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HYDROLYSIS OF LIGNOCELLULOSE USING ENZYMES
FROM PELLETIZED TRICHODERAIA REESE]
F ERM ENTATION

presented by

Ying Liu

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HYDROLYSIS OF LIGNOCELLULOSE USING ENZYMES FROM
PELLETIZED TRICHODERMA REESE] FERMENTATION
By

Ying Liu

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Biosystems Engineering

2010

ABSTRACT

HYDROLYSIS OF LIGNOCELLULOSE USING ENZYMES FROM
PELLETIZED T RICHODERJWA REESE] FERMENTATION

By

Ying Liu
Pelleted T richoderma reesei is a desirable morphology for industrial fungal fermentation
to obtain stable cellulase production. Two common cellulosic feedstocks, corn stover and
switchgrass were pretreated by acid, alkaline and ammonia fiber expansion (APEX)
methods. The pretreated cellulosic feedstocks were used as fermentation substrates for
enzyme production. It has been proved in this study that 15 g/L AF EX corn stover in
fermentation medium could stimulate the fungi to yield relatively high cellulase (1.08
U/mL at 93 hours) and xylanase (2.52 U/mL at 24 hours) activities. The enzyme
cocktails from pelletized T richoderma reesei fermentation were applied on the pretreated
cellulosic feedstocks to produce mono-sugars. A comparison of enzyme cocktails from
difl'erent pretreated feedstocks and two enzymes sources was conducted to elucidate the
effects of pretreated cellulosic materials on enzyme production and enzymatic hydrolysis.
The enzyme cocktail (10 U/ g dry mass) generated from AF EX com stover has better
enzyme compositions that enhance its hydrolysis especially working on the alkaline

pretreated switchgrass which pretreated under lhour, 121°C, 1% sodium hydrate solution.

TABLE OF CONTENTS

List of Tables ................................................................................... v
List of Figures ................................................................................ vi
1. Introduction ................................................................................ 1
1.1. Current Transportation Fuels ................................................................. 1
1.2. Objectives of This Study ....................................................................... 3
1.3. Pretreatment ........................................................................................... 3
1.4. Enzyme production and enzymatic hydrolysis of lignocellulose .......... 7
1.5. Problem and possible solution ............................................................... 9
2. Materials and Methods ............................................................ 11
2.1. Microorganism ..................................................................................... l 1
2.2. Substrate .............................................................................................. 11
2.3. Medium ................................................................................................ 12
2.4. Enzymes ............................................................................................... 12
2.5. Cellulase and Xylanase Production ..................................................... 13
2.6. Process for Pretreatment and Enzymatic Hydrolysis .......................... 13
2.7. Ethanol fermentation ........................................................................... 15
2.8. Analysis methods ................................................................................. 16
2.9. Statistical analyses ............................................................................... 18
3. Enzyme Production .................................................................. 19
3.1. Morphology evaluation ....................................................................... 19
3.2. Cellulase and xylanase productions on basic chemical defined medium
.................................................................................................................... 1 9
3.3. Comparison of basic chemical defined medium and modified medium
.................................................................................................................... 19
3.4. Comparison of CaCO3 and Tris-maleate buffer solution as the
neutralizer in cellulase and xylanase production ........................................ 20
3.5. Effects of different concentration of CaCO3 on cellulase and xylanase
production ................................................................................................... 21
3.6. Comparing different agricultural residues with purified cellulose in
cellulose production .................................................................................... 21
3.7. Effects of different concentrations of AF EX corn stover in cellulases
production ................................................................................................... 22

3.8. Comparing cellulases production with the optimal condition on both

chemical defined medium and APEX corn stover medium ....................... 23
4. Enzymatic Hydrolysis .............................................................. 24
4.1. Enzymatic hydrolysis of lignocellulosic materials .............................. 24
4.2. Ethanol Fermentation .......................................................................... 26
5. Conclusion and Perspectives ................................................... 28

List of Tables

Table 1. Correlation between biomass features and enzymatic digestibility of biomass . 30

Table 2. Key factors for an effective pretreatment of lignocellulosic materials ............... 31
Table 3. Selected diluted acid pretreatment and enzymatic hydrolysis research .............. 32
Table 4. Composition of different lignocellulosic materials (% dry wt) .......................... 33
Table 5. Comparison of different substrates using in enzyme production ....................... 34
Table 6. Effect of various pretreatment methods on the chemical composition ............... 35

Table 7. Comparison of different substrates using in enzyme production (96hours) ....... 36

Table 8. Components in different pretreated lignocellulosic materials ............................ 37
Table 9. Optimization of enzymatic hydrolysis using commercial cellulase ................... 38
Table 10. Optimization of enzymatic hydrolysis using commercial cellulase ................. 39
Table 11. Relatively high activity enzymes working on high solid content ..................... 4O

List of Figures

Figure 1. Typical morphologies of T. reesei using the two different inoculum sizes ....... 41

Figure 2. Cellulase and xylanase production time course test for 5 days with the basic
chemical defined medium ................................................................................................. 42

Figure 2. con’t Cellulase and xylanase production time course test for 5 days with the
basic chemical defined medium ........................................................................................ 43

Figure 3. Cellulase and xylanase production with the carbon source modified chemical

defined medium ................................................................................................................ 43
Figure 3. can’t Cellulase and xylanase production with the carbon source modified '

chemical defined medium ................................................................................................. 45
Figure 4. Enzyme production with both neutralizers added in the basic medium ............ 47

Figure 4. con’t Enzyme production with both neutralizers added in the basic medium... 47

Figure 5. The time course of cellulase and xylanase production in flasks with 4g/L of
CaCO3, lOg/L of CaCO3 or without CaCO3 added in the carbon source modified
chemical defined medium ................................................................................................. 48

Figure 5. can’t The time course of cellulase and xylanase production in flasks with 4g/L
of CaCO3‘, lOg/L of CaCO3 or without CaCO3 added in the carbon source modified

chemical defined medium ................................................................................................. 49
Figure 6. Cellulase 48 hours ............................................................................................. 50
Figure 7. Cellulase 72 hours ............................................................................................. 51
Figure 8. Cellulase 96 hours ............................................................................................. 52
Figure 9. Xylanase 48 hours ............................................................................................. 53
Figure 10. Xylanase 72 hours ........................................................................................... 54
Figure 11. Xylanase 96 hours ........................................................................................... 55

Figure 12. Comparing different concentrations of APEX treated corn stover as the
substrates working on cellulases fermentation. ................................................................ 56

Figure 13. Comparing different concentrations of AF EX treated corn stover as the
substrates working on cellulases fermentation ................................................................. 57

vi

Figure 14. The modified medium ..................................................................................... 58

Figure 15. Comparing the enzymes activity produced on 15 g/L APEX corn stover and
chemical defined medium ................................................................................................. 59

Figure 16. Comparing the enzyme productivity based on 15 g/L AF EX corn stover and
chemical defined medium ................................................................................................. 60

Figure 17. Different ratio of xylanase over cellulase in both Accellerase and cellulase
enzyme cocktails ............................................................................................................... 61

Figure 18. Hydrolysis of alkaline treated switchgrass, acid treated switchgrass, APEX
treated corn stover and acid treated corn stover with different enzyme cocktails ............ 62

Figure 19. Hydrolysis of alkaline treated switchgrass, acid treated switchgrass, AF EX
treated corn stover and acid treated corn stover with different enzyme cocktails ............ 63

Figure 20. Hydrolysis of alkaline treated switchgrass, acid treated switchgrass, AP EX
treated corn stover and acid treated corn stover with different enzyme cocktails ............ 64

Figure 21. Hydrolysis of alkaline treated switchgrass, acid treated switchgrass, APEX

treated corn stover and acid treated corn stover with different enzyme cocktails ............ 65
Figure 22. Effects of Accellulase working on lhour, 105C, 0.1 /O.5/ 1% NaOH solution
pretreated switchgrass. ...................................................................................................... 66
Figure 23. Effects of Accellulase working on lhour, 121C, 0.1 /0.5/ 1% NaOH solution
pretreated switchgrass. ................................................................................................... 68
Figure 24. Effects of Accellulase working on 2hour, 105C, 0.1 /0.5/ 1% NaOH solution
pretreated switchgrass. ...................................................................................................... 68
Figure 25. Effects of Accellulase working on 2hour, 121C, 0.1 /0.5/ 1% NaOH solution
pretreated switchgrass. ...................................................................................................... 69
Figure 26. Effects of cellulase and Accellulase working on lhour, 105C, 0.5% NaOH
solution pretreated switchgrass. ........................................................................................ 70
Figure 27. Effects of cellulase and Accellulase working on lhour, 121C, 0.5% NaOH
solution pretreated switchgrass. ........................................................................................ 71
Figure 28. Effects of cellulase and Accellulase working on lhour, 105C, 1% NaOH
solution pretreated switchgrass. ........................................................................................ 72
Figure 29. Effects of cellulase and Accellulase working on lhour, 121C, 1% NaOH
solution pretreated switchgrass. ........................................................................................ 73
Figure 30. Effects of different hydrolysis substrates on sugar concentration. .................. 74

vii

Figure 31. Compared the effects of different hydrolysates based on 2 different enzyme
sources working on ethanol fermentation. ........................................................................ 75

viii

1. Introduction

1.1. Current Transportation Fuels

1.1.1. Fossil Fuels

Since fossil fuels have been commercially produced in the early twentieth century, they
have dominated the transportation energy market due to their availability, high energy
density, and easy distribution. As the world population has increased in the past several
decades, and more developing countries are on the way towards industrialization, the
supply of fossil fiiels has shrunk. This petroleum shortage could severely hurt the
development of the world economy that is powered by the petroleum based fiiels. In
addition, the consumption of non-renewable petroleum also leads to environmental

impacts, such as green house gas emission. The 2010 C0penhagen Conference illustrated
that carbon dioxide as one of the major green house gases; it is responsible for at least 50%
of the causes to the global climate change (BRDI. 2006). In the United States, around 33%
US. carbon dioxide emissions is attributed to petroleum-based transportation fuels

(Wang et a1. 2002). Thus, considering both economic and environmental impacts,
renewable energy sources are urgently needed to diversify our energy resources and

reduce the negative environmental impacts of fossil fuels.

1.1.2. Starch Ethanol
Ethanol is one of the approved renewable transportation fuels that are currently available
in the marketplace. Fuel ethanol, from starch, has been commercially produced since the

19805. In the past 20 years, starch ethanol production steadily increased. The fuel ethanol

industry in the US. is currently producing around 20 billion gallons per year, and it is
anticipated that the demands for fuel ethanol would be more than 24 billion gallons/year
by 2020 (BRDI 2006). However, starch crops are major food sources. Ifmore starch
crops are used for fuel, less starch is available for food production. Following the steady
increase of the world population, starch-based crops (such as corn, wheat, cassava, rye
and barley) are still not sufiicient to satisfy their demands as animal and human foods.
Using starch-based crops to increase fuel ethanol production is obviously neither

sustainable nor practical (Keshwani et a1. 2009).

l. 1.3. Lignocellulosic ethanol

Li gnocellulose, the most abundant renewable resource on the planet, provides an
untapped reserve for bio-fuel production. Agricultural residues, such as crop residues,
grasses, sawdust, and wood chips, are the major sources of it (Hofiichter et a1. 2002). Due
to their high carbohydrate contents, relatively low cost, and worldwide availability,
agricultural residues attract significant interest regarding fuel ethanol production. It was
estimated that utilizing lignocellulosic materials produce bioethanol could replace up to
40% of the fuel consumption in the United States market (Zheng et a1. 2009).
Approximate 80-90 billion gallons of fuel ethanol could be annually produced from 1.3
billion tons of available lignocellulosic residues available in the United States. In addition,
lignocellulosic ethanol as an alternative fuel also would reduce 86% of the greenhouse

gas emission currently generated from fossil fuel consumption (Sun et a1. 2002).

1.2. Objectives of This Study

In response to the need of lignocellulosic fuel ethanol production, an environmentally
friendly biological process to achieve the highly efficient conversion of carbohydrates in
agricultural residues to fermentable sugars for ethanol production needs to be developed
Thus, this study will investigate cellulase and xylanase production using pelletized fimgal
fermentation, and subsequently apply the enzymes to convert pretreated lignocellulosic
biomass into mono-sugars for ethanol production.

The objectives of the study mainly focused on enhancing cellulase and xylanase
production on diluted alkaline treated switchgrass and APEX (Kumar and Wyman)
treated fiber using pelletized fimgal culture, and improving enzymatic hydrolysis on

pretreated cellulosic feedstocks (Esteghlalian, Hashirnoto et al.).

1. 3. Pretreatment

l. 3.1. The composition of lignocellulosic material

Fifty to eighty percent of Li gnocellulosic materials consist of three polymers of cellulose,
hemicellulose and lignin. The three polymers tightly pack together to form a stable and
recalcitrant matrix structure. Cellulose is a linear polymer of D-glucose units that linked
by beta-l, 4 glycosidic bonds. The polymer has different levels of polymerization degree
and crystallinity. Higher polymerization degree and crystallinity, less soluble and
degradable cellulose is. Hemicellulose is a complex carbohydrate that links lignin and
cellulose fibers. It mainly consists of two polymers of pentoses (D-xylose, D-arabinose)
and hexoses (D-mannose, D-glucose, D-galactose). The ratios of pentoses and hexoses in

hemicellulose are varied with respect to different sources, for instance, hardwood and

agricultural plants contain more xylan, while softwood has more glucomannan.
Hemicellulose is also a linear polymer with short branches. The differences of structure
and composition make hemicellulose much easier to be hydrolyzed than cellulose. Lignin
is another major component of lignocellulosic materials. It is an amorphous
heteropolymer that consists of three units (p-coumaryl, coniferyl and sinapyl alcohol). In
a plant, lignin is closely bound to cellulose and hemicellulose makes which make the
plant structure stiff, and be able to prevent the plant from microbial attack and enzymatic

degradation (Sun et a1. 2002).

1. 3.2. Pretreatment

Due to the facts of complicated composition and structure that lignocellulosic materials
have (Table 1.), a pretreatment step must be implemented prior to enzymatic conversion
of cellulose and hemicellulose components into sugars and further fermentation of
ethanol production. Thermal and chemical pretreatment methods were widely used to
break down the matrix structure and remove the lignin fraction of 1i gnocellulosic
materials. This section presented a brief description of different thermal and chemical
pretreatment methods such as steam explosion, ammonia fiber expansion (AF EX), carbon
dioxide expansion, acid and alkaline treatments, and ozonolysis etc based on the key

factors of selecting effective pretreatment methods for lignocellulosic materials (Table 2.)

1.3.2.1 Steam explosion
High-pressure steam is applied on lignocellulosic materials for a short time period (Sun et
a1. 2002). At the end of treatment, the pressure will be suddenly released that causes an

explosive destruction of the material, which leads to a relatively loose structure. Enzyme

and other catalysts can easily access to attack cellulose and hemicellulose to generate

mono-sugars.

1.3.2.2 Ammonia fiber expansion (AF EX)

The ammonia fiber expansion (APEX) method applies liquid ammonia at mild
temperature and high pressure for a short period of time, followed by a sudden release of
the pressure (Gao et a1. 2009). APEX process solubilizes 1i gnin and increase surface area
that will significantly enhance the following enzymatic conversion of cellulose and
hemicellulose into mono-sugars. Even though, the AF EX process was not very effective
for the biomass with high lignin content, such as woody biomass, it can effectively
improve the enzymatic hydrolysis for most of agricultural lignocellulose such as

switchgrass and corn stover.

1.3.2.3 Carbon dioxide expansion

C02 expansion is another high pressure treatment similar with APEX and steam
explosion (Sun et al. 2002). It has advantages of using non-toxic chemicals and
generating non-inhibitory compounds compared to APEX and steam explosion. The
carbonic acid produced during the carbon dioxide expansion, to some degree, helps
increase the reaction rate of following enzymatic hydrolysis. However, in terms of
hydrolysis performance, C02 expansion is still not as effective as APEX and steam

explosion.

1.3.2.4 Ozonolysis
Ozonolysis is a chemical method to pretreat lignocellulosic materials (Sun et a1. 2002). It

can remove lignin under the mild condition like room temperature and pressure. The

reaction is only limited to lignin, and has no negative impacts on cellulose and
hemicelluloses (Sun et a1. 2002). The ozonolysis reaction has a relatively high efficiency
to remove lignin. However, the requirement of large amount of ozone leads to a high cost

and makes this technology less feasible.

1.3.4.5 Dilute acid treatment

Dilute acid treatment has been widely used by industry to pretreated lignocellulosic
materials. It removes the majority of hemicellulose and break down the recalcitrant
structure of lignocellulose in order to make enzyme able to attack cellulose to produce
mono-sugars (RJ et a1. 1999). The dilute acid treatment is often teamed up with steam
expansion to improve performance of the pretreatment. The dilute acid treatment can
facilitate the following enzymatic hydrolysis to reach up to 70% conversion rate of
cellulose. Due to the fact of use of low concentration of acid, the method has relatively

less impact on the environment.

1.3.4.6 Alkaline treatment

Similar with dilute acid treatment, alkaline pretreatment is another chemical treatment
method that has been widely used to treat cellulosic materials (Keshwani et a1. 2009). The
mechanism of this method is that alkaline saponifies intermolecular ester bonds between
hemicellulose and other components (1i gnin and cellulose), and alkaline also extracts the
released hemicellulose and lignin so that both hemicellulose and lignin contents are
significantly reduced in the treated samples. The dilute alkaline treated lignocellulosic

materials can reach a 70% sugar conversion rate (Sun et a1. 2002).

1.4. Enzyme production and enzymatic hydrolysis of lignocellulose

Enzymatic conversion of lignocellulosic materials to fermentable sugars is one of the
critical steps for lignocellulose utilization of bioethanol production It has been widely
reported that most of 1i gnocellulose enzymes are generated from fimgal species such as T.
reesei, A. niger, and P. chnisosporium. etc (Dashtban et al. 2009). A unique enzyme
production of pelletized fungal fermentation and enzymatic hydrolysis were discussed in

this section.
1.4.1. Enzyme production

1.4.1.1. Stains

Filamentous fungi have been widely used in commercial cellulase production. In the past
several decades, numerous studies have been focused on finding different fugal species to
improve the efficiency of enzyme production T richoderma reesei is one of the strains
that have been identified and commercially used for cellulase production. T. reesei is

used by this study in the same capacity.

1.4.1. 2. Pelletized fungal fermentation technology of enzyme production

Pelletization of filamentous fungi is one of methods that can be applied to improve the
oxygen mass transfer and reactor performance. Compared to clump-like fungal
morphology, pelletized fungal biomass has several benefits, such as a much larger
specific surface area which reduces the mass transfer limitations, better fermentation
broth rheology which in return affects momentum, mass, and heat transfer in the reactor;
consequently, efficiency of mixing and aeration are enhanced. It has been reported that

higher yields and productivity were obtained using pelletized morphology (Liao et a1.

2002). Another advantage of fungal pellet fermentation is that the pellet makes it possible
to perform high biomass concentration cultures to enhance the productivity (Liao et al.
2002). In addition, pellet morphology not only significantly improves the culture
rheology, which results in better mass and oxygen transfer into the biomass, and lower
energy consumption for aeration and agitation, but also makes fungal biomass reuse
possible. Thus, the pelletized fungal fermentation technology developed by Drs. Liu and

Liao was adopted to fulfill the enzyme production for this study.

It has been reported that lignocellulosic materials have often been used as both the
substrate and inducers in the fermentation processes for cellulase production (Lee and
Fan, 1982). Lower solid concentrations also have been shown to yield higher cellulase
production (Szengyel et al, 1997). The highest cellulase yield from T. reesei culture was
around 150 filter paper units (PPU)/g cellulose using Solka Floc as the cellulose substrate.
The productivity of 55 FPU/L-hr was achieved in the same culture (Xia et al. 2006). This
study used different agricultural residues (APEX corn stover, alkaline treated corn stover,
acid treated switchgrass, alkaline treated switchgrass, acid treated anaerobically digested
manure fiber, and alkaline treated anaerobically digested manure fiber) as the substrates
to compare cellulase production from T. reesei culture. Recently, due to the considerable
increase of feedstock cost (starch and mono-sugars), agricultural residues as the
fermentation substrates for cellulase production could have potentials to reduce

production cost, and enhances the productivity.

1.4.2. Enzymatic hydrolysis

Main enzymes fi'om fungal fermentation include cellulase system ( l, 4-B-D-glucan
glucanohydrolase, l, 4-fi-Dglucan cellobiohydrolyase and B-glucosidase) and xylanase
system (endo-B-l, 4-xylanase, exoxylanase, and B-xylosidase). The function of the
cellulase system is: 1) endoglucanase randomly cleaves cellulose chains to form glucose,
cellobiose and cellotriose; 2) exoglucanase attacks the non-reducing end of cellulose to
release cellobiose units; and 3) Cellobiase cleaves cellobiose units into fermentable
glucose units. However, most fungal cellulase systems are lack of B-glucosidase activity
that is critical to convert cellobiose into glucose. B-glucosidase fiom A. nigers is often
added into the hydrolysis system to enhance the conversion of cellobiose. The xylanase
system has similar function with cellulase system. Endo-B-l , 4—xylanase primarily targets
the internal B-l , 4 bonds between xylose units, exoxylanase releases xylobiose units, and

B-xylosidase convert xylobiose to xylose (Zheng et al. 2009).

1.5. Problem and possible solution

Current issues of lignocellulosic ethanol production are: 1) strong thermal or chemical
methods for feedstock pretreatment; 2) high cost of fungal enzymes production; and 3)
relatively low efficiency of enzyme systems. In order to facilitate finding solutions for
these issues, this study systematically investigated three components of fungal enzyme
production, feedstock pretreatment and enzymatic hydrolysis of sugar production. An
integrated solution of pelletized fungal fermentation on APEX corn stover for enhanced
enzyme production, dilute alkaline treatment with less chemical loading, and improved

hydrolyzibility with optimized enzyme system was concluded from this study, which

would significantly alleviate the barriers that lignocellulosic biorefineries are

encountering.

10

2. Materials and Methods

2.1. Microorganism

The strain for cellulase and xylanase production was T richoderma reesei ATCC 56765. It
was cultured on potato dextrose agar slants at 28 °C for 5-7 days to form spores. The
spores were collected by using sterile distilled water to wash the agar. A sterile cheese
cloth was used to remove mycelia. The concentration of spore suspension was 1x109

spores /ml. The spore solution was stored at 4 °C.
2.2. Substrate

2.2.1. Substrate for Enzyme Production

Purified cellulose powder was from Sigma-Aldrich(a-D-G1ucose, anhydrous, 96 %).

AF EX pretreated corn stover was provided by 'Dr. Bruce Dale at Department of Chemical
Engineering of Michigan State University. It contained: 34.4% (dry basis) of glucan, 22.4%
(dry basis) of xylan, 4.2% (dry basis) of arabinan, 0.6% (dry basis) of mannan, 1.4% (dry
basis) of galactan, 3.8% (dry basis) of uronyl, 11% (dry basis) of lignin and 5.6% (dry

basis) of acetyl content.

2.2.2. Substrate for Enzymatic Hydrolysis

Switchgrass was used as the substrate for the enzymatic hydrolysis. It was harvested in

the farm at Michigan State and the component is cellulose 36.75 %, hemicellulose 28.76 %
and lignin 17.93 %. It was grinded to 4.0 mm prior to biological pretreatment and

hydrolysis.

11

The APEX corn stover for the enzymatic hydrolysis provided by Dr. Bruce Dale at
Department of Chemical Engineering of Michigan State University with the following
components : 34.4% (dry basis) of glucan, 22.4% (dry basis) of xylan, 4.2% (dry basis) of
arabinan, 0.6% (dry basis) of mannan, 1.4% (dry basis) of galactan, 3.8% (dry basis) of

uronyl, 11% (dry basis) of lignin and 5.6% (dry basis) of acetyl content.

2.3. Medium

The medium for spore culture was potato dextrose broth (Sigma-Aldrich, 24 g/L).

Two media for cellulase and xylanase production by T. reesei were used. One is the basic
chemical defined medium that contained (1 L): glucose 10.0 g, cellulose 10 g, xylan 10g,
lactose 10 g, yeast extract 0.3 g, peptone 0.75 g, KH2P04 15 g, (NH4)2SO4 5 g,
MgSO4-7H20 1.23 g, CaClZ'ZHZO 0.8 g, CaC03 4 g, Tween 80 0.5 g, FeSO4-7H20
0.00271 g, MnSO4-H20 0.0016 g, ZnSO4-H20 0.0014 g, CoC12-2H20 0.0036. The
other is the modified chemical defined medium that 15 g/L AF EX corn stover has been
used instead of glucose, cellulose, yeast extract and peptone in the previous basic

chemical defined medium, and other salts and trace elements are kept as the same.

2.4. Enzymes

Accellerase® is commercially available through Genencor. The cellulase and xylanase
activity are 26 FPU/mL and 20 FPU/mL, respectively. It has been used in this study as a

contrast to compare with the cellulases system produced by the laboratory.

12

2.5. Cellulase and Xylanase Production

One mL T. reesei spore suspension was inoculated into 50 mL of PDB medium in a 125
mL Erlenmeyer flask. After being cultured at 27 °C for 24 h on a rotary shaker at 170 rpm,
10% (v/v) of seeds was inoculated into another 50 mL PDB medium for an extra 24 h.
Consequently, the pelletized seeds were inoculated into the production medium with an
inoculum size of 10% (v/v). Cellulase and xylanase were produced in 50 mL

fermentation medium in a 250 mL flask at 180 rpm, 27°C for 96-120 hours. After that,

the culture broth was centrifuged at 3500 rpm for 10 min (AllegaraTM X-12R, Beckman

Coulter) and the supernatant was used as cellulose enzyme solution.
2.6. Process for Pretreatment and Enzymatic Hydrolysis

2.6.1. Dilute Alkali Pretreatment of Switchgrass

Switchgrass was treated using an autoclave with various sodium hydroxide concentration
(0.1%, 0.5%, 1%, wt), reaction time (1 hour, 2hours) and temperature (105°C, 121°C).
Fiber concentration was fixed at 6% dry mass. After the treatment, the reaction solutions
were adjusted to pH 4.0-5.0 using 20% sulfuric acid solution. The treated samples were
centrifuged, and solid residues were washed 4-5 times using deionized water. The solid
residues were alkaline treated switchgrass, and stored in a freezer of -20 °C. The
composition of the alkali treated switchgrass was measured to be 61.18% (dry basis) of

cellulose, 17.02% (dry basis) of xylan, 9.14% (dry basis) of lignin.

13

2.6.2. Dilute Acid Pretreatment of Switchgrass

Switchgrass was treated using an autoclave with various sulfuric acid concentration (1%,
wt), reaction time (1 hour) and temperature (130 °C). Fiber concentration was fixed at 6%
dry mass. After the treatment, the reaction solutions were adjusted to pH 4.0-5.0 using 20%
sodium hydroxide solution. The treated samples were centrifuged, and solid residues

were washed 4-5 times using deionized water. Solid residues were stored in a fi'eezer of -
20 °C. The composition of the acid treated switchgrass was measured to be 60.74% (dry

basis) of cellulose, 24.38% (dry basis) of lignin.

2.6.3. APEX Pretreatment of Com Stover

The APEX corn stover was pre-milled (passed through a 10 mm sieve) with 60%
moisture (kg water/kg dry biomass) and added into a high-pressure Parr reactor. Heated
liquid ammonia (1 kg of ammonia! kg of dry biomass) was filled up the reactor vessel
resulting in immediate rise in temperature to 130°C. The reactor was maintained at 130°C
for 15 min by an external heating mantle (within i10°C). At the end of 15 min, the
pressure was reduced to the atmospheric level and the temperature decreased rapidly. The
instantaneous pressure drop in the vessel caused the liquid ammonia to vapor, then
cooling the biomass down to less than 30°C. The pretreated materials were left under a
hood overnight to ensure the residual ammonia volatile completely. The APEX-treated
stover was kept in room temperature until fin'ther use. Its composition was measured to

be 34.4% (dry basis) of cellulose, 28.60% (dry basis) of xylan, 11% (dry basis) of lignin.

14

2.6.4. Dilute Acid Pretreatment of Com Stover

The corn stover was treated using an autoclave with sulfuric acid concentration (1%, wt),
reaction time (1 hour) and temperature (120°C). Fiber concentration was fixed at 6% dry
mass. After the treatment, the reaction solutions were adjusted to pH 4.0-5.0 using 20%
sodium hydroxide solution. The treated samples were centrifuged, and solid residues
were washed 4-5 times using deionized water. The solid residues were stored in a freezer
of -20 °C. The composition of the acid treated corn stover was 59.31% (dry basis) of

cellulose, 0.3% (dry basis) of xylan, 19.74% (dry basis) of lignin.

2.6.5. Enzymatic Hydrolysis of Pretreated Switchgrass

The enzymatic hydrolysis system contains 2 g alkaline pretreated switchgrass (dry basis),
20 FPU cellulase from T. reesei produced in the lab or Accellulase® from Genencor, and
0.5 mL 2 M sodium citrate buffer, the total weight of reaction solution was brought to 40

g by adding DI water. The reaction solutions were incubated at 50 °C for 48 hrs at 140

rpm.

2.7. Ethanol fermentation

Saccharomyces cerevisiae D5A, obtained from American Type Culture Collection
(ATCC, Manassas, VA), was used in the ethanol production from fermentable sugars.
Initial seeds were cultured for 15 h at 30°C in a 250mL flask on ATCC Medium No. 1245
(10 g/L yeast extract, 20 g/L bacto peptone, and 20 g/L glucose). The seed culture
solution was centrifuged to collect yeast biomass for next step of ethanol fermentation.
The inoculum size was fixed at 10% (v/v) and then the biomass was mixed with an

autoclaved nutrition solution (10 g/L of peptone, 5 g/L of yeast extract, and glucose from

15

hydrolysates) to conduct the fermentation. Samples were taken at the beginning and end

of a 26-28h fermentation process for both glucose and ethanol analysis using HPLC.
2.8. Analysis methods

2.8.1. Fiber Content Analysis

Neutral detergent fiber (NDP), acid detergent fiber (ADP), and acid detergent lignin
(ADL) of samples were analyzed using the Van Soest Fiber Analysis System (Goering
and Van Soest, 1970). NDF, ADP and ADL were used for calculate cellulose,
hemicelluloses and lignin contents. Cellulose and hemicelluloses can be determined by
the different of %ADF - %ADL and %NDP - %ADF, respectively. Li gnin content was

expressed by ADL.
2.8.2. HPLC Analysis

2.8.2.1. Enzyme fermentation sample analysis

The supematants of all the enzyme samples were filtrated through the 0.22 pm filter.
Sugar (cellobiose, glucose, xylose and mannose) analysis was performed by high
performance liquid chromatography (HPLC) on SHIMADZU LC 20AD series equipped
with RI detector using an Aminex HPX-87P column (7.8 X300 nm; 5 pm) at 65 °C. The

mobile phase utilized Millipore pure water at a flow rate of 0.6 mUmin.

2.8.2.2. Enzymatic hydrolysis sample analysis

All of the enzymatic hydrolysis reactions were finished at 48 hours and each reaction
solution was diluted to 100 ml in a volumetric flask. The supematants were filtrated
through the 0.22 pm filter, and then analyzed for cellobiose, glucose, xylose and mannose

concentrations by HPLC.
1 6

2.8.3. Enzyme Activity Analysis

Cellulase activity (measured as filter paper activity), the reaction mixture (4.5 mL)
contained 4 mL of 0.05 M citric acid-sodium citrate buffer Solution (pH4.8), 0.5mL
enzyme solution and a l x 5 cm stripe of Whatman No.1 filter paper. The reaction was
incubated at 50 °C for 60 min, and stopped the reaction by adding 3 mL DNS Solution
and the concentration of the reducing sugar in the reaction mixture was determined by the
dinitrosalicylic acid (DNS) method (Ghose, 1987). One unit of cellulase activity was
defined as the amount of enzyme needed to produce 1 mol of glucose per min at 1 hour
of reaction time and 50 °C of reaction temperature.

Xylanase activity of T richoderma reesei was determined by Mandel’s method (Mandel et
al.). The reaction mixture (2.5 mL) contained 1 ml citrate-NaOH buffer solution (50 mM,
pH4.8), 0.5 mL enzyme solution and lml substrate (it was prepared by dissolving xylan
in citrate buffer (1%, w/v) and removing any insoluble material by centrifuging for 10
min). The reaction was incubated at 50 °C for 30 min, and then the reaction was stopped
by adding 3 ml dinitrosalicylic acid (DNS) reagent. The samples were heated in a boiling
water bath, and then cooled, diluted with 20 ml water, and the absorbance read at 545 nm.
Blank, enzyme and substrate controls were also carried out under the same conditions.
One unit of xylanase activity was defined as the amount of enzyme needed to produce 1
mo] of xylose per minute under 1 hour of reaction time and 50 °C of reaction

temperature.

17

2.9. Statistical analyses

All experiments with at least two replicates were performed by completely random
design (CRD). Analyses of Variance (ANOVA) and Fisher’s least significant difference

(LSD) test were used to analyze the data.

18

3. Enzyme Production

3.1. Morphology evaluation

The influence of spore concentration on the pellets was studied using potato dextrose
broth (Sigma-Aldrich, 24 g/L). Two different levels of inoculum size were set for 109/m1
and 107/m1. Figure 1. shows the pellets after 2 generations of culture, the fungal pellets
fi'om higher inoculum size had a smaller diameter of 1.7-1.9 mm A smaller diameter can

facilitate mass transfer and fiirther enhance the cellulase’s production.

3.2. Cellulase and xylanase productions on basic chemical defined

medium

Cellulase activity was increased following the increase of the culture time, while
xylanase activity was dropped in the first 48 hours, and then increased for the rest of
culture time (Figure 2.). After the first three days, enzyme production for both cellulase
and xylanase reached their peaks. Cellulase activity was 0.9 U/ml, and xylanase activity
was 2 U/ml. In the basic chemical defined medium, 10 g/L glucose was the main carbon
source for strain growth and produce secondary metabolic products, and 10 g/L lactose

was added as an inducer for enzyme production.

3.3. Comparison of basic chemical defined medium and modified

medium

According to Figure 3, cellulase activities on both media were increased within first 3
days. There was a significant improvement on cellulase activity on the modified medium

at the third day". the activity reached 1.5 U/ml which was twice than the activity in basic

19

chemical defined medium (0.7 U/ml). Meanwhile, the xylanase activities on both media
were also increased in first 2 days. The xylanase activity on modified medium reached
the peak value of 1.3 U/ml at 48 hours. After 2 days of culture, the xylanase activities on
the modified medium were leveled off, while the xylanase activities on the basic
chemical defined medium were still increased

The enhancement of cellulase and xylanase activity was largely due to the carbon sources
of cellulose and xylan in the modified medium. Both substrates also played a role to
induce the enzyme production. However, both enzyme activities in the modified medium
were leveled off at the 4th day. It was much earlier than the trend of chemical defined
medium fi'om previous culture (Figure 2). The main reason was that pH of culture on
modified medium was decreased much faster than the chemical defined medium, which
means that T. reesei on modified medium grew better than chemical defined medium.
More metabolic products including various organic acids released into the broth led to a
quicker drop of pH on the modified medium. Thus, pH had to be controlled in order to

further enhance enzyme production on the modified medium.

3.4. Comparison of CaC03 and Tris-maleate buffer solution as the

neutralizer in cellulase and xylanase production

The impact of neutralizers on enzyme production was presented in Figure 4. The data
indicated that both CaC03 and buffer solution had positive impacts on enzyme
production. The figure also elucidated that CaC03 was a better neutralizer than buffer
solution. The maximmn enzyme activities for both enzymes were obtained at the fourth

day.

20

Acidity measurements and enzyme activity tests firrther demonstrated the impacts of pH
on enzyme production. The pH values of the medium with CaC03 as the neutralizer did
not change during the entire cultivation, while the pH values of the medium with buffer

solution started dropping at the 3rd days.

3.5. Effects of different concentration of CaC03 on cellulase and

xylanase production

Different concentrations (0 g/ L, 4 g/L and 10 g/L) of CaC03 have been used to study the
effects of CaC03 on enzyme production. Figure 5. showed that there were no significant
difference on enzyme activities between 4 g/L of CaC03 and 10 g/L of CaC03, while

enzyme activities on the medium without CaC03 were significantly lower than other two

CaC03 concentrations.

Besides maintaining the broth system under a stable pH level, the enhanced formation of
pellets in the fermentation also indicated that CaC03 as a neutralizer not only prevented
pH from dropping, but was also a favorable factor for the fimgal pelletization and
biomass accumulation The calcium ion has been reported as a metal inducer to facilitate
mycelial aggregation during fungal growth, which has been verified by this study. The
medium with CaC03 produced smoother and more homogeneous pellets than those

without it.

3.6. Comparing different agricultural residues with purified cellulose in

cellulose production

Although the relatively high enzyme activity has been achieved using the carbon source
modified medium, the purified glucose and cellulose powder were still not the optimal

21

feedstock for enzyme production at commercial scale. More abundant and less expensive
lignocellulosic feedstocks such as agricultural residues should be compared (Table 5. ,
Table 6.).

Seven different lignocellulosic feedstocks (APEX corn stover, alkaline treated corn
stover, acid treated corn stover, alkaline treated switchgrass, acid treated switchgrass, and
acid treated anaerobically digested fiber, and alkaline treated anaerobically digested fiber)
were compared with the modified medium to evaluate the impacts of them on enzyme
production.

The results of cellulase and xylanase production in 250 m1 flasks all using 15 g/l
lignocellulosic substrates were shown Compared with dilute alkaline treated substrates,
all the dilute acid treated substrates had less enzyme production on both cellulase and
xylanase. Alkaline pretreatment did not remove all of hemicellulose in the sample, which
might be a reason to enhance both cellulase and xylanase production. Among the alkaline
treated samples, APEX corn stover presented the highest activities on both enzymes, so it

was selected as the substrate to produce enzymes for the following study.

3.7. Effects of different concentrations of AFEX corn stover in cellulases

production

A further study of effects of concentration of APEX com stover on enzyme production
was conducted (Figure 12, 13). Cellulase activities were increased for all three
concentrations with the increase of culture time, while xylanase acitivities reached the
peak at 69 hours. Four different concentrations of APEX corn stover were tested, and the
concentration of 15 g/L showed the highest cellulase activity, while the concentrations of

15 g/L and 30 g/L presented the highest xylanase activities. Considering both cellulase

22

and xylanase activities, the APEX corn stover of 15 g/L and cultivation time of three days

were chosen as the optimal condition for enzyme production (Figure 14).

3.8. Comparing cellulases production with the optimal condition on

both chemical defined medium and AFEX corn stover medium

Enzyme production on the medium with 15 g/L of APEX treated corn stover was
compared with the chemical defined medium (Figure 15). The data showed that 20% and
40% increase were achieved by APEX medium on xylanase and cellulase, respectively.
In addition, enzyme productivity showed even larger improvement by AP EX corn stover
(Figure 16). APEX corn stover obtained 100 % enhancement on cellulase productivity
and 79 % enhancement on xylanase fermentation. The increased enzyme activities could
be benefits on the enzymatic hydrolysis of 1i gnocellulosic materials (discussed in Section

4).

23

4. Enzymatic Hydrolysis

4.1. Enzymatic hydrolysis of lignocellulosic materials

4.1.2. Comparing different enzymes and substrates combinations

Nine enzyme cocktails produced from previous fungal fermentation were applied on four
pretreated corn stover and switchgrass samples to elucidate the effects of enzyme
cocktails from different culture medium on the hydrolysis (Figure 18, 19, 20, and 21).
The enzyme cocktail fi'om the culture on the APEX corn stover had the best hydrolysis
performance on all pretreated fibers among all nine cocktails, particularly on APEX
treated corn stover and alkaline treated switchgrass, which the enzyme cocktail had the
cellulose conversion rate of 70% and 60%, respectively.

Furthermore, according to the fiber analysis results (Tables 8. through 10.), alkaline
treated switchgrass has highest cellulose content of 45% among the treated fiber
feedstocks. More cellulose in the sample, more glucose might be produced in the
following enzymatic hydrolysis. Thus, alkaline treated switchgrass was chosen as the

lignocellulosic material to fulfill the study of enzymatic hydrolysis.

4.1.3. Enzymatic hydrolysis of alkaline pretreated switchgrass using Accellerase®

Ten FPU Accellerase®/ g substrate was used to conduct the hydrolysis of 5 % pretreated
switchgrass. The switchgrass was pretreated by dilute alkaline using a completely
randomized design (CRD). Two pretreatment times (1 and 2 hours), two temperatures
(105 and 121°C), and three NaOH concentrations (0.1 %, 0.5 %. 1.0 %) were tested to

compare the effects of pretreatment on enzymatic hydrolysis.

24

The data of reducing sugar (glucose and cellobiose) from twelve treatments (Figure 22,
23, 24 and 25) demonstrated that 0.1% NaOH was too low to break down the matrix
structure, while both pretreatment conditions (105°C and 1 hour; 121°C and 1 hour) at the
elevated alkaline concentrations of 1% reached the highest sugar concentration of 29 g/L.
Longer reaction times had negative impacts on sugar concentrations. In addition, there
were no significant differences on sugar concentrations between two pretreatment
temperatures (105 and 121°C). Thus, alkaline concentrations (1 and 2%) and pretreatment
temperatures (105 and 121°C) were used to conduct the following the experiment of

enzyme comparison.

4.1.4. Comparison of different levels of Accellerase® and cellulase on pretreated
switchgrass

Commercial cellulase and cellulase cocktail produced from APEX corn stover were used
for the experiment. Two different levels of enzyme activities (5 U and 10 U cellulase/ g
fiber (all based on dry matter)) were tested Other experimental conditions were listed in
Table 10.

The data indicated that at the same enzyme activity levels the cellulose cocktails
performed significantly better than commercial Accellerase (Figure 26, 27, 28 and 29). In
addition, relatively high alkaline concentrations and high temperatures showed better
sugar conversion. Enzymatic hydrolysis of pretreated switchgrass from the treatment of
121°C, 1.0 % NaOH and 1 hour generated 40 g/L and 30 g/L of sugars for cellulose

cocktail and commercial cellulase, respectively.

25

High substrate concentrations under the optimal pretreatment conditions was applied to
further study the effects of different enzyme sources on the hydrolysis (Table 11.), the
cellulase cocktail has a better hydrolysis performance than Accellerase. The highest sugar
concentration of 60 g/L was obtained on switchgrass using the cellulase cocktail (Figure

30).

The possible reasons of better performance of cellulase cocktail might be: 1) Effects of
xylanase supplementation: based on the enzyme activity measurements, the activity ratio
(0.65) of xylanase and cellulase in Accellerase® was much lower than that (2.0) from
cellulase cocktail. It has been reported that glucose release is improved by hemicellulose
removal. 2) The synergistic action among other untested enzymes: a recent published
study indicated that there were more than 5 enzymes co-existed in the broth with
cellulase and xylanase. They can synergistically work with cellulases and xylanase, and
facilitate the conversion of cellulose into glucose.

In addition, another major difference between cellulase cocktail and Accellerase was that
cellulase cocktail was production on lignocellulosic materials. The lignocellulosic
materials can also induce the strain to release certain secondary metabolic products,

which led the enzyme to have better hydrolysis performance.
4.2. Ethanol Fermentation

In order to further evaluate the ethanol production yield from diluted alkaline pretreated
switchgrass, an enzymatic hydrolysis at high solid contents (10 % dry basis) followed by
ethanol fermentation was conducted. A yeast strain S. cerevisiae D5A was used to carry
out the ethanol fermentation. A 68% ethanol yield (ethanol yield [%] = ethanol produced

[g]/ (0.51 * 1.1 1*cellulose in sample [g]*100)) was obtained on the hydrolysate from

26

cellulase cocktail treated switchgrass, which was higher than 58% of Accellerase treated

switchgrass (Figure 31).

27

5. Conclusion and Perspectives

This study has concluded that pelletized fungal fermentation on AF EX corn stover
significantly improved the enzyme production and enzyme activity, and consequently
enzymatic hydrolysis on switchgrass demonstrated high sugar conversion rates.

Fungal morphology is a very important factor in fungal fermentation of cellulose
production Small fungal pellets enhanced enzyme production. Various lignocellulosic
materials as carbon sources had different effects on enzyme activities and ratios of
cellulase to xylanase. Cellulase and xylanase activities from the cultures on APEX corn
stover were significantly higher than cultures on other lignocellulosic materials. Cellulase
production from a culture on 15 g/L AF EX corn stover reached the highest activity of
1.08 U/ml at 93 hours, while xylanase also reached the highest activity of 2.52 U/ml at 72
hours.

The experiments of consequently enzymatic hydrolysis using the enzyme cocktail
produced form the pelletized fungal fermentation elucidated that the cocktail from the
APEX corn stover culture had better sugar conversion rates on all 4 pretreated fibers
compared to the enzyme from chemical defined culture. Considering both sugar
concentration and conversion rate, enzymatic hydrolysis on alkaline treated switchgrass
showed better performance than other lignocellulosic materials. A complete randomized
design on alkaline treatment concluded that 121°C, 1 % NaOH and 1 hour reaction time
is the optimal conditions to treat switchgrass.

The future studies should focus on investigating enzyme compositions of enzyme
cocktails from cultures on different lignocellulosic materials, and discovering the

interactions of different enzymes during fermentation and following hydrolysis.

28

Consequently the impacts of various enzymes in the cocktails on hydrolysis can be
concluded, which will lead to a mechanistic understanding of synergistic functions of
enzymes on hydrolysis, and further make it possible to intentionally design enzyme

cocktails with respect to different lignocellulosic materials.

29

 

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