OXIDATION OF SUGARCANE BAGASSE USING A COMBINATION OF

HYPOCHLORITE AND PEROXIDE

A Thesis

Submitted to The Graduate Faculty of the

Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Master of Science

in

The Department of Food Science

by
Yong-Jae Lee
B.Sc., Chonnam National University, 2003
December, 2005
 ACKNOWLEDGMENTS

I would like to thank God. He always protects and helps me. I am able to make this

work with him.

I would like to express my sincere appreciation to my advisor, Dr. Donal F. Day for

his advice, motivation and support throughout this research.

I also would like to thank everyone at the Audubon Sugar Institute especially Dr.

Chang-Ho Chung and Dr. Giovanna De Queiroz for their valuable discussion and help since I

started my work.

I would like to extend my committee members Dr. Godber, J. Samuel and Dr. Janes,

Marlene, E. in the Department of Food Science.

Finally, I would like to dedicate this work to my wife, Yoon-Jung, Jung, our son,

Daniel, my families. Their love and patience have made this work possible.

ii
 TABLE OF CONTENTS

ACKNOWLEDGMENTS.ii

LIST OF TABLES..v

LIST OF FIGURES...vi

LIST OF ABBREVIATIONS..viii

ABSTRACT..ix

INTRODUCTION.1

1. LITERATURE REVIEW..3
1.1. Overview3
1.1.1. Sugarcane Bagasse as Biomass..3
1.1.2. Cellulose.5
1.1.3. Hemicellulose.8
1.1.4. Lignin..9
1.2. Pretreatment Methods for Lignocellulosic Biomass...9
1.2.1. Physical Pretreatment Methods....12
1.2.1.1. Mechanical Pulverization.12
1.2.1.2. Pyrolysis...12
1.2.2. Physico-Chemical Pretreatment14
1.2.2.1. Ammonia Fiber Explosion (AFEX)..14
1.2.2.2. Autohydrolysis (Steam Explosion)...14
1.2.3. Chemical Pretreatment.15
1.2.3.1. Ozonolysis15
1.2.3.2. Acid Treatment.15
1.2.3.3. Alkali Treatment...15
1.2.3.4. Oxidative Delignification.15
1.2.4. Biological Pretreatment16
1.3. Chemical Oxidations.16
1.4. The Reaction of Hydrogen Peroxide and Sodium Hypochlorite...19
1.4.1. Sodium Hypochlorite19
1.4.2. Hydrogen Peroxide...20
1.4.3. Mixing Sodium Hypochlorite and Hydrogen Peroxide (Ox-B solution)..21
1.5. Degradation of Cellulose...21
1.5.1. Acid Hydrolysis22
1.5.2. Enzymatic Hydrolysis...22
1.6. Goal of This Study.26

2. MATERIALS AND METHODS..27

2.1. Preparation of Bagasse..27
2.2. Ox-B Solutions..27
2.3.Treatment of Bagasse with Ox-B...27
2.3.1. Preparation of Ox-B Solutions..27
2.3.2. Effect of pH...27
2.3.3. Effect of Temperature...28

iii
 2.3.4. Effect of Ratio of Solid to Treatment Liquid28
2.4. Time of Treatment.28
2.5. Sequential Treatments...28
2.6. Treatments of Bagasse with Ox-B, Bleach, or Caustic.....29
2.7. Effect of Ratio on Ox-B Activity..29
2.8. Determination of the Weight Loss.....30
2.9. Determination of Bagasse Composition30
2.10. Saccharification of Cellulose and Hemicellulose31
2.11. Sugar Analysis by HPLC.31
2.12. Calculations of Cellulose Digestibility...31
2.13. Microscopic Changes of Bagasse upon Ox-B Treatment32
2.14. Analysis of Total Phenolic Compounds (TPC) after Treatment for Determination
of Lignin Removal.32
2.15. Statistical Analysis..32

3. RESULTS.34
3.1. Compositional Analysis of Sugarcane Bagasse after Ox-B Treatment.34
3.2. Comparison between Ox-B and NaOCl Treatment of Bagasse34
3.2.1. Effect of the Ox-B or NaOCl on Bagasse.34
3.3. Time and pH on Effectiveness of Ox-B Treatments.39
3.3.1. Effect on Time on Ox-B Treatment..39
3.3.2. pH changes during Ox-B Treatment.39
3.4. Effect of Ratios (Hypochlorite : Peroxide) on Ox-B Activity against Bagasse...39
3.5. Effect of Sequential Ox-B Treatments..42
3.6. Microscopic Changes in Bagasse upon Ox-B Treatment.42
3.7. Effect of Caustic Wash.42
3.7.1. pH Effect on Ox-B Treatment Followed by Caustic Wash..47
3.7.2. Temperature Effect on Treatment using Ox-B followed by Caustic47
3.7.3. Effect of Solids Ratio (v/w; total volume/sample weight) on
Treatment with Ox-B followed by Caustic Wash.51
3.8. Determination of Lignin Removal by Analysis of Total Phenolic Compounds51

4. DISCUSSION..54

REFERENCES.59

VITA.65

iv
 LIST OF TABLES

1. Primary production of biomass derived chemicals4

2. Approximate composition of different lignocellulosic materials...6

3. Methods for pretreatment of lignocellulosic biomass..13

4. Energy states of oxygen molecule17

5. Oxidation potential of several oxidants18

v
 LIST OF FIGURES

1. Secondary cell-wall (CW) structure of cellulose, hemicellulose,

and lignin in lignocellulosic materials....7

2. The partial structure of cellulose..10

3. General structure of hemicellulose...10

4. Partial structure of lignin..11

5. The activation states of oxygen18

6. Activation of hydrogen peroxide..23

7. Speculation scheme for the generation of reactive oxygen species

from the sodium hypochlorite (NaOCl) and hydrogen peroxide (H2O2).24

8. A diagram of the reaction of cellulase in cellulose hydrolysis.25

9. A flow diagram of treatment procedures of dry ground bagasse used in this research33

10. Composition of solids obtained from sugarcane bagasse after treatment with Ox-B35

11. Weight loss of sugarcane bagasse treated for 30min with Ox-B or NaOCl...36

12. Removal of Klason lignin by Ox-B or NaOCl with a 30min treatment37

13. Effect of treatment on enzyme hydrolyzability..38

14. Effect of treatment time.....40

15. pH change...40

16. Cellulose digestibility as a function of ratios hypochlorite to peroxide in Ox-B

solutions used to treat bagasse...41

17. Comparison of a single Ox-B treatment with sequential treatments of bagasse43

18. Microscopic observations of structural changes in sugarcane bagasse

with time on treatment with 5.0% Ox-B (50,000 ppm sodium hypochlorite :
5,000 ppm hydrogen peroxide)..44

19. Weight loss of sugarcane bagasse on treatment with Ox-B and NaOCl
for 30min by a 0.6% NaOH caustic wash..45

20. Cellulose digestibility of bagasse treated with NaOCl or Ox-B followed

with a 0.6% NaOH wash46

21. Cellulose digestibility as a function of % chemical (wgt/wgt dry)

vi
 used to treat bagasse...48

22. Weight loss and cellulose digestibility from bagasse after Ox-B and caustic
treatment conducted at different pH values49

23. Weight loss and cellulose digestibility of bagasse after Ox-B and caustic
treatment at different temperatures.50

24. Percent weight loss and cellulose digestibility after Ox-B and caustic
treatment at different solid ratios52

25. Percent lignin removal and cellulose digestibility after bagasse treatments..53

vii
 LIST OF ABBREVIATIONS

Abbreviation Term

Alpha
Beta
C Carbon
Cl2 Chlorine
Cl Chlorine radical
ClO3- Chlorate ion
g Gram
H Hydrogen
h Hour
H2O2 Hydrogen peroxide
HOCl Hypochlorous acid
HOO- Perhydroxyl ions
KJ/mol Kilo Jule per mole
L Liter
min Minute
NaOCl Sodium hypochlorite
O Oxygen
1
O2 Singlet oxygen
3
O2 Triplet oxygen
O2- Superoxide
OCl- Hypochlorite ion
OCl2- Chlorite ion
OH- Hydroxyl ion
OH Hydroxyl radical
Ox-B Mix with hydrogen peroxide and sodium hypochlorite
ROS Reactive oxygen species
TPC Total phenolic compounds
v/w Volume per weight
w Weight
% Percent
1
g+ Higher singlet oxygen state
1
g Lower singlet oxygen state

viii
 ABSTRACT

Sugarcane bagasse is a source of lignocellulosic biomass. It is a potential renewable

energy source for ethanol production. It is naturally cheap, plentiful and has high cellulose

content. The sugarcane bagasse contains 34.5% cellulose, 24% hemicellulose, and 22-25%

lignin.

Reactive Oxygen Species (ROS), singlet oxygen (1O2), superoxide (O2-), hydroxyl

radicals (OH), and hypochlorite ion (OCl-), were found to remove both hemicellulose and

lignin from sugarcane bagasse. Ox-B (Day. 2004, US Patent 6,866,870), a solution of sodium

hypochlorite and hydrogen peroxide, was studied for its effectiveness as a pretreatment for

lignocellulosic biomass. The cellulose structure of the bagasse was easily separated from the

hemicellulose and lignin by filtration after Ox-B treatment. The remaining solids on a wet

basis, were 76.2% digestible by cellulases, after a 20:1 treatment (v/w) with an Ox-B solution

(10,000 ppm sodium hypochlorite : 500 ppm hydrogen peroxide). At a constant pH 8, 38.6%

weight loss and 97.4% cellulose digestibility were observed. Temperature did not affect

weight loss or cellulose digestibility. Above a 2% Ox-B treatment, cellulose digestibility was

100%. Cellulose digestibility increased with time for up to 3 h on treatment with Ox-B.

Sequential treatments improved cellulose digestibility at lower concentrations of Ox-B.

Treatment with Ox-B followed by a caustic wash produced solids that were between 80 and

100% digestible by cellulases.

Our studies indicate that Ox-B is a powerful room temperature chemical oxidant that

increases cellulose digestibility of sugarcane bagasse. Oxidation of bagasse, by sequential

Ox-B treatments for 30 min, at a pH of 8 and room temperature, followed by a caustic wash

may have industrial potential.

ix
 INTRODUCTION

In the 20th century, the world economy has been dominated by technologies that

depend on fossil energy, such as petroleum, coal, or natural gas to produce fuels, chemicals,

materials and power (Paster, 2003. Sun, 2002). However, fossil energy sources are not

infinite. Worldwide crude oil production is forecast to decline before 2010, from the present

25 billion barrels to about 5 billion barrels in 2050 (Campebell and Laherrere, 1998). A

search for other energy sources is advisable.

Biomass is a potential renewable energy source that could replace fossil energy for

transportation (Paster, 2003). Biomass is solar produced organic matter such as wood,

agricultural waste, and other living-cell materials, ie., algae, sewage etc. Biomass is naturally

plentiful and renewable. In the United States, about 500 to 600 million tons of biomass

resources can be produced annually (Paster, 2003).

Lignocellulosic biomass can be used to produce ethanol, an alternative liquid fuel

source. However, the production cost for ethanol from biomass is relatively high with current

technologies, primarily because of low yields, and high cost for cellulose separation and

hydrolysis. Much of the problem is a function of the structure of lignocellulosic biomass (Sun,

2002). Lignocellulosic biomass is composed mainly of cellulose, hemicellulose, and lignin

(Fox, 1987). The cellulose fibers are usually embedded in an amorphous matrix of

hemicellulose and lignin (Klass, 1998). The presence of lignin in the biomass lowers the

biodegradability both of the cellulose and hemicellulose (Pareek, 2000). The ideal

pretreatment would remove only the lignin portion without loss of hemicellulose or cellulose.

An effective pretreatment must improve the availability of sugars, prevent degradation of

carbohydrate, reduce unfavorable by-products, and be low cost (Sun, 2002). Numerous

pretreatment methods including physical, physico-chemical, chemical, and biological

methods have been developed for separation of lignocellulosic to cellulose, hemicellulose,

and lignin.

This research focuses on the use of Ox-B (a combination of hydrogen peroxide and

sodium hypochlorite) for pretreatment of sugarcane bagasse. Bagasse pretreatment using Ox-

B was optimized and the products evaluated for composition and relative cost compared with

other pretreatment technologies.

 1. LITERATURE REVIEW

1.1. Overview

Biomass is a potential renewable energy source. Generally, it is any plant material

produced by photosynthesis. Biomass is abundant in nature. Many types of biomass exist,

such as wood, sugarcane bagasse, corn fibers, rice, wheat straws, and algae (Ct, 1982).

Most biomass is either not utilized or burnt as fuel. Utilizing the biomass as a renewable

energy source can reduce dependence on fossil fuels (Hu, 2002). The major components of

lignocellulosic biomass are cellulose (C6 sugars), hemicellulose (C5 sugars) and lignins

(polyphenols). Glucose produced from cellulose can be used as feedstock for ethanol

production, while C5 sugars, from the hemicellulose, can be used to produce furfural or

ethanol. Lignin can be a potential source of a wide range of phenolic compounds (Klass,

1998). The summary of chemicals obtained from lignocellulosic materials is given in Table 1.

1.1.1. Sugarcane Bagasse as Biomass. Sugarcane (Saccharum officinarum) is a grass that is

harvested for its sucrose content. After extraction of sugar from the sugarcane, the plant

material that remains is termed bagasse. Currently, the bagasse production in the United

States is about 8.6 million tons per year. About 1.4 million tons are available for other uses as

most of the bagasse is burned to produce steam power (USDA, accessed July 2005).

Sugarcane bagasse found at sugar mills contains both relatively easy and hard to degrade

materials. The easily degraded materials appear to be from the leaf matter and the hard to

degrade from the rind (Fox, 1987). Bagasse has several advantages for use in ethanol

production. Most importantly, unlike corn stover, bagasse is collected as part of the sugar

production process, so it does not require a separate harvest. It is also physically ground as

part of the extraction process (Fox, 1987). Furthermore, bagasse is cheap, readily available,

and has high carbon content (Martn, 2002). Its disadvantage is that it already has value as

fuel to the producer, so any product values must exceed that of the fuel value.

Table 1. Primary production of biomass derived chemicals (Methods of dominant processing are chemical (C), fermentation (F), enzymatic (E),
and natural processes (N) (Klass, 1998).

Biomass
C5 sugars C6 sugars Cellulose Lignins Others
C F C E C C N&C

Furfural Ethanol Glycerol Fructose Cellulose acids Phenols, Alginates

Xylitol Acetaldehyde Hydroxymethylfurfural Cellulose esters Vanillin Alkaloids
Ethanol Acetic acid Sorbitol Cellulose nitrates Lignin Glycerides
Acetone Cellulose ethers sulfonates Gutta
Glycerol Cellulose Phenols
n-Butanol xanthogenates Resins
n-Butyric acid Rubber
Amyl alcohols Saponins
Oxalic acid Sterols
Lactic acid Tall oils
Citric acid Tannins
Amino acids Terpenes
Antibiotics Waxes
Vitamins

4
 Lignocellulosic degradation is relatively difficult to deal with. The components exist

in compact structures tied together with strong intermolecular bonds. A summary of

approximate compositions of various lignocellulosic materials proposed as ethanol feedstocks

is given in Table 2. Sugarcane bagasse is composed approximately of 40% cellulose, 24%

hemicellulose, and 25% lignin. In plant cells, including sugarcane plants, a secondary wall,

consisting of three layers (S1, S2 and S3) is surrounded by a thin primary wall. The

secondary wall is surrounded by lignin. The S1 and S3 layers contain mainly amorphous

cellulose and hemicellulose. The S2 layer contains crystalline cellulose. Crystalline regions

are shaped by linear forms hydrogen bonded in a highly ordered manner. However,

amorphous regions also exist in the cellulose. Amorphous cellulose, hemicellulose, and lignin

are present between the layers (S1, S2 and S3) (Fox, 1987). An illustration of the structure of

the cell wall with its component organization is shown in Figure 1.

1.1.2. Cellulose. Cellulose can be used as a feed stock for fuel alcohol production (Bezerra,

2004). Cellulose is a water insoluble polyglycan that composes about half the weight of a

plant (Klass, 1998) Cotton is almost pure cellulose whereas sugarcane bagasse contains only

forty percent cellulose. Cellulose may existent in crystalline or amorphous forms. Amorphous

structures are less ordered than crystalline regions. The crystalline structures are highly

ordered and poorly depolymerized by cellulases.

Chemically, cellulose (C6H10O5)n molecules are linear glucans ranging from 300,000

D to 500,000 D in molecular size (Klass, 1998. Bertran, 1986. Segal, 1971). Figure 2 shows

partial structures of cellulose. Cellulose consists of linear macromolecular chains of glucose,

linked by -1,4-glucosidic bonds between the number one and number four carbon atoms of

the adjacent glucose units. Partial hydrolysis of cellulose produces a range of

oligosaccharides including cellobiose, cellotriose, and cellotetrose (Sjstrm, 1981. Segal,

1971. Feller, 1986).

Table 2. Approximate compositions of different lignocellulosic materials (Saha, 2003. Sun,
2002).
Lignocellulosic Composition (%, dry basis)

Materials Cellulose (%) Hemicellulose (%) Lignin (%)

Corn fiber 15 35 8

Corn cob 45 35 15

Corn stover 40 25 17

Rice straw 35 25 12

Wheat straw 30 50 20

Sugarcane bagasse 40 24 25

Switchgrass 45 30 12

Coastal Bermuda grass 25 35 6

Hardwoods stems 40-55 24-40 18-25

Softwood stems 45-50 25-35 25-35

Grasses 25-40 35-50 10-30

Paper 85-99 0 0-15

 20-22% Lignin
S1

Hemicellulose
25-28%

S2

S3

Cellulose 38-40%

Figure 1. Secondary Cell-Wall(CW) Structure of cellulose, hemicellulose, and lignin in

lignocellulosic materials. At the sugarcane bagasse, the basic composition is 40% cellulose,
24% hemicellulose, and 25% lignin. S1 and S3 layers have more amorphous cellulose and
hemicellulose contents. S2 layer has more crystalline cellulose regions (Bidlack, 1992).

 Intra-molecular hydrogen bonds form between hydroxyl groups on the same

cellulose chains producing the high viscosity, and rigidity of cellulose polymers. The

hydroxyl groups at the ends of each cellulose chain have different chemical properties. The

number one carbon on the end of a cellulose chain contains an aldehyde hydrate group with

reducing activity. On the terminal end of a cellulose chain, the number four carbon is an

alcoholic hydroxyl with non-reducing activity. In reverse orientation, inter-molecular

hydrogen bonding between contiguous cellulose molecules, results in a parallel super-

molecular structures forming the cellulose fibers. Each glucose unit of a cellulose chain has

three hydroxyl groups that interact with others in another chain by secondary valence bonds.

The bond strength is 25KJ/mol, which is almost one hundred times stronger than Van der

Waals forces (about0.15KJ/mol), but less than one-tenth the strength of the O-H covalent

bond (460KJ/mol) (Krssig, 1993). Intra- and inter-molecular hydroxyl group interactions are

the forces that maintain cellulose structures.

1.1.3. Hemicellulose. Hemicellulose is present in plant cell walls and is associated with the

cellulose. It is a complex polysaccharide that is soluble in both alkali and acid solutions. Its

chemical formula is (C5H8O4)n, and in some cases (C6H10O5)n. Hemicellulose is a

heterogeneous polymer, unlike cellulose, and is usually composed of 50 to 200 monomeric

units of a few simple sugars. Xylose, a C5 sugar, is the most abundant component in

hemicellulose. Xylan contains of D-xylose units and is linked from the number one to the

number four carbon of each residue. Arabinose is normally the next most plentiful component

in hemicellulose. Minor components, including mannose, galactose, and uronic acids may

also be present. Some hemicelluloses contain both glucomannans and galactoglucomannans.

The glucomannans contain D-glucose and D-mannose units in a ratio of 30:70. The

galactoglucomannans contain D-galactose, D-glucose, and D-mannose units in a ratio of

2:10:30 (Klass, 1998. Saha, 2003. Paster, 2003). A summary of a general hemicellulose

structure is shown in Figure 3. Hemicellulose utilization is considered difficult because the

branched structure of hemicellulose slows enzymatic hydrolysis.

1.1.4. Lignin. Lignins are a highly branched polymers formed in plant cell walls (Klass, 1998.

Pareek, 2000). Lignin resists the growth of microorganisms and stores more solar energy than

either cellulose or hemicellulose (Hu, 2002). The structure of lignin is complex, disordered,

random, and consists mainly of ether linked aromatic ring structures, which adds elasticity to

the cellulose and hemicellulose matrices (Paster, 2003). In pulp industries, the emphasis is

removal not utilization of lignin. However, lignin is in the spotlight as a useful source for

phenolic compounds for plastics and other materials (Wallis, 1971. Falkehg, 1975. Klass,

1998. Hu, 2002).

Lignin is mainly composed of phenylpropane or C9 units. Three different types of C9

units are present in lignin. These are p-hydroxyphenylpropane, guaiacylpropane and

syringylpropane units (Gratzl, 2000. Freudenberg, 1968. Campbell, 1996). Figure 4 shows a

partial lignin structure. In hard wood, lignin consists mainly of guaiacylpropane and

syringylpropane units with a small amount of p-hydroxyphenylpropane units. Lignin is

composed principally of guaiacylpropane units with traces of p-hydroxyphenylpropane units

in soft wood (Baucher, 1998). In grasses, lignin is composed of both guaiacylpropane and

syringylpropane units. p-Hydroxyphenylpropane units exist as a minor component of lignin

in grasses (Grabber, 2004).

1.2. Pretreatment Methods for Lignocellulosic Biomass

Pretreatments are designed to open the structure of lignocellulosic biomass prior to

enzyme hydrolysis, to allow efficient production of C5 and C6 sugars. Hydrolysis of the

major component, cellulose has received the most attention, as it can be used to produce

ethanol by fermentation. Cellulose exists in nature as a compact and complex matrix with

lignin and hemicellulose (Ct , 1982). The cellulose is highly ordered and crystalline

Figure 2. The partial structure of cellulose.

Xylose Arabinofuranose Mannofuranose Galactose

Figure 3. General structure of hemicellulose (Lee, 1996).

Figure 4. Partial structure of lignin (Lee, 1996).

with amorphous regions. It is surrounded by lignin which acts as a physical barrier (Fox,

1987) and is associated with the hemicellulose. Obviously, reduction of crystallinity of

cellulose and removal of lignin and hemicellulose are important goals for any pretreatment

process (Wu, 1997). According to Tsao (1978), the -1,4-glucosidic bonds in cellulose are

easier to cleave than the -1,4-glucosidic bonds of starch. He makes clear that the main

problem in cellulose hydrolysis of biomass cellulose is due to the secondary and tertiary

structures, not the primary linkage structure.

A number of pretreatment methods have been developed to improve cellulose hydrolysis

from lignocellulose. They include mechanical pulverization, pyrolysis, concentrated acid,

dilute acid, alkali, hydrogen peroxide, autohydrolysis, ammonia fiber explosion (AFEX),

wet-oxidation, lime, CO2 explosion, organic solvent treatment, etc. Each method, in some

way, decreases the size of the biomass and opens its physical structure. A summary of

pretreatment methods is given in Table 3. Each pretreatment method has distinct advantages

and disadvantages (Saha, 2003. Martn, 2002. Klass, 1998. Ramos, 1996).

1.2.1. Physical Pretreatment Methods

1.2.1.1. Mechanical Pulverization. Lignocellulosic biomass can be pulverized by chipping,

grinding, shearing, or milling. The goal of mechanical pulverization is to reduce the particle

sizes of the biomass, as increased surface area leads to improved cellulose hydrolysis. Some

of the crystalline structure of cellulose is also destroyed using these methods, though the

amount varies according to the type of biomass and power applied in milling or grinding. A

vibration ball mill is the most effective mechanical tool for breaking the crystalline structure

of cellulose (Millet, 1976). Mechanical pulverization methods generally are high cost and do

not remove the lignin or hemicellulose.

1.2.1.2. Pyrolysis. Lignocellulosic biomass can be rapidly decomposed to gas, bio-oil, and

char when heated to temperatures above 300oC in the absence of oxygen. Biomass pyrolysis

Table 3. Methods for pretreatment of lignocellulosic biomass (Saha, 2003. Sun, 2002).

Pretreatment method Example

Physical Mechanical pulverization Chipping, grinding, milling, shearing,

extruder

Pyrolysis

Physico-chemical Autohydrolysis Steam pressure, steam explosion,

supercritical carbon dioxide explosion

Ammonia fiber
explosion(AFEX)

Chemical Ozonalysis

Acid hydrolysis Dilute acid(H2SO4, HCl), concentrated

acid(H2SO4, HCl)

Alkali hydrolysis Sodium hydroxide, ammonia, alkaline

hydrogen peroxide
Oxidative delignification
Organosolve process Methanol, ethanol, butanol, phenol

Biological Brown, White, and Soft-rot fungi

is conducted at 400-600 oC. At these temperatures, the biomass structure separates randomly

(Kilzer, 1965. Shafizadeh, 1979). Presence of oxygen, zinc chloride and sodium carbonate

can accelerate the decomposition of cellulose, reducing temperature requirements

(Shafizadeh, 1975. 1979) but producing CO2 + H2O. However, pyrolysis does not allow

recovery of sugars that can be used to make products such as ethanol.

1.2.2. Physical-Chemical Pretreatment

1.2.2.1. Ammonia Fiber Explosion (AFEX). AFEX treatment is similar to the

autohydrolysis process described below. AFEX is usually conducted at 90oC for 30min (Sun,

2002). It is simple and has a short process time. It is effective for the treatment of corn stover.

However, against aspen chips, which contain higher lignin content than sugarcane bagasse,

the AFEX process was less effective (McMillan, 1994). The AFEX process requires efficient

ammonia recovery to be economical due to the high cost ammonia. A possible approach is to

recover the ammonia after the pretreatment by evaporation (Holtzapple, 1992).

1.2.2.2. Autohydrolysis (Steam Explosion). Autohydrolysis is an efficient pretreatment

method for some lignocellulosics. Biomass is heated to 205oC, for 10min and then the

pressure is released rapidly. Hemicellulose and lignin degradation increase the ability to

enzymatically hydrolyze the cellulose (Ramos, 1996. Martn, 2002. Grous, 1986).

Temperature, moisture content, and treatment time all affect steam explosion (Duff, 1996).

Even though autohydrolysis requires less energy than mechanical pulverization and does not

require recycling, unlike AFEX, it may produce aliphatic acids, furaldehydes, and phenolic

side products that are inhibitors of fermentation (Holtzapple, 1989, Martn, 2002. Clark, 1987.

Mackie, 1985). To reduce deleterious by-product formation and increase enzymatic

hydrolysis yield, catalysts like sulfur dioxide or sulfuric acid have been added prior to steam

explosion (Morjanoff, 1987). Autohydrolysis is considered to be one of the most effective of

the pretreatment processes.

1.2.3. Chemical Pretreatment

1.2.3.1. Ozonolysis. Ozone is a powerful oxidizing agent. Lignin can be removed very

effectively using ozone, without by-product formation (Vidal, 1988). Ozonolysis can be

conducted at room temperature and atmospheric pressure, and removes about 60% of the

lignin from wheat straw. However, this process has a high costs and requires large amounts of

ozone.

1.2.3.2. Acid Treatment. Acid treatments normally aim for high yields of sugars from

lignocellulosic biomass (Ct, 1982). Acid usually has a greater effect on the hemicellulose

and lignin than does alkali (Fox, 1987). There are many methods for acid treatments

including use of phosphoric acid (Ramos, 1996), sulfuric acid (Ct, 1982. Camacho, 1996),

hydrochloric acid (Ct, 1982), or peracetic acid (Sakai, 1966).

Reacting biomass with dilute sulfuric acid alters the crystalline nature of the cellulose

structure by expanding the surface area of the biomass, allowing water penetration into the

crystalline structure. Dilute sulfuric acid treatment improves ease of solubilization of biomass

and formation of glucose (Millet, 1976. Tsao, 1978). Concentrated sulfuric acid pretreatment

solubilizes cellulose by breaking down the hydrogen bonds (Camacho, 1996). According to

Lai (1968), peracetic acid can also be use to treat lignocellulosic biomass. Peracetic acid is a

powerful oxidizing agent that removes lignin, in a manner similar to ozone treatment. It can

also cleave the aromatic molecules in lignin.

1.2.3.3. Alkali Treatment. Alkali treatment reduces the lignin and hemicellulose content in

biomass, increases the surface area, allowing penetration of water molecules to the inner

layers, and breaks the bonds between hemicellulose and lignin-carbohydrate (Gratzl, 2000,

Fox, 1987. Tarkow, 1969). Dilute sodium hydroxide is usually used for alkali treatment (Fan,

1987).

1.2.3.4. Oxidative Delignification. Hydrogen peroxide can enhance the hydrolysis of

biomass. Hydrogen peroxide generates hydroxyl radicals and superoxides that attack organic

materials. The primary reactions of hydroxyl radicals and superoxides are described in

Chapter 1.3. Chemical Oxidations. About half of lignin and most hemicellulose in biomass

can be solubilized with hydrogen peroxide (Azzam, 1989).

1.2.4. Biological treatment

Microorganisms, including the brown, white, and soft-rot fungi, attack

lignocellulosic biomass, breaking down both lignin and hemicellulose (Schurz, 1978). White

and soft rot fungi attack cellulose and lignin. On the other hand, brown rot fungi usually

attack only cellulose. The white rot fungi produce powerful lignin degrading enzymes (Fan,

1987). Phanerochaete chrysosporium, a species of white rot fungi produces both lignin

peroxidases and manganese-dependent peroxidases for lignin degradation (Boominathan,

1992. Waldner, 1988). Polyphenol oxidases, laccases, H2O2 producing enzymes and

quinosine-reducing enzymes also degrade lignin (Blanchette, 1991). Biological treatment

requires low energy and normal environmental conditions but the hydrolysis yield is low and

requires long treatment times (Sun, 2002).

1.3. Chemical Oxidations

Chemical oxidations are produced by radicals with unpaired electrons. In most cases,

these radicals have high reactivity and short life. Some radicals are stable enough to be long-

lived. A radical combines with another molecule in order to attain stability, obtaining a

missing electron. Radicals such as singlet oxygen, superoxide, and hydroxyl radicals cause

damage to DNA, proteins, and lipids. They also can react with lignocellulosic biomass

(Christophersen, 1991). In air, oxygen exists in a ground state as triplet oxygen (3O2). Triplet

oxygen is characterized by two unpaired electrons with parallel spins in the outermost orbital.

Triplet oxygen can change to a singlet state when it absorbs enough energy to reverse the spin

of one of its unpaired electrons. The excited states of singlet oxygen (1O2) are in equilibrium

between a first excited state (1g) and a second excited state (1g+). Singlet oxygen (1g) has a

pair of electrons with antiparallel spins in the same orbital, unlike triplet oxygen

(Christophersen, 1991). Its excitation energy is 22 kcal/mol above that of the triplet oxygen.

The higher singlet oxygen state (1g+) derives from the spin pairing of electrons in different

orbitals (Halliwell, 1982). The 1g+ state has a shorter lifetime than 1g state because it is

more reactive. A summary of the different energy states of two singlet oxygen states is given

in Table 4.

Table 4. Energy states of oxygen molecule (Ameta, 1990).

Oxygen molecule states Energy above ground state Possession of highest orbitals

Ground state (3g-)

First excited state (1g) 22 kcal / mol

Second excited state (1g+) 37 kcal / mol

If triplet oxygen accepts a single electron, it is transformed to superoxide (O2-).

Superoxide can react as either an oxidant by accepting electrons or a reductant by donating

electrons. As an oxidant, superoxide can oxidize sulfur, ascorbic acid or NADPH.

Cytochrome C and metal ions can be reduced when it acts as a reductant (Gebicki, 1981).

Sometimes, superoxide can be transformed to singlet oxygen by organic acceptors or metal

cations, and dismutation (Khan, 1991). As a reductant, superoxide can produce hydrogen

peroxide, hydroxyl radical and water. Hydroxyl radical is one of the strongest known

oxidizing agents and reacts with organic molecules at diffusion-limited rates (hydroxyl

radical damage) (Gebicki, 1981). It has strong oxidation potential compared to chlorine and

chlorine dioxide (Table 5). Figure 5 illustrates the activation states of oxygen.

 Singlet oxygen (O-O:)

Superoxide (O-O:) Perhydroxyl radical (O-O:H)

+22 +7.6
-21.7
Triplet oxgen (O-O)

Hydrogen peroxide (H:O-O:H)

-8.8

Hydroxyl radical (H:O) Water (H:O:H)

+

Free energy
Kcal/mol -53.7

Water (H:O:H)

Figure 5. The activation states of oxygen (Gebicki, 1981).

Table 5. Oxidation potential of several oxidants (USperoxide, accessed July 2005).

Oxidant Oxidant Potential (Volt)

Fluorine 3.0

Hydroxyl radical 2.8

Ozone 2.1

Hydrogen peroxide 1.8

Chlorine dioxide 1.5

Chlorine 1.4

1.4. The Reaction of Hydrogen Peroxide and Sodium Hypochlorite

Oxidation increases the positive valance state of a molecule by removal of one or

more electrons from an atom or ion. Several oxidizing agents, including ozone, hydrogen

peroxide, hypochlorite, chlorine, chlorine dioxide, and peracetic acid have been used for the

chemical treatment of biomass (Chapman, 2003).

1.4.1. Sodium Hypochlorite. Sodium hypochlorite is broadly used in both potable water and

waste treatment. It is often referred to as liquid bleach or soda bleach liquor (Casson, 2003).

In the United States, more than 150 tons/day is used (White, 1999). The production of sodium

hypochlorite involves the reaction of chlorine with caustic soda. The reaction is as follows:

2NaOH + Cl2 NaOCl + NaCl + H2O + heat

Caustic soda (NaOH) is used as a stabilizer. The strength of a soda bleach solution is

often shown as trade percent or percent by volume of its available chlorine content.

However, the more precise expression is the true weight percent of sodium hypochlorite. The

relationship between these values follows (White, 1999):

Trade percent (percent by volume) = g/L available Cl2

10

Weight percent available Cl2 = trade percent

Specific gravity of solution

Weight percent sodium hypochlorite = g/L sodium hypochlorite

Specific gravity of solutions 10
Commercially, sodium hypochlorite is available in strengths between 5-15%. Solid

sodium chloride is formed if it is over 15% strength. The stability of sodium hypochlorite is

affected by pH, heat, light, and heavy metal cations. The most stable solutions are 10%

hypochlorite concentration at pH 11, with an iron, copper, and/or nickel content less than

0.5mg/L and must be stored in the dark around 21oC. When the pH drops below 11,

decomposition occurs more rapidly (Casson, 2003. White, 1999). High temperatures also lead

to rapid decomposition of sodium hypochlorite (Gordon, 1997). Hypochlorite ion (OCl-)

forms chlorate ion. The reaction is as follows (Gordon, 1997):

NaOCl + H2O HOCl + Na+(OH-)

HOCl H+ + OCl-
OCl- + OCl- ClO2- + Cl- (Slower step)
OCl- + ClO2- ClO3 + Cl- (Faster step)

Below pH 11 the rate of ClO3- formation from hypochlorite increases, and the pH of

the OCl- solutions decreases over time, caused by a competing acid-producing reaction:

2HOCl + OCl- ClO3- + 2H+ + 2Cl-

To decrease the rate of ClO3- formation, the pH must be maintained above pH 11 (Gordon,

1997).

1.4.2. Hydrogen Peroxide. Hydrogen peroxide is a colorless and odorless liquid that can mix

with soluble organic solvents and water (Jones, 1999). Hydrogen peroxide is used for

sterilization and disinfection in aseptic and packaging processes (Shin, 1994). Hydrogen

peroxide has been used for pretreatment of biomass. As a chemical oxidizing agent, it can

produce oxidative delignification. Hydrogen peroxide can be produced by reduction of

superoxides. It can oxidize olefins, aromatic hydrocarbons, and alkanes. At the point of

application, hydrogen peroxide needs activation. In Figure 6, the numerous oxidants derived

from hydrogen peroxide are shown. Hydrogen peroxide separates into both hydrogen (H+)

and perhydroxyl ions (HOO-) under alkali conditions (Jones, 1999). The perhydroxyl anion

can attack electron deficient substrates such as olefins and aldehydes. Sometimes, it is

converted to a powerful oxidant by blending with electron-deficient acyl compounds or

nitriles (Perez-Benito, 2001). In strong acid environments, hydrogen peroxide is converted to

the equivalent of the hydroxyl cation shown below (Jones, 1999).

H2O2 + H+ H3O2

Hydrogen peroxide can be decomposed by light, metals, and enzymes (Fiorenza, 1997. Nicoll,

1955. Bckvall, 2004).

1.4.3. Mixing Sodium Hypochlorite and Hydrogen Peroxide (Ox-B solution). Singlet

oxygen, superoxide, and hydroxyl radicals are the important products in the reaction between

sodium hypochlorite and hydrogen peroxide. They, in turn, can react with organic molecules

such as carbohydrates, proteins, lipids and nucleic acids changing their chemical structures

and properties (DiGuiseppi, 1981. Gierer, 1996). A stable complex between hypochlorite and

H2O2 forms if they are mixed in the correct concentrations (US Patent 6,866,870).

The generation of singlet oxygen was observed by Khan and Kasha in 1963 while

measuring the red chemiluminescence spectrum from the reaction of hydrogen peroxide and

sodium hypochloride in an aqueous solution (Khan, 1991. Aubry, 1985). Hypochlorite ion

(OCl-) reacted with hydrogen peroxide and produced singlet oxygen. The reaction is as

follows:

H2O2 + OCl- 1O2 + H2O + Cl-

Superoxide and hydroxyl radicals are important intermediates between oxygen and

hydrogen peroxide. Superoxide and hydroxyl radicals can participate in the oxidative

degradation of lignin and carbohydrates (Gierer, 1996). Figure 7 shows the oxygen species

that can be generated from the reaction of sodium hypochlorite with hydrogen peroxide.

Superoxide selectively adds radicals to substrates that cause cleavage of carbon-carbon

chains, opening of aromatic rings, and cutting of aliphatic side chains. On the other hand,

hydroxyl radicals react rapidly and attack substrate molecules directly causing the opening or

cleavage of aromatic and aliphatic structures (Gierer, 1996). Hydroxyl radials are mainly

derived from hypochlorous acid (HOCl).

1.5. Degradation of Cellulose

The degradation of cellulose to glucose is an essential step for production of

renewable energy from biomass. In nature (Garves, 1996), the brown rot basidiomycetes are

able to depolymerize wood cellulosic structure without removing the lignin portions. They

degrade cellulose using the reaction of oxygen species derived from the Fenton reaction

(H2O2 + Fe2+ + H+ +H2O + Fe3+ + OH) and cellulases (Kenneth, 2002). Acid or enzymatic

hydrolysis methods are normally used for hydrolyzing cellulose in industrial processes. Acid

hydrolysis occurs rapidly and is more cost effective than enzymatic hydrolysis. However,

acid decomposes the glucose and produces undesirable degradation by-products that decrease

sugar yields and inhibit ethanol formation. Conversely, enzymatic hydrolysis produces few

by-products (Martn, 2002. Tsao, 1978. Camacho, 1996).

1.5.1. Acid Hydrolysis. Cellulose can be degraded to glucose by acid. The cellulose molecule

is characterized by -1,4-glucosidic linkages between sequential glucose units. There are

three reactive hydroxyl groups in each glucose unit. Acid can attack the -1,4-glucosidic

linkages in cellulose leading to degradation. The reaction proceeds in three steps. First, the

rapid protonation of the glucosidic oxygen atom, second, the transfer of a positive charge to

the number one carbon producing a cyclic carbonium cation and cleavage of the glucosidic

linkage, and finally, the addition of water to the carbonium ion (Kraaig, 1993). Using dilute

acid, cellulose can be degraded to glucose, but yields are low because dilute acid does not

penetrate the crystalline regions of cellulose. Strong acids can reduce the crystalline region

but degrades glucose. Acid hydrolysis is not usually considered to be a desired method for

biomass treatment (Tsao, 1978. Cte, 1982. Klass, 1998).

1.5.2. Enzymatic Hydrolysis. Cellulases are used to produce glucose from cellulose, with

little by-product formation. Cellulases are produced by a wide range of bacteria,

actinomycetes, fungi, etc. Trichoderma genus appears to produce the greatest quantity of

active cellulases. They produce three cellulases, an endo-glucanase, an exo-glucanase, and a

-glucosidase. Endo-glucanase attacks the internal structure of cellulose. It cleaves the -1,4-

glucosidic linkages of cellulose structure in random positions to produce oligomeric chain

fragments. Exo-glucanases attack specific locations on the non-reducing ends in the cellulose

Figure 6. Activation of hydrogen peroxide (Jones, 1999).

 (II)
NaOCl NaClO3+H2 H2O2 OH + OH
2/3NaCl +
1/3NaClO3

(I) (IV) H+
OH + Cl HOCl O2 HOO

H+
1/21O2
+H++ Cl-
(III) H2O2 O2-

OCl- OH
(V)
O2 + Cl-
OCl2 1O + Cl-
2 + H2O2

OCl3

Figure 7. Speculation scheme for the generation of reactive oxygen species from the
sodium hypochlorite (NaOCl) and hydrogen peroxide(H2O2). (I) HClO is decomposed to
hydroxyl radical (OH) and chlorine radical(Cl). (II) H2O2 is decomposed to OH+OH. (III)
Generated superoxide radical(O2-) acts on H2O2 and OH is generated by the Haber-Weiss
reaction (Sima, 1999). (IV) HClO extracts hydrogen ion from H2O2 and then hydroperoxyl
radical (HOO) and O2- are generated. (V) OCl- extracts singlet oxygen(1O2) from H2O2.
(Shiozawa, 2000. Gordon, 1997. White, 1999)

 Cellulose

C1/Cx

Swellen cellulose

C1+Cx

Cellodextrin
Cellobiose

C1+Cx
Cellobiose

-glucosidase
Glucose

Figure 8. A diagram of the reaction of cellulase in cellulose hydrolysis (C1: exo-

glucanases, Cx: endo-glucanases) (Krssig, 1993).

structure. They mainly produce cellobiose or glucose units. -Glucosidase converts cellobiose

to glucose (Krssig, 1993. Klass, 1998). A summary of the reaction of cellulases is showed in

Figure 8.

Trichoderma reesei produces two exo-glucanases, five endo-glucanases, as well as a

xylanase, a -glucosidase, a -xylosidase and a galactomannase which can be used to produce

glucose from cellulose as an enzyme cocktail (Krssig, 1993).

1.6. Goal of This Study

This research focuses on the effects of an oxidative solution (Ox-B) on sugarcane

bagasse and its potential for use as a pretreatment method for ethanol production from lingo-

cellulose.

 2. MATERIALS AND METHODS

2.1. Preparation of Bagasse

Sugarcane bagasse (bagasse) was gathered from a local Louisiana sugar mill and stored

in a freezer until used. The bagasse was dried in an 80oC oven to a constant weight and then

triturated using a commercial coffee grinder. Using a 20-mesh sifter (NO.20, micrometer 850,

Inches 0.0331), the triturated bagasse was sieved. The moisture content of the sieved bagasse

was about 3.0%. This sieved bagasse was used for all experimentation.

2.2. Ox-B Solutions

Ox-B solutions were made by combining sodium hypochlorite and hydrogen peroxide,

according to US patent 6,866,870. Six percent sodium hypochlorite (The Chlorox Co.,

Oakland, CA) and fifty percent hydrogen peroxide (Hach Company, Ames, Iowa) solutions

were used to prepare Ox-B Solutions. Ox-B solutions are defined in w/v % where a gram of

Ox-B (concentration of sodium hypochlorite plus hydrogen peroxide in a 10:1 ratio) is added

per 100ml solution. Fresh Ox-B solutions were prepared prior to each experiment.

2.3. Treatments of Bagasse with Ox-B

2.3.1. Preparation of Ox-B Solutions. Two percent (w/v) Ox-B solutions, 20,000 ppm

sodium hypochlorite : 2,000 ppm hydrogen peroxide (Weight ratio 10:1) were prepared from

six percent sodium hypochlorite and fifty percent hydrogen peroxide stocks. All solutions

were mixed at room temperature into either distilled water or sodium bicarbonate (0.1M)

solution. The sodium hypochlorite was added to prior to the addition of hydrogen peroxide.

2.3.2. Effect of pH. Two and half gram samples of dry ground bagasse were suspended in

100ml of 2% Ox-B (w/v ratio 40:1). The suspension was continuously stirred for 30 min at

room temperature and the pH was maintained at a fixed value (4, 6, 8, 10, or 12) by

automated addition of concentrated HCl or NaOH, as appropriate, using a pH controller (New

Brunswick Scientific Co. New Jersey). The treated bagasse was recovered by filtration

(Whatman #1) and washed with 100ml distilled water. The washed bagasse was then

suspended for one hour in 0.6% NaOH, at 25oC, filtered (Whatman #1) and washed with

100ml distilled water. The weight loss and enzyme digestibility of the treated solids were

determined.

2.3.3. Effect of Temperature. Samples maintained at pH 8 were treated as described above

at 25oC, 50oC, 70oC, or 90oC.

2.3.4. Effect of Ratio of Solid to Treatment Liquid. Two and half gram samples of dry

ground bagasse were treated with 25ml, 50ml, or 100ml of 2% Ox-B solutions. Ratios of Ox-

B to bagasse were 40:1, 20:1, and 10:1. A pH of 8 was maintained automatically by addition

of HCl or NaOH. After 30 min, the liquid portion was removed by filtration and the solids

recovered and washed with 100ml distilled water. The solids were then suspended for 1hr in

0.6% NaOH, at 25oC, filtered and washed with 100ml distilled water and analyzed for weight

loss and enzyme digestibility.

2.4. Time of Treatment

Ox-B solutions at a concentration of 0.1% Ox-B (1,000 ppm sodium hypochlorite : 100

ppm hydrogen peroxide), 0.2% Ox-B (2,000 ppm sodium hypochlorite : 200 ppm hydrogen

peroxide), 0.5% Ox-B (5,000 ppm sodium hypochlorite : 500 ppm hydrogen peorixde), or 1%

Ox-B (10,000 ppm sodium hypochlorite : 1,000 ppm hydrogen peroxide) were used to treat

dry ground bagasse (2.5g) for 0.5, 3, 24, or 100h. As previously described, after treatment,

the solids were recovered, washed with 100ml distilled water and analyzed.

2.5.Sequential Treatments

Dry bagasse (2.5g) was treated with two, 15min, sequential, Ox-B (0.1, 0.2, 0.5, 1%)

treatments, at room temperature, with filtration between treatments. The pH was maintained

at 8 with 0.1M sodium bicarbonate. The solids were removed by filtration through a 0.45m

filter and then washed once with 100ml distilled water. All samples were analyzed for weight

loss and enzymatic digestibility as described below.

2.6. Treatments of Bagasse with Ox-B, Bleach, or Caustic

Two and half gram samples of dry ground bagasse were treated with 100ml of solutions

of 0.2% Ox-B (2,000 ppm sodium hypochlorite : 200 ppm hydrogen peroxide), 0.5% Ox-B

(5,000 ppm sodium hypochlorite : 500 ppm hydrogen peroxide), 1% Ox-B (10,000 ppm

sodium hypochlorite : 1,000 ppm hydrogen peroxide), 2% Ox-B (20,000 ppm sodium

hypochlorite : 2,000 ppm hydrogen peroxide), 3% Ox-B (30,000 ppm sodium hypochlorite :

3,000 ppm hydrogen peroxide), 3.72% Ox-B (37,200 ppm sodium hypochlorite : 3,720 ppm

hydrogen peroxide), 5% Ox-B (50,000 ppm sodium hypochlorite : 5,000 ppm hydrogen

peroxide) or 1%, 2%, 3.72%, or 5% bleach (10,000, 20,000, 37,200, 50,000 ppm NaOCl) for

30 min, at a pH of 8. After 30 min, the solids were removed by filtration and washed with

100ml distilled water. This was followed by treating the residues with 0.6% NaOH for 1h at

25oC. In addition, treatments in the opposite order were conducted, that is 0.6% NaOH

(caustic wash) treatment for 1h at 25oC followed by treatment with Ox-B for 30min. The

treated solids were recovered by filtration and washed with 100ml distilled water prior to

analysis.

2.7. Effect of Ratio on Ox-B Activity

The following ratios of sodium hypochlorite to hydrogen peroxide were used to treat

bagasse; 20:1 (10,000 ppm sodium hypochlorite : 500 ppm hydrogen peroxide), 10:1 (10,000

ppm sodium hypochlorite : 1,000 ppm hydrogen peroxide), 5:1 (10,000 ppm sodium

hypochlorite : 2,000 ppm hydrogen peroxide), 1:1 (10,000 ppm sodium hypochlorite : 10,000

ppm hydrogen peroxide), 1:5 (10,000 ppm sodium hypochlorite : 50,000 ppm hydrogen

peroxide), 1:10 (10,000 ppm sodium hypochlorite : 100,000 ppm hydrogen peroxide), and

1:20 (10,000 ppm sodium hypochlorite : 200,000 ppm hydrogen peroxide). After 30 min of

treatments, samples were filtered (Whatman #1) and washed with 100ml distilled water. The

treated wet bagasse, containing water, was then analyzed for weight loss and enzyme

digestibility. Enzyme digestibility of dry bagasse was conducted after drying in an 80oC oven

to a constant weight.

2.8. Determination of the Weight Loss

Weight loss were determined by weighing bagasse before and after treatment, either by

directly weighing the bagasse after drying in an 80oC oven to a constant weight, or by using a

moisture analyzer (Computrac MAX 1000, Arizona Instrument Corporation, AZ) to correct

for retained moisture.

2.9. Determination of Bagasse Composition

The content of cellulose, hemicellulose and lignin in bagasse and treated samples were

determined using the methods recommended by the National Renewable Energy Laboratory

(NREL, 2005). For each 0.3g of sample, 3ml sulfuric acids (72%) were added and the

mixture swirled at room temperature. After 1h, 50ml distilled water was added and the

sample incubated at 120oC, in a dry oven, for 1h. After cooling to room temperature, samples

were separated into liquid and solid fractions by filtration (Whatman #1). From the solid

fraction, the lignin concentration was determined by measuring weight after drying at 80oC

for 24h. The cellulose and hemicellulose concentrations in the liquid fraction were

determined as follows. The liquid fraction was transferred to a volumetric flask and brought

to 100ml with distilled water. A 10ml sample was taken and transferred to a 250ml beaker

with a volumetric pipette. After mixing, 1.25g of Barium hydroxide was added. The pH was

measured after 5min. It was required to be over pH 11. When stabilized, the pH was then

adjusted to 7.5 with sulfuric acid. The mixture was transferred into a 25ml volumetric flask

and made to 25ml volume with distilled water. The mixture was filtered through a 0.45m

membrane filter, and analyzed by HPLC for glucose, xylose, galactose, arabinose, and

mannose.

2.10. Saccharification of Cellulose and Hemicellulose

Saccharifications were conducted using a crude Trichoderma viride (Cat. No. 9422,

Sigma Co. St. Louis. MO) cellulase preparation. The enzyme activity was measured in Filter

Paper Units (FPU/g solid) according to an NREL procedure (NREL, 2005). Samples were

incubated with enzyme (10 FPU/g of pretreated dry bagasse) at 37oC on a rotary shaker at

200 r.p.m for 72h and then hydrolysis was determined from the amount of simple sugar

released as determined by HPLC.

2.11. Sugar Analysis by HPLC

During the saccharification process, samples were collected at various time (3, 24, 48,

72h). The enzyme reactions were stopped by heating (95oC) for 5min followed by

centrifugation (6,000 r.p.m.) for 5min. The concentrations of cellobiose, glucose, xylose, and

arabinose were determined by HPLC (high performance liquid chromatograph). A Waters

HPLC with an Aminex-HPX-87K Bio-Rad column (Bio-Rad Lab., Hercules, CA) run at 85oC,

at a constant rate of 0.6 ml/min with 0.01M K2HPO4 as mobile phase was used for this

analysis. Xylose, glucose, arabinose and cellobiose were detected by Refractive Index.

Samples were filtered through a 0.45m filter and centrifuged for 5min prior to HPLC

analysis.

2.12. Calculations of Cellulose Digestibility

The digestibility of the cellulose in treated bagasse was calculated from the

concentrations of simple sugars obtained by HPLC. Using values from the gross analysis and

total carbohydrate content, the following formula was used to calculate percent concentration

(NREL, accessed July 2005).

(Glucose) + 1.053 (Cellobiose)

% Yield = 100
1.111f(Bagasse)
Where:
(Glucose) Residual glucose concentration (g/L)
(Cellobiose) Residual cellbiose concentration (g/L)

(Bagasse) Dry bagasse concentration at the beginning of the saccharification(g/L)
f Cellulose fraction in dry bagasse(g/g)
The multiplication factor, 1.053, converts cellobiose to equivalent glucose
F value is calculated from either composition analysis or weight loss.

2.13. Microscopic Changes of Bagasse upon Ox-B Treatment

Using microscopy (1010) (National DC3-163 Digital Microscope NTSC system,

produced by NATIONAL, Texas), structural changes were observed in the bagasse during

Ox-B treatment with 5% Ox-B. (50,000 ppm sodium hypochlorite : 5,000 ppm hydrogen

peroxide) treatment for 3h.

2.14. Analysis of Total Phenolic Compounds (TPC) after Treatment for Determination of
Lignin Removal

The Folin-Ciocalteu method (FC) (Folin and Ciocalteu, 1927) was used to measure

the total phenol in liquid after treatment of bagasse. Dry bagasse (2.5g) was treated with 1%

NaOCl (10,000 ppm sodium hypochlorite), 1% NaOCl followed by a caustic wash (0.6%

NaOH), 1% Ox-B (10,000 ppm sodium hypochlorite : 1,000 ppm hydrogen peroxide), 1%

Ox-B followed by a caustic wash, 0.6% NaOH, 0.6% NaOH followed by 1% Ox-B (10,000

ppm sodium hypochlorite : 1,000 ppm hydrogen peroxide), ratio 20:1, 1% Ox-B (10,000 ppm

sodium hypochlorite : 500 ppm hydrogen peroxide), and ratio 1:10, 1% Ox-B (10,000 ppm

sodium hypochlorite : 100,000 ppm hydrogen peroxide). After treatments, samples were

filtered (0.45m membrane filter) and the liquid collected. To the 2ml of liquid samples,

10ml of FCR (Folin-Ciocalteu Reagent) (Cat. No. vw3340-1, VWR Co, West Chester, PA)

diluted 1:10, and 8ml of 75g/L sodium carbonate were added. Final volume was 20ml. After

2h, absorbance was measured at 765nm using a spectrophotometer (DU 800

spectrophotometer, Beckman Coulter). Vanillin was used as standard.

2.15. Statistical Analysis

Means and standard deviation were used for data comparison.

 1. Dry ground bagasse

2. Treatment with Ox-B or bleach

with stirring

3. Filtration and washing with 100ml distilled water

4. Caustic treatment (0.6% NaOH)

for 1h at room temperature

5. Filtration and washing with 100ml distilled water

Weight loss Enzymatic saccharification Composition analysis

Figure 9. A flow diagram of treatment procedures for dry ground bagasse used in this
research.

 3. RESULTS

3.1. Compositional Analysis of Sugarcane Bagasse after Ox-B Treatment

Ground and sieved (40-mesh, micrometer 425) sugarcane bagasse contained 34.5%

cellulose, 24.5% hemicellulose, and 21.8% lignin. Removal of hemicellulose and lignin was

dependant on Ox-B concentration (Figure 10). Treatment of bagasse with 100 ml volume of

2% Ox-B produced a solid that contained 44.9% cellulose, 22.2% hemicellulose and 11.4%

lignin. Treatment with Ox-B solution of greater than 1.0% concentration removed

hemicellulose as well as lignin. After treatment with 5.0% Ox-B, the residual solids contained

72.7% cellulose, 3.9% hemicellulose, and 7.9% lignin. Ox-B solutions of 1.0% or less

removed lignin but not hemicellulose.

3.2. Comparison between Ox-B and NaOCl Treatment of Bagasse

3.2.1. Effect of the Ox-B or NaOCl on Bagasse. Weight loss, Klason lignin removal and

cellulose digestibility were measured after Ox-B or NaOCl treatments (Figure 11, 12, and 13).

Little difference was seen between Ox-B and NaOCl treatments. Up to concentrations of 2%,

weight loss was below 10%. Over 2%, weight loss increased rapidly to 40-50%. Weight loss

with 1%, 2%, 3.72%, and 5% Ox-B treatment was 5.6%, 9.2%, 43.5%, and 49.7%. At the

same concentrations of NaOCl, weight loss was 6.9%, 8.1%, 38.5%, and 51%. Klason lignin

removal increased with chemical concentration. Klason lignin removal with 1%, 2%, 3.72%,

or 5% Ox-B treatments were 21.7%, 43.1%, 62.8%, and 65.6%. With NaOCl, lignin removal

was 24.4%, 47.1%, 58.4%, and 63.3%. With either 5% Ox-B or NaOCl, about 60% of lignin

was removed. Figure 13 shows cellulose digestibility after Ox-B or NaOCl treatment.

Hydrogen peroxide treatments are shown for comparison. Hydrogen peroxide treatment

increased digestibility of ground bagasse about 10% (14.81% cellulose digestibility).

Cellulose digestibility of bagasse treated with 1%, 2%, 3.72%, and 5% hydrogen peroxide

treatment was 23.5%, 23.5%, 25.5%, and 25.5%. Ox-B and NaOCl

 80

Cellulose
Hemicellulose
60 Lignin
Composition (weight %)

40

20

0
0 1 2 3 4 5

Concentration of Ox-B (g/100ml)

Figure 10. Composition of solids obtained from sugarcane bagasse after treatment with
Ox-B. Composition analysis was conducted using an NREL method (NREL, 2005). Error
bars show the standard error of the mean. The pH of the Ox-B solutions was maintained at 8
with a 0.1M sodium bicarbonate solution. Treatment was for 30 min at room temperature.

 60

Ox-B
50 NaOCl

40
Weight loss (%)

30

20

10

0
0 1 2 3 4 5

% concentration (g/100ml)

Figure 11. Weight loss of sugarcane bagasse treated for 30 min with Ox-B or NaOCl.
Weight loss was determined by direct measurement correcting for moisture using a moisture
analyzer (Computrac MAX 1000, Arizona Instrument Corporation, AZ). Error bars show the
standard error of the mean.

 80

Ox-B
NaOCl
Removal of klason lignin (%)

60

40

20

0
0 1 2 3 4 5

% concentration (g/100ml)
Figure 12. Removal of Klason lignin by Ox-B or NaOCl with a 30 min treatment. Klason
lignin content was determined using the NREL method. Error bars show the standard error of
the mean.

 100

80
Cellulose digestibility (%)

60

NaOCl
Ox-B
40 H2O2

20

0
0 1 2 3 4 5

% concentration (g/100ml)

Figure 13. Effect of treatment on enzyme hydrolyzability. Enzymatic saccharification tests

were conducted with a crude cellulase prepariation from Trichoderma viride on solids
obtained after treatment with Ox-B, NaOCl, or H2O2. During the saccharification, samples
were collected and injected to HPLC to determine sugar release. Error bars show the standard
error of the mean.

treatments both produced 100% digestible cellulose at a 2% concentration. For a 1%

treatment, cellulose digestibility was between 65-75%.

3.3. Time and pH on Effectiveness of Ox-B Treatments

3.3.1. Effect of Time on Ox-B Treatment. Enzymatic glucose production from pretreated

bagasse plateaued after the bagasse was exposed to Ox-B for 3h. After 15h of treatment,

cellulose digestibilities for 0.1%, 0.2%, 0.5%, and 1.0% Ox-B treatments were 36.2%, 55.7%,

62%, and 100%. The results of exposure time to pretreatments on cellulose digestibility are

shown in Figure 14.

3.3.2. pH changes during Ox-B Treatment. Changes in pH during Ox-B treatment are

shown in Figure 15. The initial pH for each sample was about 12.3. Commercial NaOCl

(bleach) is kept above pH 11 for stability (White, 1999). For the lower concentrations of Ox-

B the pH dropped rapidly during the first 30min and then stabilized. For 5% Ox-B treatment,

pH continued to drop for 5h.

3.4. Effect of Ratios (Hypochlorite : Peroxide) on Ox-B Activity against Bagasse

Digestibility of the cellulose in bagasse after treatment with solutions containing

different ratios of hypochlorite to peroxide is shown in Figure 16. Treatments on both oven

dried and un dried (wet) bagasse with ratios of 20:1 (10,000 ppm sodium hypochlorite : 500

ppm hydrogen peroxide), 10:1 (10,000 ppm sodium hypochlorite : 1,000 ppm hydrogen

peroxide), 5:1 (10,000 ppm sodium hypochlorite : 2,000 ppm hydrogen peroxide), 1:1

(10,000 ppm sodium hypochlorite : 10,000 ppm hydrogen peroxide), 1:5 (10,000 ppm sodium

hypochlorite : 50,000 ppm hydrogen peroxide), 1:10 (10,000 ppm sodium hypochlorite :

100,000 ppm hydrogen peroxide), and 1:20 (10,000 ppm sodium hypochlorite : 200,000 ppm

hydrogen peroxide) produced cellulose digestibilities of 76.2%, 66.2%, 51.4%, 27.7%, 26.8%,

26.1, and 24.6% for wet bagasse and 61.7%, 51.9%, 35.7%, 10.5%, 15.8, 17.8, and 16.6% for

dry bagasse. The cellulose digestibility of bagasse treated with equivalent concentrations of

 100
Control
0.1% Ox-B
80 0.2% Ox-B
0.5% Ox-B
Cellulose digestibility (%)

1.0% Ox-B

60

40

20

0
0 2 4 6 8 10 12 14

Time (h)

Figure 14. Effect treatment time. Bagasse was treated for different times one a range of
concentrations of Ox-B, with the pH maintained at 8. Error bars show the standard error of
the mean. The control was treatment with distilled water.

14

0 .5 % O x -B
12 2 .0 % O x -B
5 .0 % O x -B

10
pH

2
0 2 4 6 8 10 12 14

T im e (h )

Figure 15. pH change. pH was monitored using a pH meter. Treatments were conducted at
25oC and at a ratio of 40:1(2.5g of bagasse/100ml of Ox-B solution). Error bars show the
standard error of the mean.

 100

80 Wet basis
Dry basis
Cellulose digestibility (%)

60

40

20

0
A B C D E F G H I

Ratio

Figure 16. Cellulose digestibility as a function of ratios hypochlorite to peroxide in Ox-

B solutions used to treat bagasse. Enzymatic saccharification was conducted using a crude
cellulose enzyme from Trichoderma viride after treatment with different ratios of Ox-B
solution. Dry basis samples were dried in 80oC dry oven over night to a constant weight after
treatment. The ratios were (A) treatment only sodium hypochlorite, (B) 20:1 (10,000 ppm
sodium hypochlorite : 500 ppm hydrogen peroxide), (C) 10:1 (10,000 ppm sodium
hypochlorite : 1,000 ppm hydrogen peroxide), (D) 5:1 (10,000 ppm sodium hypochlorite :
2,000 ppm hydrogen peroxide), (E) 1:1(10,000 ppm sodium hypochlorite : 10,000 ppm
hydrogen peroxide), (F) 1:5 (10,000 ppm sodium hypochlorite : 50,000 ppm hydrogen
peroxide), (G) 1:10 (10,000 ppm sodium hypochlorite : 100,000 ppm hydrogen peroxide),
(H) 1:20 (10,000 ppm sodium hypochlorite : 200,000 ppm hydrogen peroxide), and (I)
treatment only hydrogen peroxide. The total concentration was 1% (wgt/solution). Error bars
show the standard error of the mean.

sodium hypochlorite or hydrogen peroxide treated were 73.1% and 23.5% for wet bagasse

and 58.7% and 15.5% for dry bagasse. As hydrogen peroxide concentration increased,

cellulose digestibility decreased. Overall, wet bagasse was more easily degraded than dried

bagasse.

3.5. Effect of Sequential Ox-B Treatments

The results of individual or sequential treatments of bagasse with Ox-B are shown in

Figure 17. Sequential treatments with Ox-B improved cellulose digestibility two or three fold

when Ox-B concentrations were below 0.5% and by 40% for higher concentrations. Cellulose

digestibility of sequentially treated bagasse with 0.1%, 0.2%, 0.5%, or 1.0% Ox-B was 43%,

63.5%, 70.2%, and 100%. A single treatment with 0.1%, 0.2%, 0.5%, or 1.0% Ox-B

concentrations produced cellulose digestibilities of 20.8%, 22.4%, 40.8%, and 66.2%.

3.6. Microscopic Changes in Bagasse upon Ox-B Treatment

Structural changes were visible in bagasse during the course of Ox-B treatment. The

results are shown in Figure 18. Initially, a compact fiber like structure existed. During the

course of treatment, there was visible opening of the structure and water penetration. In

appearance the samples become amorphous. Individual fibers separated from the gross

structure after 30minutes exposure to Ox-B.

3.7. Effect of Caustic Wash

Weight loss and cellulose digestibility of bagasse were determined on bagasse

samples after Ox-B or NaOCl treatment followed by a 0.6% NaOH caustic wash, or a 0.6%

NaOH caustic wash followed by an Ox-B treatment (Figures 19 and 20). The weight losses

were similar for Ox-B and NaOCl treatments that were followed by caustic washes, however,

Ox-B treatment differentiated from NaOCl treatment when cellulose digestibility was

examined. In the opposite treatment order, caustic followed by Ox-B treatment gave weight

losses and cellulose digestibilities that were lower than other treatments if the Ox-B

 100

Sequential treatment
Single treatment
80
Cellulose digestibility (%)

60

40

20

0
0.0% 0.1% 0.2% 0.5% 1.0%

Concentration of Ox-B (g/100ml)

Figure 17. Comparison of a single Ox-B treatment with sequential treatments of bagasse.
The time for a single treatment was 30min. Sequential treatments were for 15 min for each
segment with intermediate filtration. Total treatment time was the same. Error bars show the
standard error of the mean.

 A B

C D

E F

Figure 18. Microscopic observations of structural changes in sugarcane bagasse with

time on treatment with 5% Ox-B (50,000 ppm sodium hypochlorite : 5,000 ppm
hydrogen peorixde). A: untreated sugarcane bagasse. B: treated for 5 min. C: treated for 1 h
30 min. D: treated for 2 h 30 min. E: treated for 3 h. F: treated for 30 min with stirring.

 80

60
Weight loss (%)

40

20
NaOCl+NaOH
Ox-B+NaOH
NaOH+Ox-B

0
0 1 2 3 4 5

Concentration (%)

Figure 19. Weight loss of sugarcane bagasse on treatment with Ox-B and NaOCl for
30min followed by a 0.6% NaOH caustic wash. Weight loss was measured directly and
moisture constantly using a moisture analyzer. Error bars show the standard error of the mean.

 100

80
Cellulose digestibility (%)

60

40

NaOCl+NaOH
20 Ox-B+NaOH
NaOH+Ox-B

0
0 1 2 3 4 5

Concentration (%)

Figure 20. Cellulose digestibility of bagasse treated with NaOCl or Ox-B followed with a
0.6% NaOH wash. Enzymatic saccharification was conducted using a crude cellulose
enzyme from Trichoderma viride after treatment with Ox-B+NaOH and NaOCl+NaOH.
During the saccharification, samples were collected and injected into HPLC for measuring
the cellulose digestibility. Error bars show the standard error of the mean.

concentration was above 1%. Weight losses for 0.5%, 1%, 2%, 3.72%, and 5% Ox-B

treatment followed by caustic washes were 23.2%, 32.8%, 42.5%, 52.8%, and 58.8% and for

NaOCl treatments followed by caustic washes they were 18.3%, 29.6%, 45.2%, 55.4%, and

71.3%. Weight losses for identical caustic concentrations followed by Ox-B treatment were

22%, 27.3%, 26.5%, 31.8%, and 34.4%. A weight loss over 70% indicated that some

cellulose may have been solubilized, as bagasse contains only 34.5% cellulose. Cellulose

digestibilities for 0.5%, 1%, 2%, 3.72%, and 5% Ox-B treatments followed by caustic washes

were 76.3%, 91.5%, 99.5%, 99.6%, and 94.7%; NaOCl treatment followed by caustic washes

produced digestibilities of 41.9%, 73%, 87%, 83.6%, and 72.2%, and with NaOH treatments

followed by Ox-B they were 40.6%, 62%, 75.7%, 74%, and 68.4%. At 2% concentration

treatment, cellulose digestibility for Ox-B followed by NaOH was 99.5% but NaOCl

followed by NaOH treatment was only 87%. Lower concentrations of Ox-B than NaOCl were

required to produce equivalent cellulose digestibility. There was more than a 30% increase in

cellulose digestibility with a 0.5% concentration treatment when Ox-B was used (Figure 21).

At a 1% concentration, Ox-B followed by NaOH treatment produced 91.5% digestability,

NaOCl followed by NaOH treatment only gave 73%, and NaOH followed by Ox-B treatment

gave 62%.

3.7.1. pH Effect on Ox-B Treatment Followed by Caustic Wash. Treatments were

conducted at different pH values (4, 6, 8, 10, and 12). The results are summarized in Figure

22. Weight loss and cellulose digestibility were optimum between pH 6 and 8. At a pH of 6,

weight loss and cellulose digestibility were 37.5% and 97% respectively and at pH 8, it was

38.6% and 97.4%. A pH of 10 to 12 produced lower weight losses and lower cellulose

digestibilities. At pH 10 to 12, weight losses were 26.1% and 25.6%, and cellulose

digestibilities were 78.2% and 86%. In general, weight loss and cellulose digestibility were

greater at acid or neutral pHs than at alkali pH.

 100

80
Cellulose digestibility (%)

60

40

NaOCl+NaOH
Ox-B+NaOH
20 NaOCl
Ox-B
NaOH+Ox-B

0
0 1 2

Concentration (%)

Figure 21. Cellulose digestibility as a function of % chemical (wgt/wgt dry) used to treat
bagasse. Enzymatic saccharification was conducted by a crude cellulose enzyme from
Trichoderma viride after treatment with Ox-B, NaOCl, Ox-B+NaOH and NaOCl+NaOH.
During the saccharification, samples were collected and injected into HPLC for measuring
the cellulose digestibility. Error bars show the standard error of the mean.

 100

80

60
Percent (%)

40

20

0
4 6 8 10 12

pH weight loss
cellulose digestibility

Figure 22. Weight loss and cellulose digestibility from bagasse after Ox-B and caustic
treatment conducted at different pH values. The pH of Ox-B solutions, sodium
hypochlorite and hydrogen peroxide solutions, were adjusted from 4 to 12 with concentrated
HCl or NaOH. Caustic washes were conducted after Ox-B treatments. 2% Ox-B solutions
(20,000 ppm sodium hypochlorite : 2,000 ppm hydrogen peroxide; ratio 10:1) were tested at a
ratio of 40:1(gram of bagasse/volume). Error bars show the standard error of the mean.

 100

80

60
Percent (%)

40

20

0
25 50 70 90

Temperature (oC) weight loss

cellulose digestibility

Figure 23. Weight loss and cellulose digestibility of bagasse after Ox-B and caustic
treatment at different temperatures. The pH of Ox-B solutions, sodium hypochlorite and
hydrogen peroxide solutions, were adjusted to 8 with concentrated HCl or NaOH.
Temperatures were tested across a range from 25oC to 90oC using a 2% Ox-B treatment for
30min. Caustic washes were conducted after Ox-B treatment. 2% Ox-B solutions (20,000
ppm sodium hypochlorite : 2,000 ppm hydrogen peroxide; ratio 10:1) were used tested at a
ratio of 40:1(volume/gram of bagasse). Error bars show the standard error of the mean.

3.7.2. Temperature Effect on Treatment using Ox-B followed by Caustic. Treatments

were conducted at different temperatures (25, 50, 70, and 90oC). The results for 2% Ox-B

treatments followed by caustic washes are shown in Figure 23. Weight loss and cellulose

digestibility improvements appear to be temperature independent. At room temperature

(25oC), weight loss was 45.9% and cellulose digestibility was 98%.

3.7.3. Effect of Solids Ratio (v/w; total volume/sample weight) on Treatment with Ox-B

followed by Caustic Wash. The results of different solid loadings on treatment effectiveness

are shown in Figure 24. A ratio of 40:1 produced the largest weight loss and greatest cellulose

digestibility. Cellulose digestibility at a ratio of 20:1 was 97%, almost the same as seen with

the 40:1 ratio, but the weight loss was 33.2%. This meant that after a 30% weight loss it was

possible to achieve 100% cellulose digestibility. Normally, the sum of the cellulose and

hemicellulose content in sugarcane bagasse is 66% (Saha, 2003. Sun, 2002). Lignin and other

components in bagasse are considered to be completely removed after a 30-40% weight loss.

3.8. Determination of Lignin Removal by Analysis of Total Phenolic Compounds

Lignin removals as measured by the Folin Cocialteau (FC) method are shown in

Figure 25. Lignin removal on treatments with 1% NaOCl (10,000 ppm sodium hypochlorite),

1% NaOCl followed by a caustic wash (0.6% NaOH), 1% Ox-B (10,000 ppm sodium

hypochlorite : 1,000 ppm hydrogen peroxide), 1% Ox-B followed by a caustic wash, 0.6%

NaOH, 0.6% NaOH followed by 1% Ox-B (10,000 ppm sodium hypochlorite : 1,000 ppm

hydrogen peroxide), ratio 20:1, 1% Ox-B (10,000 ppm sodium hypochlorite : 500 ppm

hydrogen peroxide), and ratio 1:10, 1% Ox-B (10,000 ppm sodium hypochlorite : 100,000

ppm hydrogen peroxide) were 0.33%, 6.23%, 0.33%, 7.04%, 8.79%, 0.26%, 0.38%, 0.23%.

Cellulose digestibility for same treatments were 73.1%, 73%, 66.2%, 91.5%, 41.4%, 62%,

76.2%, 26.1%.

 100

80

60
Percent (%)

40

20

0
10/1 20/1 40/1

Ratio (v/w) weight loss

cellulose digistibility

Figure 24. Percent weight loss and cellulose digestibility after Ox-B and caustic
treatment at different solid ratios. The pHs of Ox-B solutions, sodium hypochlorite and
hydrogen peroxide solutions, were adjusted to 8 with concentrated HCl or NaOH using an
automatic pH controller. Pretreatment was conducted at room temperature using 2% Ox-B
treatment for 30 min.Caustic wash was conducted after Ox-B treatment for 1h. Cellulose
digestibility was determined after enzymatic saccharification. Error bars show the standard
error of the mean.

 100
Lignin Removal X10
Cellulose digestibility
80

60
Percent (%)

40

20

0
A B C D E F G H

Figure 25. Percent lignin removal and cellulose digestibility after bagasse treatments. To
determine of lignin removal by analysis of total phenolic compounds, Folin-Chicalteu method
was used. A : 1% NaOCl (10,000 ppm sodium hypochlorite), B : 1% NaOCl followed by
caustic wash, C : 1% Ox-B (10,00 ppm sodium hypochlorite : 1,000 ppm hydrogen peroxide),
D: 1% Ox-B followed by caustic wash, E : 0.6% NaOH, F : 0.6% NaOH followed by 1% Ox-
B, G : ratio 20:1, 1% Ox-B (10,000 ppm sodium hypochlorite : 500 ppm hydrogen peroxide),
and H : ratio 1:10, 1% Ox-B (10,000 ppm sodium hypochlorite : 100,000 ppm hydrogen
peroxide). Error bars show the standard error of the mean.

 4. DISCUSSION

Ox-B is a highly oxidative solution formed by mixing sodium hypochlorite and

hydrogen peroxide. When it was used to treat lignocellulosic biomass it separated the lignin

from the cellulose and hemicellulose. When used with no other preparation it produced

affects similar to hypochlorite, yielding solids similar to that produced by bleach pulping.

Sugarcane bagasse contains 34.5% cellulose, 24% hemicellulose, and 22-25% lignin

(Saha, 2003, Sun, 2002). Composition of bagasse solids changed on Ox-B treatment, the

percentage of cellulose increased, and that of hemicellulose and lignin decreased. Ox-B

selectively removed lignin at a low concentration and both hemicellulose and lignin at higher

concentrations. As might be expected, this made the cellulose more available for hydrolysis.

The effectiveness of Ox-B was concentration dependant, treatment with 5% concentration

solution of Ox-B (50,000 ppm sodium hypochlorite : 5,000 ppm hydrogen peroxide),

increased the percent cellulose in the pulp to 72.7 and hemicellulose and lignin decreased to

4% and 8%, respectively. Treatment with a 2% concentration of Ox-B (20,000 ppm sodium

hypochlorite : 2,000 ppm hydrogen peroxide) increased the cellulose to 45% of the solids,

and hemicellulose and lignin were 23% and 11%. Lignin, but not hemicellulose, was

removed by 1.0% Ox-B. At Ox-B concentrations above 1.0%, hemicellulose was also

removed, probably because the Ox-B solution oxidized the hemicellulose structure. Wet

oxidation has been considered to be an efficient method for opening up the crystalline

structure of cellulose, solubilizing the hemicellulose fractions, and degrading lignin to CO2,

H2O, and carboxylic acids (McGinnis, 1983). According to Gould (1984), hydrogen peroxide

removes about 50% of the lignin present in wheat straw and yields a cellulose-rich insoluble

residue that can be converted from cellulose to glucose. Hypochlorite attacks the lignin

network in the presence of cellulose (David, 1985).

Reactive Oxygen Species (ROS), such as singlet oxygen (1O2), superoxide (O2-)

hydroxyl radicals (OH), and hypochlorite ion (OCl-), are generated by Ox-B and they will

react with bagasse. Singlet oxygen can be produced from the chemical reaction between

hypochlorite and peroxide. Ox-B releases singlet oxygen when in the presence of organic

matter (Day, D.F, US patent 6866870). The chemical structure of the complex that composes

Ox-B has not been determined, but it has been established that its stability is reduced by heat,

acid, UV exposure and the presence of organic matter (White, 1999). Superoxide (O2-) and

hydroxyl radicals (OH) produced by Ox-B are probably involved in the modification of

bagasse. These radicals can participate in the oxidative degradation of both lignin and

carbohydrates. Hydroxyl radicals react rapidly, attacking aromatic and aliphatic molecules of

lignocellulosic biomass causing cleavage and opening structures (Gierer, 1996). Hydroxyl

radicals can be derived from hypochlorous acid (HOCl) (White, 1999). It is one of the

strongest oxidizing agents known (Gebicki, 1981). Hypochlorous acid (HOCl) is known to be

a more effective disinfectant than other chlorine compounds including hypochlorite ion (OCl-

), chlorite ion (OCl-2), and chlorate ion (ClO-3) (White, 1999). Ox-B probably produces not

only singlet oxygen but also hypochlorous acid on degradation. No significant differences

were seen between Ox-B and NaOCl treatment of bagasse based on weight loss and cellulose

digestibility. At 2% chemical concentration, cellulose digestibility reached 100%. Treatment

with only hydrogen peroxide produced low cellulose digestibility, about 20%. The greatest

increase in cellulose digestibility was seen after Ox-B treatment was followed by a caustic

wash. Obviously Ox-B had an action different than hypochlorite. It might be expected that

Ox-B altered the structure of cellulose making it spongy and more swollen than did

hypochlorite. This would lead to expanded surface area and greater penetration of water

molecules into the crystalline structure of the bagasse. Chemical concentrations above 2%

reduced the cellulose digestibility to enzyme attack. The high concentration could cause

structural collapse of the bagasse reducing enzyme accessibly to the cellulose. Reversing the

order of treatment, 0.6% NaOH treatment followed by Ox-B, caused both weight loss and

cellulose digestibility to be lower than with either Ox-B or NaOCl, above a 1% concentration.

Treatment order was important both for maximizing weight loss and enzyme digestibility.

Sodium hypochlorite is stable at pH 11 with iron, copper, and nickel content, stored

in the dark at a 21.1oC. If the pH drops, the stability of sodium hypochlorite decreases (White,

1999). At a pH 9 and at 25oC, 97% of the titratable available chlorine in NaOCl will consist

of the OCl- ion and at a pH 7 and 25oC, 77.5% will consist of HOCl. At pH 8 and 25oC, the

conditions used in this study, HOCl is about 26% (White, 1999). When the NaOCl contacts

organic molecules, the pH drops quickly, because NaOCl decomposes to hypochlorite ion

(OCl-) (Casson, 2003. White, 1999). However, pH only drops about 0.5 units over 30 min

when buffered (0.1M sodium bicarbonate). After one hour, pH increased to the starting pH of

8. It may indicate an increase in the HOCl during treatment for 30 min and then a loss of

HOCl. Ox-B may have more latent power to make hydroxyl radicals than hypochlorite ion

because hydroxyl radicals can be produced by HOCl.

A 20:1 ratio of sodium hypochlorite to hydrogen peroxide (10,000 ppm sodium

hypochlorite : 500 ppm hydrogen peroxide) was most effective in producing digestable ligno-

cellulose on wet enzymatic saccharification. Probably more ROS was produced at this ratio.

Wet bagasse was more readily degraded than bagasse that had been dried. It is possible that

drying collapsed the structures and reduced enzyme penetration.

The effectiveness of Ox-B was not pH dependant, working well over the 6-8 range.

At a constant pH 8, a 38.6% weight loss and 97.4% cellulose digestibility were observed for

bagasse treatment, compared to 26.1% weight loss and 78.2% cellulose digestibility at a pH

of 10. According to David (1985), weight loss, lignin degradation, and enzymatic hydrolysis

yield of Eucalyptus wood treated with NaClO-HClO were at maximum at pH 8. At alkali pH,

especially 11, sodium hypochlorite is most stable. However, when the pH decreases below 11,

there is a loss in stability but a gain in activity by increased concentrations of hypochlorite

(OCl-) and chlorate ion (ClO3-) (Gordon, 1997. White, 1999). The effect of pH on Ox-B

solution is probably due to the pH effect on bleach.

Temperature (25-90oC) did not affect the bagasse treatment. In Goulds research

(1984), at both 25 oC and 60 oC, cellulose digestibility of lignocellulosic crop residues treated

with alkaline peroxide were not significantly different. However, at temperatures over 200 oC,

increasing the temperature increased the glucose yield from biomass (Kim, 2001).

The effectiveness of Ox-B depends on the solids loading of the solution. At the 40:1

ratio (100ml solution : 2.5g of bagasse), weight loss was 45.8% and cellulose digestibility

was 98.4%. This value was higher than for 20:1 or 10:1 loadings. Sufficient chemical is

needed to complete the lignin removal for effective pretreatment.

The cellulose digestibility increased with treatment time up to 3h. According to

Chesney (1991), oxidation of ascorbic acid by HOCl at pH 7.2 occurs more rapidly than

oxidation by H2O2 under the same condition. It indicates that hypochlorite reaction occur

rapidly while H2O2 reactions occur slowly.

Sequential treatments were more effective in improving cellulose digestibility than

single treatments. The results of sequential treatment for 30 min were almost the same as a

single treatment for 3h. It might be expected that initial treatment of Ox-B removes lignin and

weakens structural bonds in the bagasse. Filtration keeps the lignin from reprecipitating on

the cellulose. The second treatment with Ox-B probably removes some hemicellulose and the

lignin not removed initially.

Total Phenolic Compounds (TPC) in the extraction liquids were high after treatments

with 0.6% NaOH. For either Ox-B or NaOCl treated bagasse, TPC was low in extracted

liquids. It indicates that both Ox-B and NaOCl removes lignin less effectively than NaOH,

such that TPC could not be detected by the FC method after Ox-B or NaOCl treatment.

However, according to the compositional analysis, Ox-B or NaOCl treatment was more

effective than NaOH. At 1% Ox-B, 20:1 ratio (10,000 ppm sodium hypochlorite : 500 ppm

hydrogen peroxide), cellulose digestibility by analysis of HPLC and TPC by the FC method

were highest.

The Ox-B method offers several advantages compared to other pre-treatment

methods. First, it can be conducted at the room temperature and atmospheric pressure. There

is no energy input required. It is not necessary to heat to high temperatures, unlike steam

explosion or AFEX treatments. Steam explosion requires heating to 205oC (Martn, 2002) and

AFEX is usually conducted at 90oC (Sun, 2002). Ox-B treatment is a rapid process. A 30 min

treatment was sufficient to achieve maximum cellulose digestibility using sequential

treatments. Both acid and alkali treatments require a minimum of two hours at room

temperature. At high temperatures, these treatment times can be reduced (Fox, 1987). High

cellulose digestibility can be achieved with Ox-B treatment. Above a 2% individual Ox-B

treatment, cellulose digestibility reached 100%. Ox-B treatment has potential uses for

producing by-products such as bio-oil, paper, plastic, detergents, etc (Godshall, 2005). Finally,

it can be broadly used on other biomass such as corn, soy, wheat, cotton, rice, wood, etc.

However, Ox-B treatment has a price disadvantages, sodium hypochloride and hydrogen

peroxide costs are high relative to the sale price of ethanol.

This study has shown the Ox-B treatment produced a high digestiable bagasse.

However, the chemical cost must decrease to justify its use for alcohol production.

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 VITA

Yong-Jae Lee was born in Gwang-Ju, Republic of Korea, on May 28, 1977. After

graduating from Mun-Sung High School, Gwang-Ju, in 1996, he entered Chonnam National

University from 1996 to 2003 where he obtained a Bachelor of Science degree in food

science and technology in 2003. During school he served the army for 26 months and

attended to study about histamine in Auburn University in 2001. He attended Louisiana State

University in Baton Rouge, Louisiana, in January 2004. In December of 2005, he will receive

the degree of Master of Science in food science.