There are

two main types of hemicellulose: xylan (more common in hardwood and agricultural residue)

and glucomannan (more common in softwood). There is a much stronger interaction between

hemicellulose and lignin's functional groups due to the covalent bonds formed (Keshwani 2009).

In addition, due to the presence of side-chain substituents, toxic derivatives may be produced

when hemicellulose is chemically pretreated or processed. Removal of such byproducts is crucial

in order to ensure efficient operation of downstream processes.


6

Lignin is a highly cross-linked aromatic polymer with phenylpropane units linked by carbon-

carbon and carbon-oxygen bonds. The monomeric building blocks, shown in Figure 2-4, are

coniferyl alcohol (guaiacyl units, labeled G in Figure 2-1) which is most prominent in softwood;

sinapyl alcohol (syringyl units, labeled S) which is present in almost the same mass percent as

coniferyl alcohol in hardwood; and p-coumaryl alcohol (p-hydroxyphenol, labeled H), which is

more common in grasses. Due to its high degree of polymerization, lignin is highly resistant to

degradation. In most cases, it has to be removed or modified prior to downstream processes.

Figure 2-4 The Building Blocks of Lignin (Adapted from Rubin 2008)

Woody biomass comes from two sources: hardwood and softwood. The proportion of cellulose,

hemicellulose, and lignin differs greatly between species, as shown in Table 2-1.

Table 2-1 Typical Woody Biomass Composition (Mass Basis)

Hardwood Softwood Agricultural Residue

Cellulose 40 - 53% 41 - 44% 32 - 44% Hemicellulose 27 - 40% 25 - 30% 27 - 32%

Lignin 16 - 24% 29 - 33% 19 - 24%

2.1.1 Agricultural Residues

Agricultural residues can be classified into two categories: crop residues and processing residues.

The main advantages of using these as a feedstock are their abundance, their low cost, and


7

availability for energy production; currently, however, most of the residues are discarded as

waste or used as a direct fuel source (combustion).

2.1.2 Hardwood

Hardwoods are angiosperms that have broad leaves and usually display deciduous

characteristics. They are widely distributed across the globe, and are often fast-growing. This

makes them an ideal choice for biofuel production. Examples include poplar, willow, aspen, and

oak. For Canada, utilizing residual hardwood may not be the best option due to the low

availability, as shown in Figure 2-5.

Figure 2-5 Hardwood Roadside Harvest Residue Distribution Across Canada


8

2.1.3 Softwood

In contrast to hardwood, softwoods are gymnosperms usually known as evergreens. They are most

prominent in the northern hemisphere, at 92% of all softwood forest, and have been the main source

for timber (Wang and Keshwani 2010). In Canada, around 67% of the forested land is coniferous,

which is about 234 million hectres. Figure 2-6 illustrates the distribution of Canada's forested area,

where majority of the softwood available is located in BC, and southern Ontario and Quebec.

Softwood has historically provided cellulose used in pulp and paper because of its longer fibre

length. But as the pulp and paper industry diminishes, Canada's abundant softwood lumber stands

serve as an ideal feedstock source for biofuel production.

Figure 2-6 Canada's Total Forested Area (Hardwood and Softwood)


9

Another advantage is the well-documented wood chemistry available from the extensive work from

pulping. Specifically, in addition to the mass quantity of growing softwood, it is essential to dispose

of pine-beetle ravaged pine on the west coast of Canada. In addition, when compared to hardwood,

the vast amount of softwood residue, as shown in Figure 2-7, also serves as a good source for the

biofuel production.

Figure 2-7 Softwood Roadside Harvest Residue Distribution Across Canada

Figure 2-8 shows a typical composition for pine. The low xylose and other pentose content is

advantageous because more aggressive processing conditions can be used to remove the lignin

without having to compromise for toxic inhibitor production. This feedstock also has the potential to

produce mainly monomeric hexose sugars, which are much easier to ferment than pentose sugars.


10

Figure 2-8 Dry Pine Composition by Mass (w/w%) (Galbe and Zacchi 2007)

2.2 Bioconversion Processes

Figure 2-9 summarizes the four main steps used in the biomass conversion process.

Figure 2-9 Bioconversion Process

The conversion of lignocellulose into monomeric building blocks requires intensive energy use.

Enzymatic hydrolysis reduces the energy requirement by lowering the activation energy required

to break the bonds within the structure, specifically the β-1,4 glycosidic bonds. Nonetheless,

43.3

10.7 5.3 1.6

2.9

28.3

7.9

Glucose Mannose Xylose Arabinose Galactose Lignin Other


11

some energy must be expended during pretreatment in order to render the feedstock amenable for

enzyme hydrolysis.

However, due to crystalline cellulose’s complex structure, shown in Figure 2-3, multiple

enzymes with specific functions must be used. Enzymatic hydrolysis followed by fermentation is

preferred over thermochemical conversion because of the lower energy requirement. However,

the main disadvantages of enzymatic hydrolysis are the low conversion, the high cost associated

with enzyme production, and the long time (up to 72hrs) required for the reaction. To overcome

the low hydrolysis conversion, certain pretreatments are effective in increasing the fibre

reactivity, and if used in combination, may raise the conversion. This may ultimately make the

process feasible for commercialization.

2.3 Pretreatment

The main objective for pretreatment is to increase the reactivity of biomass fibres, specifically to

the enzymes used during hydrolysis. Figure 2-10 illustrates the changes in the structure of

biomass components when subjected to pretreatment. Pretreatment can be classified into two

categories: chemical and physical. Chemical pretreatment is typically more effective, but it may

result in expensive downstream clean-up. Physical pretreatment does not usually produce by-

products, but it is more energy intensive. It is often necessary to use a combination of the two

(physicochemical methods) to maximize the pretreatment effectiveness. Key parameters that

affect the pretreatment include temperature, pressure, particle size of substrate, and the presence

of catalysts (including catalyst concentration). Temperature affects the degree of lignin removal

in two ways; it impacts the structure of lignin, depending on the glass transition point which is

varies between types of lignin, and it affects the kinetics of the delignification process. In


12

addition, high temperature generally favours the dissolution of hemicellulose but can also result

in degradation of these sugars, which is detrimental to enzymatic hydrolysis and fermentation.

Pressure affects the phase in which the pretreatment occurs; liquid phase will require higher

pressure, commensurate with the required temperature for the same performance in terms of

delignification, hemicellulose removal, and cellulose activation. Lastly, the addition of catalysts

allows the pretreatment to be carried out at lower temperatures; this is beneficial because it may

lessen the degree of generation of degradation products.

Figure 2-10 Schematic Representation of Effect of Pretreatment on Biomass (Adapted from

Mosier et al. 2005)

The main factors for an effective pretreatment process include sugar recovery yield, chip size

required, and low energy use; ultimately, a good pretreatment will allow optimum enzymatic

hydrolysis yield and will not adversely affect downstream fermentation (Yang and Wyman

2008).


13

2.3.1 Physical Treatment

2.3.1.1 Mechanical Milling

Mechanical milling uses shear to reduce the particle size of the biomass. Often, the biomass is

received in chipped or chopped form. However, it may be necessary to further grind the chips to

increase the surface area and ensure proper impregnation of chemicals. The main tradeoff that

needs to be evaluated is the energy consumption versus the degree of size reduction. In addition,

negative impacts from extensive milling were observed due to collapse of cellulose fibre, in

addition to coagulation of fine particles (Talebnia et al. 2010).

2.3.2 Chemical Treatment

2.3.2.1 Acid Treatment

Acid treatment comes in two forms, concentrated and dilute. The acid functions as a catalyst and

has a stronger interaction with hemicellulose and lignin compared to cellulose. Concentrated acid

leads to high glucose yield during enzymatic hydrolysis, but creates problems in terms of

equipment protection, and generation of highly inhibitory products. From a process point of

view, dilute acid is more favourable compared to concentrated acid. The main advantage is the

solubilization of lignin and hemicellulose while potentially hydrolyzing xylan into xylose; with

less xylan to dissolve, more enzymes can work towards breaking down the cellulose that is still

in polymeric form.


14

2.3.2.2 Alkaline Treatment

Alkaline treatment focuses on lignin removal while minimizing the sugar degradation observed

when acid treatment or hydrothermal treatment is used (Tomás-Pejó et al. 2011). It is believed

that the saponification of intermolecular ester bonds that cross-link xylan and lignin is the main

pathway for the solubilization of lignin.

A common alkaline agent is sodium hydroxide (NaOH). In addition to solubilization of lignin,

NaOH causes cellulose fibre to swell, thus increasing surface area while decreasing cellulose's

crystallinity (Taherzadeh and Karimi 2008).

The main advantage of alkaline treatment is that it can be optimized between two process

parameters, namely residence time (between 1hr and 24hr) and temperature (room temperature

or elevated above 100oC). Recently, it was observed that even cold (-5oC to -20oC) NaOH

solutions increased hydrolysis yield (Li et al. 2010).

2.3.2.3 Organosolv

Organosolv utilizes solvents to extract lignin without reacting with the sugars, and often

produces highly digestible fibre from all types of biomass (Tomás-Pejó et al. 2011). This

treatment usually occurs between 100oC and 250oC. Common solvents used are ethanol and

acetone; addition of acid catalysts has been shown to increase rate of delignification and

cellulose reactivity (Zhao et al. 2009). It is possible to obtain non-degraded lignin by using

organosolv treatment; the lignin-solvent can then be separated and the isolated lignin can be used

to make valued-added products. Drawbacks include additional energy requirement to completely

remove the solvent prior to enzymatic hydrolysis.


15

2.3.2.4 Ionic Liquid

Ionic liquids are salts that are composed of a large organic cation and a small inorganic anion.

Examples include 1-n-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium

acetate. They have received much attention due to their capability to break down the hydrogen

bonds within the cellulose fibre, thus reducing the crystallinity. Some advantages of ionic liquids

include their specific affinity to different compounds, which can be tuned to target a bond. In

addition, unlike organosolv, the low volatility of ionic liquids creates less environmental

problems during disposal. Increases in enzymatic hydrolysis yield as high as 50-fold have been

observed (Dadi, Varanasi, and Schall, 2006). However, due to its novel nature and high costs,

more research in process economics and development in recycling is required before it can be

implemented on a industrial-scale (Zavrel et al. 2009).

2.3.3 Physicochemical Treatment

2.3.3.1 Autohydrolysis (Steam Explosion)

This method is carried out at a neutral pH, with autocatalytic characteristics due to the acetic acid

released from the acetyl groupsin the biomass structure. The severity of autohydrolysis is

calculated using Equation 1, where Ro is the severity, t is the time, and T is the temperature in oC

(Galbe and Zacchi 2007).

𝐑𝐨 = 𝐭 ∗ 𝐞𝐱𝐩 �𝐓 − 𝟏𝟎𝟎𝟏𝟒.𝟕𝟓

�

Equation 1 : Severity Factor as a Function of Time and Temperature

During autohydrolysis, biomass is exposed to steam at high temperature (160oC to 260oC) and

pressure (0.69 to 4.83 MPa) in either a batch or continuous reactor. A valve is opened after a


16

desired retention time, which causes the outlet pressure to instantaneously decrease to

atmospheric. This causes the water in the biomass to expand and vapourize, thus breaking the

biomass into smaller pieces and increasing its reactivity. During autohydrolysis, hemicellulose is

solubilized and lignin is both removed and redistributed.

Even though the increase in reactivity depends on the severity, autohydrolysis under extreme

conditions is not preferred because it may lead to the collapse of fibers, an adverse effect

described in more detail in section 2.3.4.2.

2.3.3.2 Liquid Hot Water

Liquid hot water is a hydrothermal pretreatment to provoke alterations in lignin structure but

causes minimal lignin solubilization. The treatment is usually carried out at a temperature

between 160oC and 240oC, with a range of residence time from seconds to hours. Due to the high

temperature (160oC to 240oC), most hemicellulose is solubilized, which increases cellulose's

accessibility. The advantage of liquid hot water treatment is the low cost as no catalyst is

required, but additional cost is associated with additional equipment and additional energy used

to maintain water in liquid state).

2.3.4 Potential Adverse Effects of Pretreatment

2.3.4.1 Toxic Compounds Generated

The potential for toxic compound production depends on the following: raw material, type of

pretreatment and its condition, and the use of catalysts. Degradation products are classified into

three categories, namely, furan derivatives, weak acids, and phenolic compounds.


17

In general, furans interfere with enzymatic hydrolysis by acting as an inhibitor. Weak acid

produced, often acetic acid from the degradation of acetyl groups in hemicellulose, causes

localized pH gradients which need to be adjusted prior to hydrolysis; acetate has been observed

to inhibit specific types of enzymes, and some yeast during fermentation (Palmqvist and Hahn-

Hägerdal 2000; Tengborg, Galbe, and Zacchi 2001; Jing, Zhang, and Bao 2009). Phenolic

compounds, which are largely unidentified, come from the degradation of lignin. These interfere

with hydrolysis through both enzyme deactivation and inhibition.

2.3.4.2 Hornification

Hornification refers to the collapse of cellulose into a compact structure. This is detrimental to

enzyme hydrolysis as it reduces the sites for enzymes to act on, and limits access to these sites by

imposing a mass transfer barrier, thus increasing the difficulty for enzymes to carry out their

function.

2.4 Enzymatic Hydrolysis - Kinetics

Figure 2-11 shows the three different types of enzymes required for glucan hydrolysis, and their

respective substrates. First, endo-glucanases cleave the crystalline structure and the long-chain

cellulose polymer by adding a hydrogen atom onto the oxygen atom, thus reducing the structure

to shorter cellulose chains (~30 to 40 monomer units). Second, exo-glucanses further reduce the

chain length (to ~2 to 10 monomer units) by hydrolyzing the β-glycosidic bonds; this is

typically considered to be the rate limiting step (Gan, Allen, and Taylor, 2003). Lastly, β-

glucosidases hydrolyze the dimer cellobiose into monomeric glucose. As a result, cellulases must


18

have all three types of enzymes in order to successfully convert the cellulosic fraction of

lignocellulose into glucose.

Figure 2-11 Enzymes and substrates used in biomass hydrolysis (Adapted from Binod et al.

2011).

In addition, accessory enzymes are often required to break down bonds within the hemicellulose

structure. Xylanase cocktails and other supplements that contain endo- and exo-xylanases, β-

xylosidase, acetyl-xylan esterase, and mannanase are usually added as part of an enzyme cocktail

to ensure that the hydrolysis is optimized. This is often referred to as synergism between the

enzymes. It is believed that these additional enzymes help expose cellulose structure to the

glucanases by removing hemicellulose (Selig et al. 2008). Glucanases also display similar

interaction; an enzyme mixture often has higher activity than each of the individual enzymes

(Henrissat 1994).


19

2.4.1 Particle Size Reduction

Enzymatic hydrolysis of biomass is an adsorption type reaction, which can be limited by internal

and external mass transfer. It is ideal to reduce the particle size such that an optimum mixing

regime is achieved and the external mass transfer is reduced.

2.4.2 Inhibitors

In addition to the inhibitors from pretreatment that are listed in section 2.3.4.1, products from

hydrolysis can also reduce the enzyme activity (Xiao et al. 2004; Yang et al. 2011). For example,

products from each step of hydrolysis can be inhibitors. Cellobiose is a strong inhibitor of

glucanases, and thus, it is important to keep ensure sufficient β-glucosidase in the enzyme

cocktail, to ensure that glucanases can function at their optimum (Knutsen and Davis 2004; Xiao

et al. 2004).

2.4.3 Non-productive Binding

Lignin is the binding agent that holds cellulose fibres together, which in turn results in a decrease

in accessibility. In addition, lignin attracts cellulases, which results in non-productive binding.

This phenomenon is highly structure dependent, where a small change in the lignin structure may

cause the affinity between the cellulase and the enzyme to increase dramatically (Chandra et al.

2007). It has been suggested that hydrophobic interactions are the main cause for non-productive

binding (Ooshima, Sakata, and Harano 1986). Methods for reducing this interaction, such as

addition of surfactants, have been observed to reduce the impact and to increase the enzymatic

hydrolysis yield (Seo, Fujita, and Sakoda, 2011).


20

2.5 Enzymatic Hydrolysis - Scale-up

Enzyme hydrolysis of softwood is a heterogeneous reaction. There are three distinct components

in the reactor mixture: lignocellulose substrate (solid), enzyme-substrate colloid (suspended

solid), and hydrolysate (liquid). Mass transfer becomes an important factor in determining the

rate and overall conversion; due to the presence of solids, the kinetics may deviate from the

intrinsic kinetics seen in a homogeneous reaction with soluble substrates such as soluble

oligosaccharides. These effects are amplified when the process is scaled up for industrial-level

production, due to higher solids loadings and less free “water”. Therefore, it is important to

conduct scale-up studies to identify how the hydrolysis reaction changes when various reaction

parameters, such as solids loading, reactor geometry and agitation, are altered.

In addition, the hydrolysis process becomes more economical if the reactor is run at higher solids

loading: a higher solids loading should generate a higher sugar concentration in the product

stream, while reducing the amount of water required for removal. Therefore, scale-up studies

also require investigation of the enzymatic hydrolysis at higher solids loadings, and a range of

enzyme dosages.

Higher solids loadings may also increase the concentration of inhibitors in the biomass slurry.

Consequently, increased attention to enzyme inhibition is required, which may require tailoring

of the enzyme cocktail.


21

2.6 Current Status - Softwood as a Second-Generation Biofuel Feedstock

Many studies have investigated micro-scale enzymatic hydrolysis of softwood. Yields as high as

90% have been observed in reactors with working volume between 1mL and 5mL. In terms of

pretreatment, multiple methods from dilute acid to ionic liquids have been investigated.

However, minimal studies have used uncatalyzed autohydrolysis as a pretreatment method to

evaluate its effectiveness on increasing the fibre reactivity. Some of these results are summarized

in Table 2-2.

Table 2-2 Select Results from Past Investigations Using Different Softwood as a Biofuel

Feedstock (Mosier et al; 2005; Galbe and Zacchi 2007; Zhu and Pan 2010)

Type of Pretreatment Conditions Results Catalyzed Autohydrolysis – Optimum

215oC, 3min Spruce - Enzymatic Hydrolysis Yield - 70% (2% solids loading20FPU cellulase per gram of cellulose, 72hr)

Liquid Hot Water 200oC - 230oC, 15min

Sugar Extraction: 4% - 22% Cellulose Over 90% Hemicellulose

Sulfite Pretreatment to Overcome Recalcitrance of Lignocellulose (SPORL)

160oC - 190oC, 10min - 30min

Enzymatic Hydrolysis Yield - 98% (2% solids loading, 20FPU cellulase and 30CBU β-glucosidase per gram of cellulose, 48hr)

Ammonia (AFEX) 160oC - 180oC, 14min,

Enzymatic Hydrolysis Yield - 50% (2% solids loading, low enzyme loading – 10FPU/g cellulose) Considered to be less efficient in pretreating softwood than hardwood

Organosolv, with acid catalyst 200oC Enzymatic Hydrolysis Yield - 90% (2% solids loading, 20FPU cellulase and 30CBU β-glucosidase per gram of cellulose, 48hr)

A lot of focus has been placed on using sulphuric acid or sulphur dioxide as an catalyst, which is

shown to improve enzymatic hydrolysis yield dramatically. However, due to its corrosive nature,


22

acids are not preferred at an industrial setting. Therefore, if uncatalyzed autohydrolysis can

achieve the same yield, it would be much more beneficial to use instead.

In addition, various alkaline agents have been used. Ammonia (NH3) and lime (Ca(OH)2) have

both been investigated as pretreatment chemical; however, the cost associated with ammonia and

the scaling that is caused by lime makes these less attractive options compared to NaOH.

As second-generation biofuel technology matures, more research focus has been placed on

process economics and optimization. The impact of enzyme recycle, continuous hydrolysis, and

multi-stage hydrolysis have all started gaining attention. Thus, experimental data exploring these

will be invaluable when it comes to the scale-up of these processes.

Chapter 3 Research Focus and Aim

23

3 Research Focus and Aim

The objective of this study is to improve softwood hydrolysis yield by evaluating the impact of

different pretreatment conditions, enzyme cocktails, and inhibition. Specifically, a broad

screening will be conducted to determine the impact of different post-autohydrolysis treatment

on improving enzymatic hydrolysis yield.

Enzymatic Hydrolysis – Reaction Rate and Yield

It is hypothesized that the initial rate of the hydrolysis reaction will be directly proportional to

enzyme dose since enzymatic hydrolysis governs the kinetics of the reaction. Higher glucanase

concentrations will result in a higher hydrolysis rate. This will test whether or not the hydrolysis

is enzyme limited or severely impacted by enzyme inhibition. In addition, by supplementing the

enzyme cocktail with β-glucosidase which consumes cellobiose, it is hypothesized that glucanase

inhibition may be reduced.

Different enzymes, cocktail loadings and cocktail combinations will be used during enzymatic

hydrolysis and sugar yield will be used to evaluate their impact.

Enzymatic Hydrolysis - Inhibitors

Novozymes CTEC2 contains glucanases and β -glucosidases as its main hydrolysis enzymes, in

addition to accessory proteins. It is hypothesized that certain inhibitors such as lignin and hemi-

cellulose derivatives may deactivate the enzymes; in addition, some co-factor may be required

for the enzymes to function properly. Large molecules such as conjugate salts of acids may act as


24

ligands that bond to metal co-factors the enzymes required, which may indirectly affect the yield

of the hydrolysis.

Enzymatic hydrolysis slurry will be vacuum-filtered at a specific time to separate the solids from

the liquid containing both product sugars and inhibitors. Solid will be re-suspended for further

hydrolysis until the end of the trial. Fibre-washing will also be used as a method to eliminate

inhibitors.

Pretreatment – Autohydrolysis (Steam Explosion)

Pretreatment at higher severity will generate fibres with higher reactivity and smaller particle

size. However, hornification, the collapse of the cellulose fibre, may occur under extreme

conditions such as high temperature and/or long retention time. It is hypothesized that hydrolysis

yield should improve by increasing the severity of the pretreatment, as long as minimal

hornification occurs.

Autohydrolysis will be carried out under various severities and the effectiveness will be

evaluated through comparing sugar yields after enzymatic hydrolysis.

Pretreatment – Autohydrolysis (Steam Explosion) + Alkaline (NaOH) Post-treatment

Treatment of softwood fibre under alkaline conditions has been used to increase the cellulose

content by delignification, specifically the Kraft pulping process. Lignin has been shown to

cause non-productive binding of enzymes, which causes deactivation and leads to a decrease in

enzyme activity. Therefore, removal of lignin and other inhibitors prior to enzymatic hydrolysis

will be beneficial in terms of optimizing hydrolysis yield. It is hypothesized that as the reactivity


25

of fibre increases with autohydrolysis severity, a second pretreatment stage under alkaline

conditions should increase the hydrolysis yield. However, a maximum pretreatment time may

exist such that the hydrolysis yield is maximized by balancing lignin removal and inhibitor

generation.

Autohydrolyzed fibre will be subjected to alkaline treatment at different temperatures,

concentrations, and for different time durations. The effectiveness of each will be measured

through the amount of lignin removed (by analyzing the composition) and the sugar yield after

enzymatic hydrolysis.

Chapter 4 Experimental Protocol and Design

26

4 Experimental Protocol and Design

4.1 Materials

4.1.1 Substrate

The substrate used in this study comes from chipped Jack Pine (Pinus banksiana) and Loblolly

Pine (Pinus taeda). The background information on the feedstock (age, ratio of sapwood to

heartwood) was unavailable and the carbohydrate composition was obtained through NREL’s

characterization protocol outlined in section 4.3.4. Autohydrolysis of the pine chips was

conducted by Mascoma Canada. The chips were first washed then fed into a continuous Stake

autohydrolyzer operating at three different conditions: 200oC and 8min; 205oC and 10min, and

215oC and 8 minutes. Pretreatment was conducted at Mascoma Canada’s pilot plant in

Waterdown, ON. Fiber samples were collected and stored for subsequent enzyme hydrolysis

studies. The moisture content of the product fibre ranged from 30% to 55%.

In addition, autohydrolyzed Douglas Fir (Pseudotsuga menziesii) was provided by the Forest

Products Biotechnology/Bioenergy lab at the University of British Columbia. The chips were

autohydrolyzed in a batch reactor operating at 215oC and a retention time of 8min.

Pure cellulose obtained from Sigma was used for benchmark and baseline performance studies.

Pure glucose from Sigma was used as an inhibitor during enzyme performance studies.

4.1.2 Enzymes

Three main enzymes were used in this study: Cellic CTec2 (Novozymes), Novo188

(Novozymes), and AlternaFuel100 (AF100; Dyadic). CTec2 is a cellulase blend which includes


27

glucanases and β-glucosidases in addition to other accessory enzymes and binders. It is the main

hydrolysis enzyme blend with a dry matter content of 60% by mass and an active enzyme/protein

present between 75 to 150mg/g of enzyme blend.

Novo188 is also an enzyme blend, but primarily comprised of β-glucosidase. AF100 is a

xylanase blend. In addition, Cellic CTec3 (cellulase) and Alcozyme (xylanase) were also used to

compare their performance on specific sugars within the substrate.

4.1.3 Pretreatment Chemicals

Sodium hydroxide (NaOH, EMD Millipore) was used in alkaline pretreatment studies. In

addition, sulphuric acid (H2SO4, Fischer) was used for dilute acid pretreatment; isopropyl

alcohol (IPA, Caledon) was used for organosolv pretreatment, and polyethylene glycol (PEG,

Sigma-Aldrich) was used in surfactant studies.

4.2 Apparatus/Equipment

4.2.1 Pretreatment Reactor

Pretreatment below 100oC was carried out in either an Erlenmeyer flask (250mL) or a steel

reactor (1L). The flasks were placed in a shaker where temperature was maintained at 70oC, such

that pretreatment would be carried out at 65oC. The steel reactor was immersed in a waterbath to

keep the temperature at 65oC.

Pretreatment at an elevated temperature of 125oC was carried out in Ace Glass pressure tubes,

(20.3cm in length and 19mm in outer diameter (O.D.)), to maintain aqueous phase conditions


28

and prevent vapor loss. A Napco 8000 Autoclave was used as the heat source to maintain the

temperature.

4.2.2 Incubator Hydrolysis Reactions

For screening and small-scale enzymatic hydrolysis, 50mL and 250mL Erlenmeyer flasks were

used. The flasks were placed in a shaker where temperature was maintained at 58oC such that the

reactor contents were at 50oC. The rotation speed was set at 185RPM for all flask experiments.

Most screening trials that only required an initial sample and a final sample were carried out in

the 50mL flasks with a reaction volume of 25mL. All experiments that required a sugar profile to

calculate and compare the initial rate were carried out in the 250mL flasks with a reaction

volume of 125mL.

4.2.3 Jacketed Reactor

For scale-up and higher solids loading trials, three different types of reactors were used: (i) a

jacketed steel reactor with a working volume of 1L, (ii) an SBI glass reactor with a working

volume of up to 5L, and (iii) a 20L Chemglass reactor. A recirculating waterbath at 52oC was

used to maintain the hydrolysis reaction at 50oC.

Barnant mixer assemblies mounted via a stand was used for the steel reactors. Agitation was

performed through a steel shaft with a movable steel blade (2" paddle diameter) which can be

adjusted for appropriate degree of mixing; multiple blades were used in cases when the reactor

contents were large and if inadequate mixing was observed at the top of the slurry. The speed of

the agitator was kept constant by a Barnant controller at a dial setting of 1.5.


29

The SBI glass reactor was designed to maximize shear: multiple blades were attached onto the

agitator shaft in addition to stationary blades at the walls, attached to an extension from the cover

(see Figure 4-1). Due to this arrangement, the SBI reactor had the best mixing among these three

reactors.

Figure 4-1 Agitator Blade Arrangement for SBI Reactor

The Chemglass jacketed reactor (15L) was equipped with a electric stirrer motor with a glass

shaft and 2 adjustable teflon agitators: 4 high viscosity semi-circular blades made up the bottom

agitator (7.5" O.D) and 4 rectangular blades (60o incline) made up the top agitator (5" O.D.). It is

the most representative of an industrial-scale batch reactor used for enzymatic hydrolysis. The

mixer speed was set to 100RPM for both the SBI and Chemglass reactors.


30

4.2.4 Milling

Particle size reduction was carried out by using an Ultra Centrifugal Retsch Rotor Mill (model

ZM100). This mill is capable of rotating at two speeds: 14,000RPM and 18,000RPM. The mesh

sizes used were 5.0mm, 2.0mm, and 0.5mm, to produce fibre with specific size ranges.

4.3 Experimental Protocol

4.3.1 Pretreatment

Alkaline treatment was carried out at 60oC, 75oC and 125oC. First, deionized water was added to

the reactor. Then sodium hydroxide pellets were slowly added. Finally, the substrates were

loaded at 10w/v% and the reactor was either covered or sealed. Three different alkaline

concentrations were used: 1M, 2M, and 4M. The impact of pretreatment time was determined

using reaction times of 1hr, 2hr, and 24hr.

In addition to alkaline pretreatment, dilute acid (1v/v% H2SO4), surfactant (5w/v% PEG), and

hot water pretreatments were used at 125oC. The pretreatment time was 1hr for all these cases.

These pretreatments were conducted in pressure tubes in the autoclave, as described in section

4.2.1.

After pretreatment, the pretreated fibres were then filtered and washed 5 times to remove

byproducts and dissolved inhibitors. pH also adjusted by adding HCl or NaOH to the washwater.

The fibres were stored in the fridge prior to hydrolysis, to minimize microbial growth.


31

4.3.2 Enzymatic Hydrolysis

Enzymatic hydrolysis reactions were carried out at 50oC and pH of 5.2. Initial trials were

performed in a 0.15M citrate buffer. After citrate was determined to be a strong enzyme

inhibitor, pH was instead monitored every hour for the first 6 to 8hr of enzymatic hydrolysis, and

kept between 5.1 to 5.3 by the addition of NaOH.

In all the hydrolysis trials, the substrate was loaded with either the buffer or de-ionized water at

the set dry solids loading (10% or 20%) which represents the mass of dry solids added per

reaction slurry volume. The dry matter of the substrate was measured using a Mettler Toledo

HB43-S Halogen Moisture Analyzer. Next, the reactor temperature was brought to 50oC and

enzyme was added, again at a prescribed enzyme loading and cocktail composition. In this case,

enzyme loading refers to the mass of enzyme solution added per mass of either dry matter or dry

cellulose within the substrate. Samples were collected throughout the experiment at regular

intervals and kept frozen until analysis using HPLC.

4.3.3 Multistage Enzymatic Hydrolysis

Multistage enzymatic hydrolysis was carried out in the same way as outlined in the previous

section. Additional solid-liquid separation was performed at the desired time by collecting the

reactor contents, and then dewatering the sample using a vacuum filter. The solid was re-

suspended in fresh buffer solution or de-ionized water and hydrolysis was allowed to run with

fresh enzyme addition until the end of the experimentation. To remove soluble inhibitors, fibres

were washed with de-ionized water when necessary until a Brix reading of less than 0.2 was

reached.


32

4.3.4 Solids Characterization

Untreated chips, autohydrolyzed fibre, and residual cake from hydrolysis were characterized

using the protocol given in NREL Determination of Structural Carbohydrates and Lignin in

Biomass - Laboratory Analytical Procedure (NREL/TP-510-42618).

4.4 Sample Analysis

4.4.1 High Performance Liquid Chromatography (HPLC)

Samples were collected using a 2mL micro-centrifuge tube. These were first centrifuged, then

filtered through a 0.22µm syringe filter and collected in an HPLC vial. The hydrolyzate samples

were then analyzed using an Agilent 1200 Series HPLC equipped with a Bio-Rad Aminex HPX-

87P column (lead resin). A set of 5 calibration standards were analyzed for every 10 samples to

create and verify the calibration curve used to calculate sugar concentration.

The same technique using Bio-Rad's Aminex HPC-87H column was used when analyzing for

organic acids or other byproducts of hydrolysis.

4.4.2 Brix Refractometer

In some experiments, a refractometer (Brix meter) was used to rapidly determine the sugar

concentration. This method provided a quick measurement of the sugar concentration by

correlating the refractive index to a known sugar concentration. However, it was not possible to

determine the exact composition of the sample, as the refractometer provides an aggregate

measure of refractive index, and does not differentiate between the sugars. The refractometer

was only used for screening and preliminary trials.


33

4.5 Methods to Evaluate Enzyme Performance

4.5.1 Enzyme Hydrolysis Yield

For all enzymatic hydrolysis trials, the overall yield is used for comparison. Equation 2, from

NREL SSF Experimental Protocols – Lignocellulosic Biomass Hydrolysis and Fermentation –

Laboratory Analytical Procedure (NREL/TP-510-42630), was used to calculate the yield relative

to the initial dry cellulose present in the substrate, where concentrations are expressed in g/L and

f is the cellulose fraction in the dry biomass (g/g). The correction factors (1.111, 1.053) account

for the mass gain when hydroxyl groups are added to cellulose and its oligosaccharides during

hydrolysis.

% 𝐆𝐥𝐮𝐜𝐨𝐬𝐞 𝐘𝐢𝐞𝐥𝐝 =[𝐆𝐥𝐮𝐜𝐨𝐬𝐞] + 𝟏.𝟎𝟓𝟑 ∗ [𝐂𝐞𝐥𝐥𝐨𝐛𝐢𝐨𝐬𝐞]

𝟏.𝟏𝟏𝟏 ∗ 𝐟 ∗ [𝐁𝐢𝐨𝐦𝐚𝐬𝐬]∗ 𝟏𝟎𝟎%

Equation 2 : Enzymatic Hydrolysis Yield Calculation – f is the fraction of cellulose in dry

substrate

Alternatively, when enough residual cake from hydrolysis was available, yield was calculated by

the change in glucan in the fibre, determined by biomass characterization.

4.5.2 Graphical Analysis of Rates

Comparison between the kinetics for each experimental condition was carried out by plotting the

sugar generation curve. The production is usually fast initially, followed by a plateau due to

enzyme inhibition or enzyme deactivation. Figure 4-2 illustrates typical sugar generation curves.

The purple line represents a fast initial rate with a high yield; the blue line represents a fast initial


34

rate with a moderate yield; the red line represents a moderate initial rate with a high yield; and

the green line represents a slow initial rate with a relatively low yield.

By plotting different data sets against each other, the initial rate, final yield, and other special

phenomena (degradation, fermentation, etc) that might have occurred during hydrolysis can be

compared.

Figure 4-2 Typical Sugar Generation Curves

4.5.3 Enzyme Performance Index

The enzyme performance index (EPI), proposed by Di Risio et al. (2011), is used to compare

trials that have different enzyme and substrate loadings. It is essentially a normalization of the

enzymatic hydrolysis yield, with the total amount of enzymes added, and is calculated using

Equation 1, where [sugar] is the total reducible sugar released after enzymatic hydrolysis and

[enzyme] is the grams of enzyme solution per gram of total sugars in the fibre used for

hydrolysis. This is especially useful when comparing different solids loadings during enzymatic

hydrolysis.

0 5

10 15 20 25 30 35

0 20 40 60 80

Amou

nt o

f Sug

ar

Prod

uced

(g/L

)

Time (hr)


35

𝐄𝐏𝐈 =[𝐬𝐮𝐠𝐚𝐫]

[𝐞𝐧𝐳𝐲𝐦𝐞]

Equation 3 : Enzyme Performance Index Calculation

Chapter 5 Observation and Results

36

5 Observation and Results

5.1 Pretreatment Conditions

5.1.1 Autohydrolysis

Preliminary investigation focused upon two pretreatment conditions: 200oC/8min and

205oC/10min. It was found that there was particle size reduction from the original chip size.

Figure 5-1 shows the fibres obtained from these two conditions. It was observed that the lower

severity resulted in a coarser fibre. This could increase the mass transfer resistance during

enzymatic hydrolysis. However, since both fibres produced a low hydrolysis yield, a higher

severity autohydrolysis was subsequently used to see if yield improved. When the operating

conditions were changed to 215oC and 8min, a finer fibre was obtained, which is illustrated by

Figure 5-2. In addition, from a qualitative perspective, this fibre had a much sweeter scent, which

could be due to the presence of glucose or other monomeric sugars that were released due to

higher severity.

Figure 5-1 Pine Autohydrolysis Product - 200oC/8min (left) and 205oC/10min (right)

1.5cm


37

Figure 5-2 Pine Autohydrolysis Product - 215oC/8min

It should be noted that all pine autohydrolysis pretreatments were carried out in a continuous

reactor (Stake II system, Mascoma Canada, Waterdown, ON). Under the same temperature and

pressure, a batch reactor produced a much coarser fibre (Figure 5-3), which contained large chip

residues (red circles).

Figure 5-3 Douglas Fir Autohydrolysis Product - 215oC/8min, batch

The fibres produced from each of these pretreatments were subsequently subjected to enzymatic

hydrolysis in flask reactors as described in sections 4.2.2 at a dry solids loading of 10% and an

1.5cm

1.5cm


38

enzyme (CTec2) loading of 5%, based on cellulose content. Results from these trials, including

pretreatment severity and hydrolysis yields, are summarized in Table 5-1.

Table 5-1 Impact of Autohydrolysis Condition on Enzymatic Hydrolysis Yield

Autohydrolysis Conditions Severity Enzymatic Hydrolysis Yield 200oC / 8min 7500 14% 205oC / 10min 12500 21% 215oC / 8min 17500 26%

215oC / 8min (Batch) 17500 7%


Four different chemicals were used during post-autohydrolysis treatments. Post chemical

treatment enzymatic hydrolysis was conducted in flask reactors at a dry solids loading of 10%

and pH of 5.2 using citrate buffer. An enzyme (CTec2) loading of 5% by cellulose content was

used, except in trials with the hydrolysis of fibres from alkaline treatment at 65oC, which had an

enzyme loading of 10% by cellulose content. The initial focus was placed on alkaline treatment.

Variables such as NaOH concentration, treatment time and temperature were investigated. In

addition, the impact of alkaline treatment on fibres produced from different autohydrolysis

severities was evaluated. The results are summarized in Table 5-2 and Table 5-3.

Table 5-2 Impact of Length of Alkaline Treatment Time (at 65oC) on Enzymatic Hydrolysis

Yield

Autohydrolysis Conditions

NaOH Concentration

Chemical Treatment Time

Enzymatic Hydrolysis Yield

200oC / 8min 1M 1hr 9% 200oC / 8min 1M 2hr 17% 200oC / 8min 1M 4hr 11% 200oC / 8min 1M 24hr 27%


39

Table 5-3 Impact of Alkaline Concentration and Autohydrolysis Severity (at 125oC) on



NaOH Concentration

Chemical Treatment Time


200oC / 8min 1M 1hr 7% 200oC / 8min 2M 1hr 12%

205oC / 10min 2M 1hr 10% 215oC / 8min 2M 1hr 5%

Milled Raw Chips (2mm) 2M 1hr 15%

As no dramatic impact was observed with alkaline treatment, further experiments using

organosolv, hot water, and dilute sulphuric acid were conducted. The impact of using organosolv

during chemical treatment is summarized in Table 5-4. The long treatment time was used to

account for the lower treatment temperature. The results from higher temperature chemical

treatment using water and sulphuric acid are presented in Table 5-5.

Table 5-4 Impact of Organosolv Treatment (at 65oC) on Enzymatic Hydrolysis

IPA Concentration (v/v%) Treatment Time Enzymatic Hydrolysis Yield

0% 24hr 27% 25% 24hr 19% 50% 24hr 18%

100% 24hr 12%

Table 5-5 Impact of Different Chemical Treatment (at 125oC) on Enzymatic Hydrolysis

Yield

Chemical Used Treatment Time Enzymatic Hydrolysis Yield Hot Water 1hr 20%

Dilute Sulphuric Acid (1w/w%) 1hr 17%


40

5.1.3 Particle Size Reduction

In this set of experiments, the impact of further particle size reduction after autohydrolysis was

investigated. Autohydrolyzed fibres were milled as described in section 4.2.4 and then subjected

to hydrolysis in flask reactors as described in section 4.2.2 at a dry solids loading of 10% and an

enzyme (CTec2) loading of 5% by cellulose content. A control study was performed using un-

milled fibres. The fibres used in this investigation were autohydrolyzed at 200oC and 8min.

Citrate buffer was used during the enzymatic hydrolysis. The results are summarized in Table

5-6. It should be noted that a greater extent of particle size reduction resulted in an increase in

dry matter due to the temperature increase during milling.

Table 5-6 Impact of Particle Size Reduction on Enzymatic Hydrolysis Yield

Mesh Size Dry Matter (Mass Basis) Enzymatic Hydrolysis Yield No Mill 55% 10% 5.0mm 58% 12% 2.0mm 63% 14% 0.50mm 77% 19%

5.2 Effect of Enzyme Loading and Cocktail

A series of trials was conducted to investigate the effect of enzyme dose and enzyme cocktail on

hydrolysis. Four different enzyme loading/cocktails were investigated, using autohydrolyzed

fibres produced from 200oC/8min and 205oC/10min conditions in flask reactors at a dry solids

loading of 10% with citrate buffer. Initial investigations focused on the necessity of adding extra

enzymes such as β-glucosidase and xylanase, to assist CTec2 in breaking down the fibre. The

various enzyme cocktails investigated are summarized in Table 5-7 and the enzymatic hydrolysis

yields are summarized in Table 5-8.


41

Table 5-7 Different Enzyme Cocktails Used

Case 1 2 3 4 CTec2 5% 5% 10% 5%

Novo188 1% 1% 1% 0% AF100 5% 0% 0% 5%

Table 5-8 Impact of Different Enzyme Cocktails on Enzymatic Hydrolysis Yield

Cocktail 200oC / 8min 205oC / 10min Case 1 6% 5% Case 2 6% 5% Case 3 8% 6% Case 4 7% 6%

The sugar generation profiles in terms of glucose yield as a percent of total glucan available are

plotted in Figure 5-4 and Figure 5-5.

Figure 5-4 Yield Profile in Terms of Total Glucan Available for Different Enzyme

Cocktails Using Autohydrolyzed Fibres from 200oC/8min Condition (Enzymatic Hydrolysis

Conditions – 125mL Reaction Volume, 10% Solids Loading, n = 1)

0

2

4

6

8

10

0 20 40 60 80

Glu

cose

Yie

ld (%

)

Time (hr)

Case 1 Case 2 Case 3 Case 4


42

Figure 5-5 Yield Profile in Terms of Total Glucan Available for Different Enzyme

Cocktails Using Autohydrolyzed Fibres from 205oC/10min Condition (Enzymatic

Hydrolysis Conditions – 125mL Reaction Volume, 10% Solids Loading, n = 1)

After it was determined that citric acid is a strong inhibitor, additional flask trials using

autohydrolyed fibres (215oC and 8min; 10% solids loading) were conducted to verify the extent

of this inhibition. Since CTec3 was available when the trial was conducted, it was also used to

see if it improved enzymatic hydrolysis yield. Figure 5-6 plots the results obtained.

Figure 5-6 Impact of Enzyme Load with No Buffer on Enzymatic Hydrolysis Yield in

Terms of Total Glucan Available (Enzymatic Hydrolysis Conditions – 125mL Reaction

Volume, 10% Solids Loading, n = 1)

0 1 2 3 4 5 6 7

0 20 40 60 80

Glu

cose

Yie

ld (%

)

Time (hr)


0

10

20

30

40

50

0 20 40 60 80

Glu

cose

Yie

ld (%

)

Time (hr)

CTEC2 5w/w% CTEC2 10w/w% CTEC3 5w/w%


43

5.2.1 Enzyme Inhibition

The preliminary experimental protocol used citric acid and sodium citrate as a buffer. However,

a side trial conducted in our laboratory subsequently suggested that citric acid might be a

inhibitor stronger than acetic acid. Further investigation found this to be the case. Switching

from a citrate buffer to a slurry in water, with regular NaOH addition to control pH caused the

yield to increase from 8% to 26%, using autohydrolyzed fibre pretreated at 215oC/8min. The

results are presented in Table 5-9.

Table 5-9 Impact of Citrate Buffer on Enzymatic Hydrolysis Yield


Enzymatic Hydrolysis Yield with Citrate Buffer

Use

Enzymatic Hydrolysis Yield with Water and Periodic NaOH for pH

Control 200oC / 8min 4% 14%

205oC / 10min 6% 21% 215oC / 8min 8% 26%

The main method used to determine the impact of product inhibition was solid-liquid separation

via vacuum filtration. A set of trials was conducted in the flask reactor at a solids loading of 10%

using fibres obtained from autohydrolysis at 215oC and 8min. The result of small scale

investigation is illustrated in Figure 5-7. No significant changes in enzymatic hydrolysis yield in

terms of total glucan available was observed, though different initial rates were seen. This led to

further investigation to see the impact of reducing enzyme inhibition by staggered addition and

the impact of washing the fibre after solid-liquid separation. Figure 5-8 illustrates the impact of

staggered enzyme addition.


44

Figure 5-7 Impact of Liquid Removal on Enzymatic Hydrolysis Yield in Terms of Total

Glucan Available (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10%

Solids Loading, n = 1)

2.5w/w% Enzyme Loading (Blue), 5w/w%+5w/w% with Liquid Removal (Purple),

5w/w%+5w/w% without Liquid Removal (Red), 10w/w% (Green)

Figure 5-8 Impact of Staggered Enzyme Addition on Enzymatic Hydrolysis Glucose

Generation (Enzymatic Hydrolysis Conditions – 1L Reaction Volume, 10% Solids Loading,

5% Enzyme Loading, n = 1)

1hr Alkaline Treatment with Staggered Enzyme Addition (Red), 1hr Alkaline Treatment

(Green), No Alkaline Treatment with Staggered Enzyme Addition (Blue), No Alkaline

Treatment (Purple)

0

5

10

15

20

25

0 20 40 60 80

Glu

cose

Yie

ld (%

)

Time (hr)

0 5

10 15 20 25 30

0 20 40 60 80

Glu

cose

Pre

sent

(m

g/m

L)

Time (hr)


45

The results of studies with no wash and an interstage wash (with water and using protocol

outlined in section 4.3.3) are shown in Figure 5-9.

Figure 5-9 Impact of Interstage Wash on Enzymatic Hydrolysis Yield in Terms of Total

Glucan Available (Enzymatic Hydrolysis Conditions – 1L Reaction Volume, 20% Solids

Loading, 5% Enzyme Loading with Addition of Half the Initial Amount after Solid/Liquid

Separation, n = 2 averaged value)

5.3 Effect of Substrate Loading and Reaction Scale

5.3.1 Reaction Scale-up

A series of trials were conducted under equivalent conditions using three different reactor

volumes: 125mL (flask reactor), 1L (steel reactor), and 10L (Chemglass reactor). The fibre used

in this investigation was autohydrolyzed at 215oC and 8min. The enzymatic hydrolysis was

carried out at a dry solids loading of 10% and an enzyme (CTec2) loading of 5% by cellulose

content, using citrate buffer. Minimal differences in yield were observed when mixing was

adequate (when SBI reactor and Chemglass reactor were used). Table 5-10 summarizes these

results.

0 5

10 15 20 25 30

0 20 40 60 80

Sing

le S

tage

Glu

cose

Yi

eld

(%)

Time (hr)

No wash Interstage wash


46

Table 5-10 Impact of Reactor Volume on Enzymatic Hydrolysis Yield

Reactor Volume Enzymatic Hydrolysis Yield 125mL 9%

1L 14% 10L 13%

5.3.2 Higher Solids Loading

All higher solids loading enzymatic hydrolysis reactions were carried out in the SBI reactor since

it provides the best mixing. The fibres used in this investigation were autohydrolyzed at 215oC

and 8min because the particle size is the smallest. Hydrolysis trials were conducted at 10 - 20%

dry solids loading, using an enzyme loading of 5% by cellulose content. No buffer was used and

pH was maintained at 5.2 by addition of NaOH. It was observed that higher solids loading gave

both higher sugar production and yield. The results are summarized in Table 5-11 and the

generation profile is plotted in Figure 5-10.

Table 5-11 Impact of Solids Loading on Enzymatic Hydrolysis Yield

Solids Loading Enzymatic Hydrolysis Yield 10% 30% 15% 35% 20% 42%


47

Figure 5-10 Impact of Solids Loading on Enzymatic Hydrolysis Yield in Terms of Total

Glucan Available (Enzymatic Hydrolysis Conditions – 1L Reaction Volume, 5% Enzyme

Loading, n = 1)

0

10

20

30

40

50

0 20 40 60 80

Glu

cose

Yie

ld (%

)

Time (hr)

10% Solids Loading

15% Solids Loading

20% Solids Loading

Chapter 6 Discussion and Analysis

48

6 Discussion and Analysis

The goal of this study is to investigate methods for improving softwood hydrolysis yield.

Specific focus was placed on four areas: impact of autohydrolysis, impact of post-autohydrolysis

treatment, impact of different enzyme cocktails, and impact of reactor operations. Using the

results presented in Chapter 1, analysis and comparison can be made in order to optimize the

enzymatic hydrolysis of softwood and identify areas for improvement. The impact of

pretreatment is summarized in Table 6-1.

Table 6-1 Summary of Impact of Pretreatment on Enzymatic Hydrolyis of Softwood Pine

Factor Impact Figure/Table Reference

Autohydrolysis Severity Significant increase in enzymatic hydrolysis yield

Table 5-1, Figure 6-1

Alkaline – Temperature Decrease in enzymatic hydrolysis yield as temperature increases

Table 5-2, Table 5-3,

Alkaline – Time Increase in enzymatic hydrolysis yield as time increases, but local maximum exists. Extended treatment time does further increase yield


Isopropyl Alcohol Concentration

Some degree of delignification; increasing concentration decreases the enzymatic hydrolysis yield

Table 5-4,Figure 6-6

Dilute Acid Some degree of delignification, higher enzymatic hydrolysis yield compared to autohydrolysis pretreatment alone

Table 5-5

Hot Water Highest increase in enzymatic hydrolysis yield when compared to all other chemical post-autohydrolysis treatment

Table 5-5

Particle Size Increase in enzymatic hydrolysis yield as particle size decreases, but increase in moisture content with increase in extent of milling



49

6.1 Autohydrolysis Pretreatment

Autohydrolysis pretreatment focuses on increasing the fibre reactivity, through increasing

accessible surface area, and removing hemicellulose by dissolving those sugars into liquid. In

this investigation, multiple autohydrolysis severities were investigated. It was hypothesized that

a higher severity will lead to an increase in enzymatic hydrolysis yield. It can be seen from the

data obtained and presented in section 5.1.1 that increasing the severity of the autohydrolysis

increased the sugar yield; Figure 6-1 illustrates this relationship. However, even the most severe

pretreatment condition did not result in significant sugar yield, which was still less than 30%.

It has been previously suggested, through comparisons of ethanol yields after fermentation, that

non-catalyzed autohydrolysis is not suited for use with coniferous woods (Wayman and Parekh,

1988). Multiple studies have indicated an increase in enzymatic hydrolysis yield when acid

catalyst is added, through impregnation with sulphur dioxide (SO2) (Söderström et al. 2002,

Öhgren et al. 2007, Kumar et al. 2010). The addition of acid catalyst can be expressed as an

increase in severity and approximated by modifying Equation 1 to include the pH, as shown in

Equation 4 (Galbe and Zacchi 2007).

Combined Severity (CS) = log�t ∗ �T − 100

14.75�� − pH

Equation 4 : Combined Severity as a Function of Time, Temperature, and pH


50

Figure 6-1 Effect of Autohydrolysis Severity on Glucose Enzymatic Hydrolysis Yield in

Terms of Total Glucan Available (Enzymatic Hydrolysis Conditions – 125mL Reaction

Volume, 10% Solids Loading, 5% Enzyme Loading)

Thus, for the case of softwood, it may be necessary to conduct autohydrolysis at even higher

temperatures and/or longer retention times to reach the same severity. The advantage is the

minimal generation of degradation products due to the low hemicellulose present in softwood.

Other modifications during the biomass conversion process can also be implemented to improve

yield.

Alternatively, the autohydrolyzed fibre may need to be washed prior to enzymatic hydrolysis.

Past studies have shown that when the liquid fraction was included in the enzymatic hydrolysis,

the yield was reduced up to 36% (Tengborg, Galbe and Zacchi 2001). In this work, similar

results were obtained using hot water as a post-autohydrolysis treatment, as described in section

6.2.2.3.

Comparing batch and continuous autohydrolysis, the latter produced a fibre that was more

reactive in terms of enzymatic hydrolysis yield. It is possible that this is due to differences

between pine (used in continuous autohydrolysis) and Douglas fir (batch AH). Previous study

y = 0.0012x + 5.3333 R² = 0.99

0 5

10 15 20 25 30

0 5000 10000 15000 20000

Glu

cose

Yie

ld (%

)

Autohydrolysis Severity


51

has suggested that different wood species, age, and proportion of sap and heart wood may have

different response to pretreatment (Boussaid et al., 2000). However, minimal difference in sugar

yield was observed when Douglas fir and pine, batch autohydrolyzed at 200oC, 4% SO2, and

5min, were used as substrates for enzymatic hydrolysis (Kumar et al. 2010). In addition,

composition analysis suggests that the cellulose, hemicellulose, and lignin content are all

comparable between Douglas fir and pine, making direct comparison possible, without the

adjusting for differences in mass of enzyme added. Previous studies using wheat-straw and other

agricultural residues also support the idea that continuous autohydrolysis is better in terms of

increasing fibre reactivity and efficiency (Ropars et al. 1992; Zimbardi et al. 1999). An

explanation, from a process point of view, for the lower fibre reactivity may be the uneven heat

transfer from the steam to the chip, which would result spatial temperature differences and

varying cook times. Batch autohydrolysis usually does not preheat the chips and thus part of the

treatment time is used to simply bring the chips to the appropriate temperature (reduction in the

actual cook time). In contrast, chips used in a continuous autohydrolysis reactor are often

preheated and will reach the operating temperature much faster. The screw conveyer used to

move the chips within the reactor also provides some degree of mixing, which will reduce

heterogeneity and temperature gradients. Ultimately, since the effectiveness of autohydrolysis is

highly dependent on the process conditions, it is necessary to accurately control the process,

which is much easier to do in a continuous setting (Nativel et al. 1992; Ropars et al. 1992). From

these studies, it can be concluded that a continuous autohydrolysis reactor is preferred when

pretreating softwood biomass, leading to increased enzymatic hydrolysis yield.


52

6.2 Post-autohydrolysis Treatment

The increase in fibre reactivity due to autohydrolysis will not only impact enzymatic hydrolysis

but also any subsequent treatment that is carried out. Because autohydrolysis does not effectively

remove lignin (Mosier et al. 2005), it is important to evaluate how different lignin removal

methods are affected by autohydrolysis, and to determine the impact these would have on

enzymatic hydrolysis.

6.2.1 Physical Treatment

The enzymatic hydrolysis is intrinsically dependent on only the substrate concentration, initial

enzyme concentration, and the affinity between the substrate and enzyme. However, since

enzymatic hydrolysis is also considered a solid-liquid reaction because the substrate is insoluble,

mass transfer will directly impact the yield of the hydrolysis. In addition, energy consumption for

mixing decreases as the size of the “particles” in the slurry decreases. Figure 6-2 illustrates the

impact of particle size reduction on glucose yield. The yield increased from 10% to 19% when

autohydrolyzed fibre was reduced to 0.5mm in size. It should be noted that more intensive

milling (greater size reduction) led to an increase in dry matter, due to heat generated. This is

unavoidable and could cause hornification, leading to a reduction in the hydrolysis yield.


53

Figure 6-2 Impact of Particle Size Reduction on Enzymatic Hydrolysis Yield in Terms of

Total Glucan Available (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10%

Solids Loading, 5% Enzyme Loading, n = 1)

The majority of studies place emphasis on the actual catalyzed hydrolysis while ignoring the

external mass transfer resistance, assuming it is insignificant (Gan, Allen and Taylor 2003). The

fact that particle size indeed affects the enzymatic hydrolysis yield and initial rate (see Figure 6-3)

indicates that there may be enough external mass transfer resistance to affect the overall

observable kinetics, which includes the movement of enzyme within the liquid phase, the

adsorption of enzyme onto the substrate, the desorption of the enzyme, and the movement of the

enzyme onto another substrate. The degree in which the initial rate is affected by the particle size

is important, as majority of the conversion occurs in during the first 24hr of hydrolysis.

Optimizing the initial rate to maximize the sugar yield will lead to better economics when the

process is being commercialized for industrial-scale production.

0

20

40

60

80

100

0

5

10

15

20

0 1 2 3 4 5 6

Dry

Mat

ter (

%)

Glu

cose

Yie

ld (%

)

Particle Size (mm)

Yield Dry Matter


54

Figure 6-3 Impact of Particle Size on Initial Amount of Sugar Generated During

Enzymatic Hydrolysis (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10%

Solids Loading, 5% Enzyme Loading, n = 1)

Direct mechanical milling may not be the best method for reducing particle size due to the

increase in temperature during milling, which may cause hornification. In addition, higher

cellulose loading (7%) during milling has been observed to reduce the specific surface area than

a lower loading (3%), resulting in a lower sugar yield (25% versus 35%) (Yeh, Huang, and Chen

2010). This suggests that substrates may not be milled at high loadings without adversely

affecting the hydrolysis yield. Thus, from a process optimization point of view, increasing

autohydrolysis severity may be the best method to decrease particle size. In addition, industrial

autohydrolysis units usually have the versatility of installing a disk-refiner, which can be used as

another method to reduce particle size should higher temperature and longer pretreatment not be

feasible. Disk refiners can also lead to fiber swelling, which should help enzyme hydrolysis.

0 0.5

1 1.5

2 2.5

3 3.5

4

0.5mm 2mm 5mm No Reduction

Amou

nt o

f Glu

cose

G

ener

ated

(mg/

mL)

Particle Size

2hr 4hr


55


6.2.2.1 Alkaline

The use of alkaline chemicals to treat biomass comes from Kraft pulping, except that alkaline

agents other than sodium sulfide (Na2S) are used. Fibre characterization showed that minimal

acid-soluble lignin is present in autohydrolyzed pine. In addition, NaOH has been shown to swell

the cellulose fibre, thus increasing the accessibility and digestibility (Zhao et al. 2008, Tomás-

Pejó et al., 2011). Accordingly, it is ideal to use alkaline conditions for delignification, assuming

that Klason lignin is the present in large quantities.

Initial investigations using NaOH treatment reported an increase in the enzymatic hydrolysis

yield when compared to using no NaOH (9% in Table 5-2 and 4% Table 5-9). However, in

terms of absolute amount, the yield was still small. When comparing length of treatment at 65oC,

2 hours gave the highest enzymatic hydrolysis yield for shorter treatment times, as seen by the

local maximum peak in Figure 6-4. This suggests that a tradeoff between length of treatment and

enzymatic hydrolysis yield should be evaluated; thus, optimization can be performed. It was also

observed that at this temperature, prolonged exposure (24hr) to NaOH gave the highest yield

(26%); this may be beneficial but not practical, as long treatment time may result in higher

amounts of degradation products and would require very large vessels, usually not favoured in an

industrial setting. This suggests an “optimum” treatment time of 2hr, while at the same time

identifying an upper limit of how effective NaOH treatment can be.


56

Figure 6-4 Impact of Duration of NaOH Treatment at 65oC on Enzymatic Hydrolysis Yield

in Terms of Total Glucan Available (Enzymatic Hydrolysis Conditions – 125mL Reaction

Volume, 10% Solids Loading, 5% Enzyme Loading, n = 1)

When comparing the impact of treatment temperature, it was found under the same treatment

time (1hr) and NaOH concentration (1M), treatment at 125oC and 65oC yielded 7% glucose and

9%, respectively, after enzymatic hydrolysis. Also, fibres that were treated at a higher

autohydrolysis severity and subsequently underwent NaOH pretreatment yield less sugar after

the enzymatic hydrolysis than those treated at a lower severity. This is opposite of what was

hypothesized, as it was expected that higher autohydrolysis severity would lead to higher fibre

reactivity, which would lead to better hydrolysis yield, partly due to higher degree of

delignification.

An explanation for this may be the condensation of lignin back onto the fibre surface after it has

been solubilized. Formation of quinone methide intermediates has been observed to contribute to

condensation of lignin which counters chemical fragmentation (Gierer 1985). During

delignification, internal nucleophiles compete for the quinone methide intermediates generated

from non-phenolic units. In basic environments, removal of a proton from the intermediates

0 5

10 15 20 25 30

0 5 10 15 20 25 30

Glu

cose

Yie

ld (%

)

Length NaOH Treatment (hr)


57

allows re-aromatization which negates the fragmentation. This does not happen in an acidic

environment, as α-substituents are protonated, enhancing phenolic units’ ability to detach from

the lignin structure. Thus, the increase in fibre reactivity after autohydrolysis may increase the

degree of condensation which creates a barrier between the enzyme and substrates, leading to the

decrease in hydrolysis yield. Observations from this study using milled raw pine chips (less

reactive than autohydrolyzed fibre) as a substrate with NaOH supports this theory, as enzymatic

hydrolysis yield improved from 4% (not treated) to 15%. However, increasing the NaOH

concentration does increase the enzymatic hydrolysis yield, which suggests that more alkaline

conditions may overcome the condensation of lignin.

From a kinetics point of view, it may be necessary to decrease the pretreatment time to account

for the increase in temperature and to minimize potential lignin condensation. Interestingly,

alkaline treatment at low temperatures has been observed to increase enzymatic hydrolysis yield.

Zhao et al. (2008) studied the impact of treating size-reduced raw spruce with alkaline (NaOH

only and NaOH and urea) solutions at a temperature of -15oC for 24hr and observed an

enzymatic hydrolysis yield of 70%, versus 20% when a treatment temperature of 20oC was used.

In the same study, it was observed that at 60oC with a treatment time of 2hr to reduce lignin and

carbohydrates dissolved, the sugar yield only increased to 25% (Zhao et al. 2008). This suggests

that additional chemicals that can reduce the re-aromatization may need to be used with NaOH to

make the treatment effective, and temperature is an important variable.

In contrast, a previous study on the impact of alkaline pretreatment on hydrolysis yield using

cereal crop as the substrate found a positive trend when the treatment temperature was increased

(Vancov and McIntosh 2011). This suggests that using an alkaline solution with only NaOH may


58

be sufficient in order to increase the enzymatic hydrolysis yield for certain lignocellulosic

biomass.

6.2.2.2 Organosolv

A different method of delignification is organosolv treatment. It was found enzymatic hydrolysis

yield increased when isopropyl alcohol (IPA) was used during chemical treatment when

compared to no post-autohydrolysis treatment (see Figure 6-5).

Figure 6-5 Comparing the Impact of Alkaline Treatment and Organosolv Treatment on

Enzymatic Hydrolysis Yield in Terms of Total Glucan Available (Enzymatic Hydrolysis

Conditions – 125mL Reaction Volume, 10% Solids Loading, 5% Enzyme Loading, n = 1)

A decrease in the hydrolysis yield was observed when more IPA was used, as illustrated by the

negative correlation in Figure 6-6. It has been documented that presence of ethanol in

hydrolysate can reduce sugar production from 12.5µg/mL (0v/v% ethanol) to 5µg/mL (7v/v%

ethanol) (Chen and Jin 2006). A low ethanol concentration of 9g/L can reduce cellulase activity

by 9% while a high concentration of 60g/L can reduce up to 65% activity (Sun and Cheng 2002).

Thus, residual solvent that did not get evaporated may be the reason for the poorer hydrolysis

0 5

10 15 20 25 30

Alkaline Treatment (1M,

24hr)

Organosolv (25w/w%, 24hr)

No Treatment

Glu

cose

Yie

ld (%

)


59

performance with higher concentrations of IPA. In addition, since the organosolv treatment ran

for 24hr, more IPA may have penetrated into the fibre, making removal more difficult. With

IPA's higher boiling point (82.5oC) relative to ethanol (78.4oC), it may be necessary to evaporate

the solvent from the treated fibre at a higher temperature and/or for a longer time. Moreover,

Yamashita, Sasaki, and Nakamura (2010) observed a decrease in fermentation yield when more

ethylene glycol (organosolv) was used to chemically treat raw red cedar. It can be seen that even

though organsolv may indeed increase the fibre's reactivity during enzymatic hydrolysis, the

presence of the solvent in excessive amount will cause problems during downstream processes.

This ultimately may hinder the use of organsolv as an efficient method for delignification.

Figure 6-6 Impact of IPA Concentration Used in Chemical Treatment on Enzymatic

Hydrolysis Yield in Terms of Total Glucan Available (Enzymatic Hydrolysis Conditions –

125mL Reaction Volume, 10% Solids Loading, 5% Enzyme Loading, n = 1)

It is worth noting that the control used during organosolv treatment investigation, treating the

fibres for 24hr with just water (Table 5-4), gave the highest hydrolysis yield. It is suggested that

during this time, lignin that has condensed onto the fibre after autohydrolysis may have been

y = -0.1371x + 25 R² = 0.9023

0 5

10 15 20 25 30

0 20 40 60 80 100 120

Glu

cose

Yie

ld (%

)

IPA Concentration Used During Chemical Treatment (v/v%)


60

solubilized and extracted by the aqueous phase. Thus, a hot water treatment may be the preferred

method for post-autohydrolysis treatment.

6.2.2.3 Dilute Acid and Hot Water

Comparing the different pretreatment chemicals used at elevated temperature (125oC), it was

observed that liquid hot water performed best, producing a fibre substrate that gave the highest

yield after enzymatic hydrolysis. Dilute acid ranked second in terms of enzymatic hydrolysis

yield, followed by the yield with autohydrolyzed fibres. The use of NaOH actually produced a

fibre that performed worst in terms of hydrolysis yield. The difference in enzymatic hydrolysis

yield is shown in Figure 6-7.

Figure 6-7 Impact of Using Different Chemical Treatment on Enzymatic Hydrolysis Yield

in Terms of Total Glucan Available (Enzymatic Hydrolysis Conditions – 125mL Reaction

Volume, 10% Solids Loading, 5% Enzyme Loading, n = 1)

Hot water treatment may be the most effective as it does not appreciably solubilize lignin, which

makes the potential of condensation through the mechanism described in section 6.2.2.1 less

likely (Zhao, Cheng, and Liu 2009). In addition, the presence of water acts as a solvent to

0

5

10

15

20

25

215C/8min/2M Alkaline

Hot Water Dilute Sulphuric Acid

Glu

cose

Yie

ld (%

)


61

dissolve any of the autohydrolyzed byproduct, including lignin and other phenolic

compounds,that may have condensed onto the fibre itself.

Studies using liquid water as a treatment chemical in wet oxidation of bagasse have shown an

increase in cellulose digestibility (Martín, Klinke, and Thomsen 2007; Zhao, Cheng, and Liu

2009). In this study, a lower temperature is used, but a longer treatment time could allow the

same performance as those observed in past studies. Even though the amount of phenolic

compounds was not quantified in this study, increased solubilization of phenolic compounds,

carboxylic acid, furfural, and hydroxymethylfurfural (HMF) has been documented to occur when

wet oxidation at 195oC and 12 bar, with a retention time of 15min, is used instead of steam

explosion (35w/w% in the liquid obtained after treatment vs. 25w/w%) (Martín et al. 2007). The

similarity between the hot water treatment used in this investigation and wet oxidation could

suggest that more phenolic compounds were dissolved in the liquid, which would improve

enzymatic hydrolysis by reducing the potential for inhibition.

In the same study, Martin, Klinke, and Thomsen (2007) observed that acid catalyzed (1w/w%)

wet oxidation helped increase cellulose conversion from 5% (no treatment) to 55% In addition,

dilute acid treatment has been documented to increase the crystallinity of the substrate by

removal of the amorphous cellulose and other lignocellulosic components (Sannigrahi,

Ragauskas, and Miller 2008). Both of these would suggest that an acidic condition is beneficial

when treating fibres for the purpose of increasing enzymatic hydrolysis yield.


62

6.3 Enzymatic Hydrolysis – Reaction Rate and Yield

The complex structure of lignocellulosic biomass and the multiple sugar composition makes the

use of multiple enzymes often necessary. Glucanases are required to break down cellulose,

xylanases are required to break down xylan, and β-glucosidases are rquired to break down

cellobiose. Many studies have emphasized the observed synergistic effect of enzyme cocktail on

increasing hydrolysis yield, specifically the release of more sugar when compared to using only a

specific enzyme (Lynd et al. 2005; Selig et al. 2008; Wen, Nair, and Zhao 2009). Thus, a

primary aim for this study is to investigate whether or not different enzyme blends have an

impact on the enzymatic hydrolysis yield of autohydrolyzed fibre.

Figure 5-4 and Figure 5-5 illustrates the enzymatic hydrolysis yield profile using the different

enzyme cocktails listed in Table 5-7. It was found that adding Novo188 did not increase yield

(comparing case 1 and case 4). This suggests that CTec2 may have enough enzyme activity such

that additional β-glucosidase is not required. In addition, later trials in the study reported minimal

to no cellobiose in the hydrolysate, suggesting that inhibition by this product may be minimal

and the activity of glucanases are not affected. Moreover, this observation reinforces the notion

that the hydrolysis of cellulose to cellobiose is the rate limiting step, as stated in section 2.4.

When using fibre obtained from autohydrolysis at 200oC and 8min, the addition of AF100, a

xylanase cocktail, does not seem to appear to have an impact on the enzymatic hydrolysis yield

(compare case 1 with case 2). However, an increase in the initial rate and final yield was

observed when AF100 was replaced, at the same mass %, with CTec2 (comparing case 1 to case

3). Though this does not completely agree with the hypothesis that the kinetics of enzymatic

hydrolysis using autohydrolyzed pine is directly proportional to the amount of enzyme added,


63

there is evidence to suggest strong dependence between yield and enzyme loading and it is clear

that the key enzymes are the endo-glucanases and exo-glucanases within CTec2.

The enzymatic hydrolysis yield from using fibre obtained from autohydrolysis at 205oC and

10min tells a different story. Again, the addition of Novo188 did not improve yield, but the

impact of the addition of AF100 (case 1) led to a hydrolysis yield similar to that obtained from

using only CTec2 (case 3). Interestingly, the impact of autohydrolysis condition seemed to affect

how well the enzyme cocktail worked. Figure 6-8 illustrates the difference in the enzymatic

hydrolysis yield within the first 24hr between the use of fibre autohydrolyzed at two different

conditions. It was observed that the addition of AF100 seem to be more effective in terms of

increasing the initial rate but not the final yield. This suggests that more enzymes are able to act

on the fibre, which agrees with the notion that autohydrolysis increases fibre reactivity. In

addition, it was observed that under certain circumstances, a lower autohydrolysis severity

produced a fibre that was more susceptible to enzymatic hydrolysis. An explanation for this

observation may be the degree of condensation of autohydrolysis product onto the fibre surface.

As higher severity produces more byproduct through delignification and hemicellulose

dissolution, the potential for these to condense, when temperature is suddenly decreased after the

explosive decompression, is much higher. This would create a barrier between the enzyme and

the substrate, thus resulting in a less reactive fibre.


64

Figure 6-8 Impact of Cocktail Blend on Enzymatic Hydrolysis Yield Within First 24hr

(Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10% Solids Loading, 5%

Enzyme Loading, n = 1)

Interestingly, when higher severity fibre was used, the difference between the cases of enzyme

cocktails used in terms of enzymatic hydrolysis yield is relatively small and no significant

difference was observed even between cases when twice the amount of enzyme was added. This

could mean that either enzymes are being strongly inhibited/deactivated, or the substrate is no

longer active. Lu et al. has observed that enzymes are still active after being through 3 rounds of

enzymatic hydrolysis using autohydrolyzed douglas fir as substrate (Lu et al. 2002). This

prompted further study of fibre reactivity after 72hr of enzymatic hydrolysis, in which

supplemental enzyme was dosed to the partially hydrolyzed slurry, to determine if further

conversion was possible. Figure 6-9 illustrates the results obtained when pine autohydrolyzed at

200oC and 8min was used as the substrate during enzymatic hydrolysis at 10% solids loading in a

steel reactor; in the first stage of hydrolysis, a 10% CTec2 loading by cellulose mass was used,

leading to. a first stage yield of 15%. After addition of another 10% CTec2, the second stage of

hydrolysis) gave a final yield of 25%. This suggests that the fibres are still active and the cause

for the low yield is most likely due to enzyme inhibition or deactivation.

0

1

2

3

4

5

6


Glu

cose

Yie

ld W

ithin

Fi

rst 2

4hr o

f Enz

ymat

ic

Hyd

roly

sis

(%)

200C/8min 205C/10min


65

Figure 6-9 Glucose Generation Profile with Second Enzyme Addition after First 72hr of

Hydrolysis (Enzymatic Hydrolysis Conditions – 125mL Reaction Volume, 10% Solids

Loading, 5% Enzyme Loading, n = 1)

Additional trials were conducted to see the difference between using an enzyme loading of 5%

CTec2 and 10% CTec2. Since there was no significant difference observed in initial rate and

final yield, it was decided that an enzyme loading of 5% by dry cellulose mass would be used for

the rest of the study, because if efficacy can be established, a lower enzyme loading would mean

a reduction in process cost.

Due to the strong inhibition effect citrate buffer has on the hydrolysis enzymes, additional trials

were conducted to verify the impact. Figure 5-6 illustrates the improvement in both the initial

rate and hydrolysis yield when no citrate buffer was used. Again, this observation tests the

hypothesis that the initial rate of the enzymatic hydrolysis is directly proportional to the enzyme

added; however, since the final sugar yield is not doubled when the enzyme loading is doubled, a

direct proportionality may not be present, but there is definitely some dependence. This suggests

that there may be an optimum enzyme loading that will maximize yield per mass of enzyme used.

Interestingly, CTec3, which is an improved version of CTec2, did not increase either initial rate

0 5

10 15 20 25 30

0 50 100 150

Glu

cose

Pro

duce

d (m

g/m

L)

Time (hr)


66

or final sugar yield. The sugar generation curves plotted show minimal difference between

enzymatic hydrolysis when CTec3 and CTec2 are used. This may be due to the fact that CTec3 is

optimized for a different type of substrate, and the cellulose structure in softwood may be

sufficiently different that hydrolysis is not improved with the new formulation. Accordingly,

CTec3 was not used in subsequent trials.

6.4 Enzymatic Hydrolysis - Inhibitors

Preliminary investigations show that enzyme inhibition and/or deactivation may play a big role

in the low yield obtained. Different methods were used to determine if this was actually the

cause, and to address and potentially rectify this phenomenon. Three methods were investigated

to address this issue: washing, multistage enzymatic hydrolysis, and staggered enzyme addition.

These three methods are commonly used in an industrial setting because of lower cost and ease

of implementation. One of the key findings in this study is the inhibition of CTec2 by citric acid.

The decrease in yield was dramatic enough to mask the impact of adding additional enzymes to

the hydrolysis reaction. One possible explanation for this inhibition is citrate acting as a

chelating agent for the metal ions which are responsible for acting as co-enzymes to help

CTec2's function. Citric acid has been shown to have a high affinity for aluminum and iron ions,

especially at low pH (Martin 1986). This is put forth in Chapter 7 as a recommendation for future

work.

6.4.1 Liquid Removal

A set of trials was conducted in the flask reactor at a solids loading of 10% using fibres obtained

from autohydrolysis at 215oC and 8min. The main comparison is made between trials with or


67

without liquid hydrolyzate removal, to determine whether hydrolyzate removal increased the

hydrolysis yield. It was determined that the removal of liquid did not increase the final yield.

This suggests that product inhibition might be minimal.

6.4.2 Staggered Enzyme Addition

A different approach was used to look at potential inhibition by pretreatment degradation

products. Since pretreatment is known to generate enzyme inhibitors and deactivating agents, the

concept of staggered enzyme addition was used to reduce the exposure of enzyme to these

inhibitors and deactivating agents within the reactor. When this strategy is combined with 1hr

alkaline treatment, it showed a dramatic increase in yield, even in the presence of a citrate buffer,

as shown by the red line in Figure 5-8. Interestingly, the same method did not work when used

with fibres that were not treated with NaOH (blue line). This suggests that there are inhibitors

within the liquid slurry and at the same time, there is a barrier preventing enzyme access to the

substrate after autohydrolysis. This observation also supports the concept that alkaline treatment

is effective at increasing the cellulose reactivity, even though inhibitors are also produced and

can adversely impact the enzymatic hydrolysis yield.

The concept of staggered enzyme addition extends to continuous addition of enzymes, which

would ultimately lead to a continuous enzymatic hydrolysis process. This concept is supported

by Brethaur and Wyman (2010) as a method to reduce costs.

6.4.3 Higher Solids Loading

In addition to product inhibition, the substrate itself may be an inhibitor. Kristensen, Felby, and

Jǿrgensen (2009) noticed a decrease in the enzymatic hydrolysis yield as the initial substrate


68

loading was increased. They noted that even though product inhibition is in play, it did not

account for the decrease in conversion. In this study, substrate loading was investigated to see

the efficacy of conducting enzymatic hydrolysis of autohydrolyzed softwood at higher solids

loading. Figure 5-10 shows the yield curve when pine autohydrolyzed at 215oC and 8min was

used as enzymatic hydrolysis substrates in the SBI reactor. It was observed that, although the

initial rate may be lower, 20% solids loading actually gave the highest yield.

A potential reason to explain the lack of observable impact of substrate might be due to the

concentration not at the threshold. Kristensen, Felby, and Jǿrgensen (2009) used filter papers, a

pure cellulose matrix, in their investigation. In this study, autohydrolyzed pine, which is much

more heterogeneous than the filter paper, is used. Thus, the enzymes may not be directly exposed

to the surface of cellulose. Therefore, even though the solids loading investigated were the same,

from a microscopic point of view, the enzymes were not exposed to the same level of cellulose

concentration due to the presence of other biomass components.

In terms of enzyme performance index (EPI), it is suggested that even though the enzyme

loading was the same across the three different substrate loadings, a much better performance is

achieved when the substrate is loaded at 20%.

Table 6-2 Enzymatic Hydrolysis Yield and Enzyme Performance Index of Different Solid's

Loading

Solids Loding Enzymatic Hydrolysis Yield Enzyme Performance Index 10% 30% 263 15% 35% 471 20% 42% 715


69

Palmqvist, Wiman and Lidén (2011) explore the impact of mixing, specifically the agitation

speed, on enzymatic hydrolysis yield. In their study, autohydrolyzed spruce (210oC and 5min

with 20min 2.5% SO2 impregnation) was used and it was found that the enzymatic hydrolysis

yield was about 30% after 72hr. Compared with the results obtained in this study, this suggests

that a good/adequate mixing may even be able to overcome a decrease in fibre reactivity due to

lower pretreatment severity.

6.4.4 Multistage Enzymatic Hydrolysis/Washing

Another method of tackling potential inhibitors/deactivating agents is to conduct enzymatic

hydrolysis in multiple stages, with fibre washing in between. A multistage process has been

documented to in allow glucanases to retain their activity by removal of products such as

cellobiose and monomeric sugars (Vanderghem et al. 2010). This has the advantage of removing

the products, but at the same time, soluble enzymes such as β-glucosidase may also be removed.

As seen from before, enzymatic hydrolysis kinetics usually becomes stagnant and plateaus after

the first 24hr of reaction. Hence, a separation of the slurry at this time seemed ideal. In this

investigation, pine autohydrolyzed at 215oC and 8min was subjected to enzymatic hydrolysis in

the SBI reactor at a solids loading of 20% and an enzyme loading of 5% by cellulose mass. After

separation and re-suspension, additional enzymes were added (half of the initial amount) and the

enzymatic hydrolysis was carried out for an additional 48h. As expected, the initial 24hr data

should be the same, as the conditions were identical. However, it was observed that washing the

cake led to a lower second stage yield. This suggests that the negative impact of enzyme removal

by washing is more severe than the advantage gained by the removal of inhibitors.


70

An issue with data analysis at high solids loading is the inconsistency between the slurry volume

and the hydrolyzate volume. The HPLC analysis determines the concentration within the

hydrolyzate, but it is often hard to back-calculate the actual concentration, and thus, the yield

will not accurately reflect the true value (Kristensen, Felby, and Jǿrgensen, 2009). A different

method using the mass of each phase was used to calculate the actual sugar yield (see Appendix

A - Calculating Yield Through Total Mass Balance For Multistage Enzymatic Hydrolysis). In

addition, enough cake was left such that characterization was possible. The results are shown in

Table 6-3. This shows a much smaller change, if any, between the yields, suggesting that

washing may not have any significant impact on enzymatic hydrolysis yield.

Table 6-3 Enzymatic Hydrolysis Yield for Multistage Hydrolysis Using Different Yield

Calculations


Enzymatic Hydrolysis Yield Using Mass

Balance

Enzymatic Hydrolysis Yield Using

Characterization No Wash 40% 24% 43% Washed 32% 22% 43%

A different consideration is the impact of using enzymes as a pretreatment. This has been shown

to increase the enzymatic hydrolysis yield (Jeon, Xun, and Rogers 2010). Using a multistage

setting, it may be worthwhile to investigate shortening the initial stage and conditioning with a

less expensive/active enzyme.

Chapter 7 Conclusion and Recommendation

71

7 Conclusions and Recommendations

Enzymatic hydrolysis of softwood pine proves to be challenging in terms of the low sugar yield.

Various pretreatments prior to enzymatic hydrolysis were evaluated. It was determined that

increasing the severity of autohydrolysis leads to an increase in fibre reactivity and ultimately,

leads to an increase in the sugar yield during enzymatic hydrolysis. In addition, due to the low

amount of xylan and hemicellulose in pine, higher severity pretreatment can be used to increase

cellulose reactivity without the risk of generating too much inhibitory or toxic compounds.

For post-autohydrolysis treatment, it was determined that between using alkaline, organosolv,

dilute acid, and hot water treatments, hot water performed best in terms of increasing the

enzymatic hydrolysis yield. Nonetheless, all of the chemical treatments increased the reactivity

of autohydrolyzed fibre and showed some degree of delignification and byproduct removal. Due

to the presence of external mass transfer resistance, particle size reduction is preferred; it is best

conducted through higher autohydrolysis severity, since milling dried the fiber, causing

hornification and results in lower enzymatic hydrolysis yield.

For the enzymes, there is minimal synergistic activity between CTec2 (cellulase) and AF100

(xylanase) when using autohydrolyzed pine as the substrate. The addition of more enzymes at the

start of enzymatic hydrolysis does increase the initial rate, but the final yield is not proportional

to the increase in enzyme loading.

Inhibition by substrate and products seems to be minimal when autohydrolyzed pine is used as a

substrate, though the latter could be due to the low amount of sugar present in the hydrolyzate.


72

However, degradation products from pretreatment can adversely impact the enzymatic

hydrolysis yield and thus methods for removal must be considered.

7.1 Recommendations

This investigation opened up a lot of options towards optimizing the use of softwood as a

second-generation biofuel feedstock. More work is required to bring the enzymatic hydrolysis

yield to a level such that it will be economical to commercialize the process. The following are

recommendations for future work:

• Investigate impact of treating softwood pine at higher severity, specifically, at 220oC or

225oC, with a retention time of 8min

This will allow a more comprehensive analysis of the impact of autohydrolysis temperature

on fibre reactivity and enzymatic hydrolysis yield.

• Investigate the addition of catalysts during post-autohydrolysis treatment

This will look at the impact of additional chemicals and their interaction during chemical

treatment; it will then elucidate which path, in terms of chemical treatment, will be most

economical by comparing the relative operating costs.

• Re-evaluate the impact of post-autohydrolysis treatment

Though improvements were observed when chemical treatments were performed after

autohydrolysis, the results were influenced by the presence of citrate buffer. The impact


73

should be re-evaluated to see if the differences without citrate buffer are more dramatic than

those observed with citrate buffer.

• Impact of higher enzyme loading (10%)

It was determined that there was minimal difference between an enzyme loading of 5% and

10% by dry cellulose mass. But this impact is masked by the presence of a strong inhibitor,

namely citrate. Further investigation is required to see if using 10% enzyme loading would

result in higher yield, especially in the larger reactors; this would help determine the

economics of scaling up the enzyme loading and the impact on the feasibility of

commercialization.

• Evaluation of other potential synergistic activities, specifically interaction between cellulase

and mannanase

The main hemicellulose in softwood is glucomannan. The minimal synergistic impact

between cellulase and xylanase may have been the result of the low xylan content; the

addition of mannanase may be more effective at increasing cellulose accessibility by

releasing the glucomannan from the lignocelluloses structure.

• Impact of continuous enzyme addition

It is hypothesized that enzyme deactivation/inhibition will be minimized if enzymes are

continuously added, in small amounts, over the period of the enzymatic hydrolysis reaction.

• For multistage enzymatic hydrolysis, determine the optimum time to stop the first stage. This

is the time at which the initial rate is maximized with the final yield.


74

• For multistage enzymatic hydrolysis using the SBI reactor, a balance should be used in order

to keep track of the actual amount of water added and slurry removed. This will allow a

better mass balance and ultimately lead to a more accurate yield calculation.

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75

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Appendix A

80

Appendix A - Calculating Yield Through Total Mass Balance For

Multistage Enzymatic Hydrolysis

Figure A-1 is a schematic representation of multistage enzymatic hydrolysis. This approach is a

mass balance around the whole process to determine the total amount of sugar present, in both

liquid and solid (attached onto the fibre). Yield is calculated based on the total mass of sugar

obtained from each stream using Equation 2 (on mass basis instead of concentration basis).

Streams 1, 2, and 7 are enzymatic hydrolysis slurry streams. Streams 3 and 8 are solid fibre

cakes. Stream 4, 9, and 10 are hydrolyzate streams. Samples from these streams are analyzed for

sugar composition. Stream 6 is the wash stream. This stream accounts for sugars that are washed

from the fibre after the slurry has been separated into hydrolyzate and cake.

The following equations were used to determine the mass of sugar in each stream.

Initial Sugar Mass in Reactor (at t = 0), or Slurry

msugar,reactor =

massliquid,added + �masssolid,added ∗ �1 − DM%100 ��

ρliquid,reactor∗ [HPLC]sugar

1000

Sugar Mass in Liquid

msugar,liquid =

massliquid,separated + �masssolid,,separated ∗ �1 − DM%100 ��

ρliquid,separated∗ [HPLC]sugar

1000

Appendix A

81

Sugar in Washwater

It was assumed that the washwater density is roughly 1000kg/m3 because 8000mL was used.

msugar,washwater = � mwashwater added,i

number of washes

i=1

∗ [HPLC]sugar,i

Sugar Mass in Attached to Solid

In cases where wash wasn't implemented, residual sugar will be present on the cake itself. A

similar method to determining sugar mass in slurry was used. The small solids sample was re-

suspended and agitated for 2hrs at room temperature. A liquid sample was collected and the

original sugar mass attached to solid was back-calculated by a dilution calculation.

[HPLC]sugar,initial =[HPLC]sugar,sample ∗ �msample ∗ �

100 − DM%100 � + mwater,added�

msample ∗ �100 − DM%

100 �

msugar,solid = [HPLC]sugar,initial ∗ mcake ∗ �100 − DM%

100�

The total sugar produced is then calculated by

𝑚𝑠𝑢𝑔𝑎𝑟 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑 = 𝑚𝑠𝑢𝑔𝑎𝑟,4 + 𝑚𝑠𝑢𝑔𝑎𝑟,6 + 𝑚𝑠𝑢𝑔𝑎𝑟,9 + 𝑚𝑠𝑢𝑔𝑎𝑟,10 − 𝑚𝑠𝑢𝑔𝑎𝑟,1 − 𝑚𝑠𝑢𝑔𝑎𝑟,6

and the yield is calculated through an adjusted version of Equation 2:

𝑦𝑖𝑒𝑙𝑑 % = 𝑚𝑠𝑢𝑔𝑎𝑟 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑

1.111 ∗ 𝑓 ∗ 𝑚𝑏𝑖𝑜𝑚𝑎𝑠𝑠∗ 100%.

Appendix A

82

Figure A-1 Block Flow Diagram for Multistage Enzymatic Hydrolysis

impact of pretreatment methods on enzymatic … · 2.1.2 hardwood ... figure 2-8 dry pine...

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