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APRIL 2011
ACCEPTANCE SHEET
The thesis attached hereto, entitled Parametric Study on Chemical and Enzymatic
Hydrolysis of Alginate from Sargassum cristaefolium C.A.Agardh (Phaeophyta)
for Bioethanol Production, prepared and submitted by Michael Angelo M. Viray in
partial fulfillment of the requirements for the degree in Bachelor of Science in
Chemical Engineering, is hereby accepted.
Date Signed
Date Signed
Dr. Jessica F. Simbahan
Panel Member
Date Signed
Date Signed
Date Signed
Prof. Rex B. Demafelis
Adviser
Date Signed
Date Signed
Date Signed
ACKNOWLEDGEMENTS
College life is one of the best things that ever happened in my life. Sobrang kakaiba
at talagang hahanap hanapin ko. But of course, there still comes a time when every chapter
of our lives has to end. At eto na nga, gagraduate na ko. Sa paglisan ko sa malawak na
mundong ito ng kolehiyo, hayaan niyong pasalamatan ko ang ilan sa mga taong di lamang
tumulong sakin para maging masaya at makabuluhan ang college life ko kundi pati na rin
sa mga tumulong para mapagtagumpayan ko ang isa sa pinakamalaking hamon para
makatapos ANG THESIS KO.
Unang una sa lahat, nagpapasalamat ako sa Kanya. Kasi, kung di dahil sa Kanya,
wagas talaga. Di siguro magpapakita saken ung mga ineexpect kong dapat magpakita sa
experiment ko. I thank God for always being there for me when there comes a time that I
really had to struggle and fight to continue this journey. I thank God for giving me patience,
wisdom and perseverance all throughout my experiment. And I thank God, mostly, for
bringing me the strength every time I had to do overly-exhausting overnights and
experimental repetitions because of de-motivating outcomes in my experiment. At talagang
de-motivating di ba? (Syempre, ikaw ba naman umulit ng tatlong beses ng buong
experiment noh. Dagdag mo pa ung pagpapalit ko ng topic nung first sem. Kumbaga itong
thesis na to, second thesis na to. Wagas talaga!) Thank you Lord!
Syempre, di mawawala dito ang aking ever-supportive and ever-dedicated Mother! I
thank my Mom for always being there for me. All these years, she was never gone. She
supported me in every decision I make, in every endeavors I wish to pursue and in every
downs that I had. Words were not enough to say how thankful I am for having her. Kahit na
minsan, pasaway talaga ako, nandyan pa din siya. Thank you Mom. I love you! And of
course, I wouldnt be able to here without my family my Dad, Ate Pajing, Kuya Paeng,
Kuya Pajun, Kuya Maki, Kuya Matyok, and Doping. I thank them for being there for me
and for supporting me in every path I take. Kahit na minsan, nakakaaway ko yung iba, cool
pa din. Hehehe. Thank you so much! I love you all!
And yes, the most instrumental people who without their presence could never have
happened this THESIS of mine my ever-supportive ADVISERS. To Sir Rex Sir, thank
for believing and trusting my ideas. Thank you for accepting me to become one of your
advisees. Thank you for supporting me to make my thinking come into reality. Youre one
of the best advisers that I had. You taught me a lot of things not just through my thesis but
also throughout my college days. Thank you so much Sir. To Mam Bonic Hi Mam!
Salamat po kasi tinanggap nio pa din akong advisee kahit na di po ako Micro. Hehehe. :P
Thank you Mam for supporting my ideas and for giving me knowledge on what to do.
Siguro po kung wala ung suggestion niyo Mam, malamang wala akong second thesis.
Thank you so much Mam. To Mam Goss- Hello Mam! Thank you for lending me your
reference materials and for giving insights regarding macroalgae. Thank you also for all
your compliments that really boost my perseverance in pursuing my research. Thank you!
To all the RAs. Thank you guys! Really, thank you talaga. Without you guys, I
would never have done my overnights and would have never been able to make up to my
deadlines. To Kuya Francis Kuya, salamat sa pagpapahiram ng magnetic stirrer.
Sobrang malaking tulong po un kasi crucial talaga ang chemical hydrolysis ko. Pati salamat
kasi nakakasama ko kayo sa pagpupuyat ko. At syempre sa mga compliments nio na talaga
namang flattering. To Kuya EJ thank you sa pagpupuyat din kasama namin, sa mga
questions mo kuya about my thesis which I am glad to answer (hehehe) at sa mga biro mong
bigla-bigla nalang. Thank you! To Kuya Peps salamat din kuya sa pagsama sa aking
overnight at sa pagiging accommodating sa aking mga pangangailangan. Hehehe. To Ate
Val the one great super scout girl. Thankful talaga ako kasi ikaw ung nag-aaccomodate ng
weekend experiment ko. Saka super thanks na rin kasi kung wala ka nun, baka hinimatay
nalang ako sa thesis lab. Thanks for being super nurse. To Kuya Pao salamat po sa
pagsama smin kumuha ng algae pati na rin sa mga encouragements para po gawin ko ung
thesis ko nung first sem. Thanks Kuya. And last but not the least, To Ate Lisa. Thanks ate
for accompanying us gather our seaweed samples and thanks as well for helping us in our
experimental needs.
Syempre, I will never ever forget my ENVI Lab Family in BIOTECH Mam Jac,
Sir Nayve, Tita B, Tita Gie, Tita Buena, Tita Dory, Tita Oyie, Tita Pat, Tito Rey, Kuya
Narsing, Kuya Athan, Kuya Renz, Ate Mylene, Ate Janice, Ate Chan, Ate Ivy, Kuya
Badz, Kuya Joel, Ate Jasmin, Allan, Sean, and Johnry. Thanks for making me become
part of your family. Salamat po sa pagiging supportive, as in super! You guys made my
experiment so happy and alive. Thanks for bringing me joy and smile every time I go to
your Lab. Thanks for all the laughter that relieves me from stressful work of thesis and
acads. Surely, I will treasure all the days that I stayed with you. This space is not enough to
thank you all for all your efforts not just in encouraging me but also for making me enjoy
my research. Syempre to my ENVI Lab thesismates Ate Jenny, Alex, Herra and Sitti.
Thank you guys for being my good friends and masayang kakwentuhan sa Lab. You guys
make my thesis days so joyful. Without you as well, thesis could have been so boring. I will
miss you all.
To my beloved organization my home and my family in UPLB the UP Alliance
of Chemical Engineering Students (UP AChES), words really are not enough to say how
grateful I am to be part of this exceptional group of people. You guys have taught me a lot of
invaluable lesson which I will treasure my entire life from leadership skills, self-esteem
enhancement, work management, camaraderie and a lot more. You guys are the best!
Thanks specially to my Ninang Irene Villanueva for being one of my model who pushes
me to achieve greater things in life, to Johans Claudine Ufano and Michelle Tortosa for
being my inaanaks in the organization (hehehe..) and my super duper galing na apo, Marky
Panganiban, you make our angkan proud! Continue that! I also would like to thank my
batchmates, STOICH (Kuya Ada, Ate Van, Kuya Joker, Kuya Paul, Kuya Noy, Kuya
Doms, Ate Eden, Ate Odeth, Jerson, Julius, Kevin, Mac, Lithlyn, Marious, Jerick, Titus
and Jam). Thank you for being a good family as well and for always being there for me.
Stoich, the best!
To the chemical engineering faculty and staffs (Mam Movi, Sir Abrigo, Sir Alf,
Mam del Barrio, Mam Parao, Mam Monet, Mam Jewel, Mam Jeanne, Sir Tengco, Sir
Jeck, Sir Butch, Sir Ram, Sir Mico, Sir Dhan, Mam Denden, Tita Otie, Tita Mila and
Tito Mert), you guys have been my family and my home as well for almost 4 years. Thank
you for imparting all your knowledge and for guiding me throughout my chemical
engineering undergraduate journey. Surely, I will make use of that knowledge rightfully.
And I will someday make you all proud. Thank you so much.
To my chemical engineering batchmates (Batch 06) and my colleagues in the
department, thank you so much for being part of my life. To my closest batchmates, you
know who you are guys. Thank you so much. Without you, college life would have been
dull and gray. Thanks for happiness and for sharing laughter with me. Thanks for being my
company in good times and in bad times. We guys rock!
Hindi ko na rin siguro palalampasin ang pagpapasalamat sa aking mga friends sa
University, the ISKULMEYTS GIRLS (Hayren, March, Abi, Joy and Ate Lala). Salamat
sa pagiging kakwentuhan pag walang magawa. Sa libreng Facebook at internet access sa
inyong shop. Hehehe. Sa aking mga discounts pag nagpapaprint. Ansaya-saya niong
kasama. Thank you so much sa chikahan at chismisan at syempre sa bonggangbonggang okrayan. Hahaha.. I will miss you guys.
And of course, to those people who I forgot to mention but who have been a part of
my success not just in this THESIS but also in my college life - you guys know who you are
- THANK YOU SO MUCH!
This chapter of my life may have ended. But it continues to travel different journey.
So long my friends. Thank you and lets continue our own lives journeys.
TABLE OF CONTENTS
Title Page
Acceptance Sheet
ii
Acknowledgements
iii
Table of Contents
iv
List of Tables
List of Figures
vi
Abstract
vii
INTRODUCTION
1.1 Significance of the Study
REVIEW OF LITERATURE
2.1 Biofuels
2.1.1 Bioethanol
2.1.2.1 Pretreatment
2.1.2.2 Hydrolysis
10
10
12
2.1.2.3 Fermentation
13
13
2.2 Macroalgae
2.2.1 Production and Use
15
16
17
17
2.2.2.1.2 Fucoidan
18
2.2.2.1.3 Cellulose
19
2.2.2.2.1 Mannitol
20
2.2.2.2.2 Laminarin
20
20
21
23
25
27
27
28
29
29
31
19
32
32
32
33
34
34
39
43
47
51
53
5
6
54
57
REFERENCES
59
APPENDICES
66
A. Standard Curves
66
70
73
76
78
F. Sample Calculations
80
G. Statistical Analysis
84
92
LIST OF TABLES
Table #
Title
Page
2.1
2.2
12
Methods
2.3
13
2.4
16
2.5
21
2.6
22
2.7
24
4.1
35
conditions
4.2
37
4.3
38
4.4
38
4.5
39
4.6
42
4.7
42
4.8
43
4.9
44
4.10
47
temperature
4.11
47
4.12
48
4.13
49
4.14
49
4.15
52
52
and 72hrs
4.17
53
LIST OF FIGURES
Figure #
Title
Page
2.1
2.2
17
2.3
18
2.4
19
2.5
23
3.1
27
3.2
28
3.3
29
3.4
30
3.5
31
3.6
32
4.1
35
4.2
36
4.3
36
4.4
39
4.5
40
4.6
41
4.7
43
4.8
44
4.9
45
4.10
46
4.11
48
4.12
50
4.13
51
4.14
53
ABSTRACT
VIRAY, MICHAEL ANGELO MELO. College of Engineering and AgroIndustrial Technology, University of the Philippines Los Baos, March 2011.
Parametric Study on Chemical and Enzymatic Hydrolysis of Alginate from
Sargassum cristaefolium C.A. Agardh (Phaeophyta) for Bioethanol Production.
Adviser: Prof. Rex B. Demafelis
Co-Advisers: Ms. Irene G. Pajares; Dr. Milagrosa Goss
Parametric study for the chemical and enzymatic hydrolysis of alginate from
seaweed, Sargassum cristaefolium was conducted to determine its potential for bioethanol
production.
The effect of time (1hr, 3hrs and 5hrs), temperature (60oC, 80oC, and 100oC) and
acid concentration (70%, 80%, and 90%) on the reducing sugar and uronic acid yield
were determined for the chemical hydrolysis. It was found out that time has no significant
effect on the reducing sugar yield but has significant effect on uronic acid yield. In terms
of the effect of temperature, reducing sugar showed a decreasing trend with increasing
temperature. For uronic acid, a peak value was observed at 80oC and further increase in
temperature resulted in decreasing uronic acid yield. In terms of the effect of acid
concentration, both reducing sugar and uronic acid exhibited a peak value at 80% acid
concentration and further increase resulted in decreasing yields. Optimum chemical
hydrolysis condition based on the highest amount of reducing sugar was found to be at
60oC, 80% acid concentration and 1hour.
The effect of time (24hrs, 48hrs, and 72hrs) and temperature (37oC, 40oC and
45 C) were investigated during enzymatic hydrolysis. Results showed an increasing
reducing sugar yield with increasing time whereas a decreasing reducing sugar yield was
observed with increasing temperature. Optimum hydrolysis condition based on the
highest reducing sugar was found to be at 37oC and 72hours.
o
The optimum conditions were evaluated on the extracted alginate. However, for
enzymatic
hydrolysis the condition applied was at 45oC and 72hours. Chemical
hydrolysis yielded 0.0005 mg/ml reducing sugar while enzymatic hydrolysis yielded
0.9915 mg/ml reducing sugar. The highest amount was used to determine the bioethanol
potential of the hydrolysates and was found to be too low to be considered for bioethanol
production. Further studies for enzymatic hydrolysis were recommended as this gave
quite meaningful results in the experiment.
CHAPTER ONE
INTRODUCTION
The
results of the hydrolysis of alginic acid/ alginate will provide us meaningful insights
regarding the further utilization of our seaweed resources for a more sustainable energy
and fuel importation independence of our country in the future.
INTRODUCTION
Page 2
production.
Specifically, this study was designed in order to:
1) develop a hydrolysis method of alginate from Sargassum cristaefolium;
2) determine the effect acid concentration, effect of temperature and effect of
reaction time on the uronic acid and reducing sugar yield of commercial alginate using
formic acid;
3) determine the effect of incubation temperature and incubation temperature on the
uronic acid and
hydrolysis;
4) determine optimum conditions for both acid and enzymatic hydrolysis of
commercial alginate based on the uronic acid yield;
5) evaluate the optimum conditions of hydrolysis to the extracted alginate and raw
seaweed material; and
6) compare the acid and enzymatic hydrolysis of the alginate.
1.3 Date and Place of Study
This study was conducted from December 2010 to March 2011.
Chemical
Page 3
INTRODUCTION
Page 4
CHAPTER TWO
REVIEW OF LITERATURE
2.1 BIOFUELS
While the science of fuel production from agricultural crops has already been
established, little studies have focused on seaweed resources for renewable energy.
Today, the world is faced with aggravating problems on fuel security and global
warming brought by the rapid growth of industrialization and population. This is mainly
because much of the energy consumption is dependent on non-renewable petroleum-fuels
which are basically derived from fossils. As of 2008, the Philippines oil consumption
reached 11.93 million tons of oil equivalent (MTOE) through which 31.15% are imported
(www.doe.gov.ph).
Aside from the very high fuel demand, attention has also been focused on the
negative impacts on the use of petroleum-fuels in the environment such as global
warming and air pollution.
With these two major problems at hand, researches have been conducted on
finding alternative resources of fuel that will not just aid in fuel scarcity but will also help
in the preservation of the environment. Among the major solutions found today are the
biofuels.
Biofuels are actually fuels derived from biomass materials such as plants, wastes
and other organic materials. And there are three categories mainly: 1) solid fuels, 2)
liquid fuels, and 3) gaseous fuels; and are produced either by biological or
REVIEW OF LITERATURE
Page 5
thermochemical methods (Goodman and Love, 1981). Among the three categories of
biofuels mentioned, liquid biofuels are the most commonly produced and currently,
receive the widest attention among researchers.
Biofuels are considered because they are non-polluting, locally available,
accessible, and sustainable (Demirbas, 2005). It is said to be non-polluting since the
biomass feedstocks used for the production of biofuel is reducing the net carbon emission
from previous cultivation. In addition to that, biomass feedstocks are locally available
and accessible because they can easily be obtained from a wide set of sources such as
wastes and plants.
Today, the country has already adopted the use of these biofuels to adrress the
aggravating concerns on fuel demand and environmental degradation. This was done
through the implementation of RA 9367 also known as the Biofuels Act of 2006 which
mandates the use of 10% bioethanol blend and 2% biodiesel blend in all petroleum
stations by 2011.
2.1.1 BIOETHANOL
Nowadays, bioethanol is the most widely used liquid biofuel along with biodiesel
and is considered a promising resource (Demirbas, 2005)
In addition, bioethanol has already been commercially produced by several
countries not only for fuel production but also for several other purposes such as solvents,
disinfectants and others. Among the major producers of bioethanol are Brazil, United
States, China and India.
REVIEW OF LITERATURE
Page 6
As a transport fuel, bioethanol has been blended to gasoline which would account
for 5% up to a maximum of 10% blend without any modification in transport engine
(www.doe.gov.ph). Aside from that, bioethanol has brought a lot of advantages not only
in terms of reduction of fuel demand but also contributed for cleaner and greener
utilization of fuel because it burns more cleanly and has almost complete combustion
thus reducing carbon emissions and the cultivation of biomass crops for bioethanol
production could reduce the carbon dioxide in the atmosphere. The table below shows a
comparison of commonly used transport fuel against bioethanol
Table 2.1 Comparison of Bioethanol against Unleaded Gasoline
Source: www.doe.gov.ph
Most of the feedstocks used for bioethanol production are agricultural crops such
as cassava, corn, sugar beet, and wheat straw together with sugar cane being the primary
source in the Philippines and some recent researches on cellulosic and lignocellulosic
feedstocks.
REVIEW OF LITERATURE
Page 7
REVIEW OF LITERATURE
Page 8
Page 9
Page 10
structure. However, in two separate studies conducted by Patle and Lal (2007) and
Jeihanipour and Taherzadeh (2009), it was found out that alkaline hydrolysis of biomass
materials can also result to the production of monomeric sugars.
2.1.2.2.1.2 DILUTE ACID HYDROLYSIS
The dilute acid process is conducted under high temperature and pressure, and has
a reaction time in the range of seconds or minutes, which facilitates continuous
processing (Demirbas, 2005). However, Badger (2002) noted that the sugar recovery
efficiency of this process is limited to around 50%. This is mainly because the two
reactions involve in the process have conditions that are same. These two reactions are
the conversion of the complex carbohydrate into sugar and the degradation of the sugar
into other chemicals. Fortunately, there is way in order to decrease the degradation of
sugar and this involves the use of a two stage process. The first stage is conducted under
mild process conditions to recover the 5-carbon sugars which are relatively faster to
degrade than 6-carbon sugars.
acid
hydrolysis
generally
involves
two
steps: first,
a decrystallization step that breaks down the crystal structure of the carbohydrates; and
a second step which involves the hydrolysis of the decrystallized fiber using a lower
acid concentration (Bayat-makooi et. al, 1985). This process is usually conducted
under relatively mild temperatures, and the only pressures involved are those
created
REVIEW OF LITERATURE
Page 11
factors needed to make this process economically viable are to optimize sugar recovery
and cost effectively recovers the acid for recycling (Demirbas, 2005). A comparison of
the advantages and disadvantages of the two acid hydrolysis processes are summarized
in the table below.
Table 2.2 Comparison between Concentrated-and Dilute-Acid Hydrolysis Methods
(Taherzadeh and Karimi, 2008)
Hydrolysis
Advantages
Disadvantages
-High acid consumption
-Equipment corrosion
Concentrated Acid - Low operating temperature
-High sugar yield
- High energy consumption for
Process
acid recovery
-Longer time of reaction
(e.g 2-6 hours)
-Operated at high
temperature
-Low sugar yield
-Equipment corrosion
Formation of undesirable byproducts
Page 12
Short
Relatively long
Reaction Time
High
Relatively low
Energy consumption
Short
Relatively long
Reaction Time
Sources: Demirbas, 2005; Taherzadeh and Karimi, 2008
Enzymatic
hydrolysis
Mild
Yes
No
Long
Low
Long
2.1.2.3 FERMENTATION
Fermentation
Page 13
fuel use, it is actually the one causing major problems on global warming due to
industrial farming methods used to cultivate the crops.
In the study conducted by Crutzen et. al. (2008), they found that fertilizers used
in farming and cultivation of the crops is increasing the greenhouse gas (GHG),
nitrous oxide, in the atmosphere. This gas is actually 300 times more insulating than
carbon dioxide which means worse conditions of global warming. In addition to that,
they also enumerated some crops which tend to contribute more in the GHG. These
crops include sugar cane which produce between 0.5 and 0.9 times GHG as ordinary
fuel gases; corn, between 0.9 and 1.5 times global warming effect as conventional
gasoline; and rapeseeds, between 1 and 1.7 times more GHG than conventional diesel.
In 2008, Tilman as cited by Inman, found that clearing of forests and
grasslands for biofuels production are forming carbon debt. He stated that when
grasslands and forest are cleared, the soil releases much of the carbon it has stored
over the years. In addition to that, forests containing decomposing plants beneath the
soil also release carbon dioxide since there are no longer plants that will trap them.
This means that even though biofuels reduce the carbon dioxide emission because of
its use of plants, the amount of carbon being released for clearing areas is too much to
compensate for the previous reduction. He further concluded that these carbon debt
would take longer years to repay that biofuels would in turn become unsustainable for
the environment. Among those clearings for biofuel production he cited are sugar cane
which would take 17 years to repay carbon debt; corn, 93 years; tropical rainforest for
biodiesel on palm, 86 years; and peatland rainforest also for biodiesel on palm, 423 years.
REVIEW OF LITERATURE
Page 14
Aside from the environmental concerns reported by Inman on the use of biofuels
from terrestrial crops, he also cited some adverse effects of cultivation of these crops on
food sectors. In his study, he found that biofuels are in fact competing with food for land
allocation. He reported that for every 5 acres (2 hectares) of land used for cultivation
of corn, more than 4 more acres (1.6 hectares) of cropland would still be needed to
provide food for the world. This need for more land then leads to more rainforest
clearance thus resulting to a cycle of adverse effects.
Crutzen et. al. (2008) suggested that in order to provide for the current demands
of energy sustainably, research should be focused more on crops utilizing low amounts
of nitrogen and those which do not have huge impact on agriculture. An example of
such is the macroalgae.
2.2 MACROALGAE
Macroalgae, more commonly known as seaweeds are one of the marine biomass
which have a huge potential to be utilized for energy production. They are generally
classified into three major groups namely the green, red and brown algae.
REVIEW OF LITERATURE
Page 15
costing to around $48,000 from 1990 to 1995. In addition to that, exports of dried
seaweeds also reached an annual amount of 26,000 dry tons and being marketed at a
value of $30,000. Lastly, seaweeds have also been utilized in the country as sources
of food,
phycocolloids
(agar,
carrageenan
and
algin),
growth
regulators,
Laminaria,
Macrocystis,
Vegetation Type
Production (kg/m2-yr)
Trees
Grasses
0.9 2.8
1.1 6.8
Terrestrial
Marine
microalgae (waste treatment ponds)
microalgae (laboratory culture)
Kelps/ macroalgae (natural beds)
REVIEW OF LITERATURE
4.5
6.8 13.5
4.9
Page 16
Page 17
The
enzymatically. Alginate lyase, the enzyme responsible for breaking the bonds between its
constituent carbohydrates (D-mannuronic and L-guluronic acids) are commonly found in
various sources including marine algae, marine mollusks, and a wide range of
microorganisms (Wong et. al, 2000).
The figure below shows the chemical constituent and overall structure of an
alginic acid.
Figure 2.3Alginate structural data: (a) alginate monomers (M vs G); (b) the alginate
polymer; (c) chain sequences of alginate polymer (Smidsrod and Draget, 1996)
2.2.2.1.2 FUCOIDAN
Fucoidan, like alginic acid, is also a polysaccharide consisting mainly of Lfucose units and sulphate ester groups (Percival and McDowell, 1967). They are mainly
derived from brown algae and are commonly used in pharmaceuticals and
medicine. Acid hydrolysis of this polysaccharide also yields various proportions of Dxylose, D- galactose and uronic acids (Mackie and Preston, 1974 as cited by Davis et.
REVIEW OF LITERATURE
Page 18
al, 2003). In addition to acid hydrolysis, fucoidan may also be degraded using
enzymes known as fucoidanases found among marine organisms.
2.2.2.1.3 CELLULOSE
Cellulose is also a polysaccharide consisting of glucose units in B-1,4 linkages
and is universally present in terrestrial plants. In brown algae, they occur in a particular
form known as Cellulose IV (Lewin, 1962).Figure 2.4 shows the chemical structure of
cellulose.
REVIEW OF LITERATURE
Page 19
2.2.2.2.1 MANNITOL
Mannitol is a 6-Carbon sugar alcohol/ polyol/ hexitol universally found in brown
algae. It is the alcohol form of the sugar, mannose. It is usually extracted from seaweeds
for
use
in
food
manufacturing and
as
sweeteners
in
dietetic
products (http://en.wikipedia.org/wiki/Mannitol)
.
species and the season of the year which usually accumulates during winter season
(Percival and McDowell, 1967).
2.2.2.2.2 LAMINARIN
Laminarin or laminaran, is a polysaccharide consisting of glucose units in B, 1-3
linkages which occurs in two forms based on their solubility in cold water: soluble and
insoluble. They are present in a majority of brown algae as a storage product. In
Laminaria, they usually exhibit high amounts of up to 25% dry weight during late
summer (Percival and McDowell, 1967).
Page 20
commonly found all over the rocky, wave exposed or sheltered areas (Montano et. al,
2006).
As previously noted, brown algae consist of high amounts of carbohydrates which
can be hydrolysed and converted into energy. These include polysaccharides such as
laminarin, cellulose, fucoidan, and alginic acid and the sugar alcohol, mannitol which are
all varying in content depending on species, season of the year and physical location.
Table 2.5 Chemical Composition of Various Sargassum specie (From Ji and Zhang, 1962)
Sargassumpallidum
S. kjellmanianum
S. thunbergii
S. fusiforme
S. hemiphyllum
S. horneri
S. siliquastrum
S. vachellianum
S. polycystum
Sargassumspp.
Mannitol
(%)
5.47 - 12.81
6.84-13.40
Alginic Acid
(%)
10.7-26.1
16.3-26.3
Crude Protein
(%)
6.82-15.83
17.61-26.50
Crude Fiber
(%)
6.51-6.66
4.35-14.43
1.64-15.48
2.45-10.25
10.9-26.2
11.1-24.5
9.97-25.28
7.95-12.13
3.2-6.27
3-4.92
6.23-11.02
1.62-13.75
17.3-23.6
25.3-31.0
9.22-12.4
14.08-17.47
5.44-5.54
6.26-6.78
9.96-13.68
1.3
22.4-25.5
26.2
10.58-17.85
12.58
5.01-6.74
9.3
1.78
1.86-10.60
14
14.1-32.5
15.45
4.42-21.46
7.79
4.59-9.04
of
been performed by the Naval Weapons Center in their Ocean Food and Energy Farm
Project since 1970s (Show, 1981; Benson and Bird, 1987).
REVIEW OF LITERATURE
Page 21
Macroal
gae
730000
40150
1010
2010
5150
6756
23400
Page 22
Figure 2.5 Pathway for processing brown seaweeds for fuel and other commercial
products (Horn and Ostgaard, 2000)
2.3 RELATED STUDIES ON HYDROLYSIS OF MACROALGAE
Little studies have been conducted regarding the hydrolysis of macroalgae for
bioethanol production here in the country. This is a very challenging study since the
macroalgae, as previously, contains not only a single polymeric carbohydrate but also
contains various types of it including sugar alcohols and sulphated polysaccharides. Yet,
this study might provide a greater innovation for cleaner and greener bioethanol industry
for our country.
REVIEW OF LITERATURE
Page 23
In 2009, two parallel studies were conducted regarding the hydrolysis of two
species of brown algae, Sargassum cristaefolium and S. kushimonte. Reyes (2009) and
Rivera (2009) were able to attain the highest sugar yield in their enzymatic hydrolysis as
compared to the chemical hydrolysis. Later in 2010, a study of similar result regarding
the hydrolysis of Turbinaria ornata was also conducted by Quiones (2010). A
summary of the results of their chemical and enzymatic hydrolysis is shown in Table 2.7.
Table 2.7 Chemical and Enzymatic Hydrolysis of Various Brown Macroalgae
Chemical Hydrolysis
Enzymatic Hydrolysis
Condition
Condition (Acid
(enzyme
Reducing Sugar
Reducing Sugar
Conc., Temp.,
loading, Temp.,
(mg/ml)
(mg/ml)
Time)
Time)
Sargassum cristaefolium
Sargassum kushimonte
Turbinaria ornata
3% HCl,
o
60 C, 300 min
3% HCl,
o
60 C,
300min
6.67% HCl,
o
80 C,
255min
Reference
0.2661
0.5 mg/g,
o
50 C, 48hrs
2.6278
Reyes
(2009)
0.3263
0.5 mg/g
o
50 C, 72hrs
2.7372
Rivera
(2009)
0.7014
0.5 mg/g,
o
50 C, 72hrs
1.6333
Quiones
(2010)
The hydrolysis experiments were conducted in the assumption that the fiber
content of each species is equal to the cellulose content of the algae. Thus, during the
study other carbohydrates present in the algae were not considered during hydrolysis.
They have recommended that in order for the brown algae to become an effective
feedstock for bioethanol production, the carbohydrates (alginic acid, laminarin and
mannitol) present in the algae should also be taken into consideration. The results of the
proximate analysis of the composition of the algae showed that carbohydrate contents
are in the range 42.89% 58.00% making consideration of these inevitable. However,
the results were only limited to proximate analysis and no detailed analysis was done on
the exact amounts of these carbohydrates.
REVIEW OF LITERATURE
Page 24
Page 25
On the other hand, although researches have already been conducted regarding the
isolation of alginases from various sources and its mechanism of action (Wong et. al,
2000), relatively few studies have been conducted on the optimization of conditions for
enzymatic hydrolysis.
REVIEW OF LITERATURE
Page 26
CHAPTER THREE
Page 27
Prior to extraction, several steps were done. First, the stored seaweed was
washed with distilled water to remove excess formalin. After that, the seaweed was
soaked in 0.5M H2SO4 for at least a night. The seaweed was again washed to remove the
excess acid.
To 100g of acidified seaweed, 500ml of 4% (w/w) Na2CO3 solution was added.
The resulting mixture was magnetically stirred for 1hour after which it was
centrifuged (10,000 x g) for 10mins at 10oC. The supernatant was stored at 4oC prior to
precipitation. The gelatinous precipitate that formed was pressed manually to remove the
liquid. Then it was dried in an oven at 35oC. The dried alginate was pulverized using
a mortar and pestle.
Page 28
with 100 ml of formic acid in varying concentrations (70%, 80%, and 90%). It was
placed in a hot plate and was stirred at varying temperatures (60oC, 80oC and 100oC) for
5 hours. Two ml (2ml) of the heated solution were obtained at 1hour, 3hours and
5hours. Then they were mixed with 10ml of distilled water. The resulting solutions
were heated again at 100oC for 2 hours. After the period, 5ml samples were obtained
and placed in a vial. Samples were stored at 4oC prior to analysis.
Page 29
alginate (pH 7.8). After cultivation, the culture supernatant was obtained by
centrifugation (10,000 x g) at 4oC for 10 mins and was used as enzyme source for
hydrolysis. Semi-purification was done to ensure the stability of enzyme activity. This
was done by slowly adding ammonium sulphate, (NH4)2SO4 to the supernatant until it
reached 75%. The resulting solution was allowed to stand overnight after which,
it was centrifuged again (10,000 x g) at 4oC for 10mins.
The enzyme activity was measured by mixing 0.5ml of enzyme solution and
1.5ml of 50 mMTris-HCl buffer (pH 8.0) containing 0.4% sodium alginate and 0.4M
NaCl. Reaction proceeded for 20mins and was then stopped by addition of 2ml of
DNS solution. The reducing sugar was measured using DNS method (See Section 3.5.1)
while the protein was determined using the method of Lowry et. al (1951) (See Section
3.5.3). One unit of enzyme activity was defined as the amount of which liberated 1umol
of D-mannuronic or L-guluronic acid per min under the above conditions.
Page 30
Page 31
Page 32
Lowry Reagent 1
5ml (0.5 % Copper Sulfate Pentahydrate, 1% Sodium or Potassium Tartrate) +
250 ml (2% Sodium Carbonate, 0.4% NaOH)
Lowry Reagent 2
12.5 ml Folin-Ciocalteau Phenol Reagent was diluted with distilled water to 25
ml solution.
Page 33
CHAPTER FOUR
RESULTS AND DISCUSSION
Macroalgae or more commonly known as seaweed is currently getting attraction
among researchers for its great potential as bioethanol feedstock mainly because of its
high carbohydrate content, absence of lignin content and its non-competing utilization
with food crops.
Alginate, one of its major components (composition could reach to about 40% of
its dry weight), is a polysaccharide consisting of monomeric units of L-guluronic and Dmannuronic acid arranged either in alternating monomeric or polysaccharide unit or in
random sequence. Hydrolysis of this polysaccharide into its monomeric unit could be
very useful since they can be utilized by several microorganisms for bioethanol
production. However, parametric study regarding the hydrolysis of alginate has not been
fully established yet towards bioethanol production.
For this research, the parametric study on the hydrolysis of alginate was first
performed using commercially available alginate samples followed by evaluation
procedures using extracted alginate. Hydrolysis treatment of the commercial alginate was
divided mainly into two parts namely: acid hydrolysis and enzymatic hydrolysis.
4.1 Acid Hydrolysis of Commercial Alginate Samples
4.1.1 Effect of Parameters on the Reducing Sugar Yield
For the acid hydrolysis, three parameters were considered for the experiment
namely: time (1hr, 3hrs and 5hrs), temperature (60oC, 80oC, and 100oC) and acid
concentration (70%, 80%, and 90%). Formic acid was used in the experiment based on a
research conducted by Chandia et. al (2001) for the total hydrolysis of alginate and for
the aim of developing a hydrolysis procedure for alginates in seaweeds. To evaluate
the effects of the parameters, reducing sugar and uronic acid concentrations were
chosen as responses for the study. Table 4.1 summarizes the values of reducing sugar
concentration
at
different
hydrolysis
conditions
acid
concentration).
RESULTS AND DISCUSSION
Page 34
70 % (v/v) HCOOH
60oC
80oC
100oC
2.4616 1.0951 0.9594
1.7669 0.7086 0.8374
1.5230 0.6787 0.7661
80 % (v/v) HCOOH
60oC
80oC
100oC
2.5963 2.2767 1.1607
2.3754 2.0700 1.1019
1.9337 1.9196 1.0009
90 % (v/v) HCOOH
60oC
80oC
100oC
0.7411 1.0242 1.3312
0.6068 0.9834 1.2256
0.5756 0.8227 0.9858
3.5000
3.0000
2.5000
2.0000
1hr
1.5000
3hrs
1.0000
5hrs
0.5000
0.0000
60
80
100
Temperature (oC)
Page 35
from hydrolysis decreased. The same was true for temperature, which was, as
temperature increased the amount of reducing sugar decreased. At this acid
concentration, reducing sugar yield ranged from 1.009 mg/ml to 2.5963 mg/ml.
3.5000
3.0000
2.5000
2.0000
1hr
1.5000
3hrs
1.0000
5hrs
0.5000
0.0000
60
80
100
Temperature (oC)
1.4000
1.2000
1.0000
0.8000
1hr
0.6000
3hrs
0.4000
5hrs
0.2000
0.0000
60
80
100
Temperature (oC)
Page 36
the previous two figures, the figure showed a different trend with regards to the
temperature. It can be clearly seen that as temperature increased, the amount of reducing
sugar yield increased and the values ranged from 0.5756 mg/ml to 1.3312 mg/ml.
Comparing the range of values of reducing sugar yield from the three acid
concentrations, it showed that 80% acid concentration yielded the greatest average
amount of reducing sugar among the three with an average value of 1.8227 mg/ml.
Meanwhile, the 90% acid concentration yielded the lowest average amount of reducing
sugar among the three with the minimum value of 0.9218 mg/ml.
To evaluate whether the effect of the three parameters on the yield of reducing
sugar was significant or not, Duncans Multiple Range Test (DMRT) with three factorial
completely randomized design (3! CRD) analysis at 5% level of significance was used
for the data. The results of the analysis for the effect of time on reducing sugar was
summarized in Table 4.2
Table 4.2 3! CRD Analysis for Effect of Time on Reducing Sugar
Level of significance,
0.05
51
0.37244
Number of means
Critical Range
0.4085
0.4301
Duncan A
groupinga
1.5162
Mean
(mg/ml)
Time1(hrs)
1.2865
1.1340
From the table above, it indicated that the effect of time on the reducing sugar
yield wasnot significant. Thus, increasing the time for the hydrolysis treatment of alginate
would have no significant effect on the amount of reducing sugar produced.
Page 37
Table 4.3 shows the 3! CRD analysis of data for the effect of temperature on the
reducing sugar yield. It showed that there was a significant effect of temperature on
reducing sugar from temperature 60oC to 80oC then remained insignificantly different
after the temperature range.
Table 4.3 3! CRD Analysis for Effect of Temperature on Reducing Sugar
Level of significance,
Error degrees of freedom
Error mean square
0.05
51
0.33855
Critical Range
0.38949
0.41006
Duncan A
groupinga
B
B
1.6200
Mean
(mg/ml)
1.2865
1.0376
60
Temperature
(oC)
80
100
For the effect of acid concentration on the reducing sugar, a summary of the 3!
CRD analysis was shown on Table 4.4. In the table, it showed that there was a significant
effect of acid concentration on the reducing sugar yield.
Table 4.4 3! CRD Analysis for Effect of Acid Concentration on Reducing Sugar
Level of significance,
Error degrees of freedom
Error mean square
0.05
51
0.24860
Number
CriticalofRange
means
0.33376
2
3
0.35139
Duncan groupinga
A
B
B
Mean (mg/ml)
1.8227
1.1996
0.9218
Acid Concentration
80
%(v/v)
70
90
Page 38
4.1.2
In addition to the reducing sugar yield, the effects of three parameters on the
uronic acid yield were also studied. Table 4.5 summarized the results of the uronic acid
yield at different hydrolysis conditions.
Table 4.5 Uronic Acid Concentration (mg/ml) at different hydrolysis conditions
Uronic Acid Concentration (mg/ml)
Time (hrs)
1
3
5
70 % (v/v) HCOOH
60oC
80oC
100oC
1.4796 1.1998 0.7402
1.2286 0.9070 0.6173
1.0648 0.6548 0.5658
80 % (v/v) HCOOH
60oC
80oC
100oC
1.1967 1.3903 1.0355
1.0448 1.2702 0.7862
0.6374 0.9906 0.7095
90 % (v/v) HCOOH
60oC
80oC
100oC
0.4768 0.7100 0.6539
0.4380 0.7029 0.6183
0.3858 0.6175 0.5993
1.6000
1.4000
1.2000
1.0000
1hr
0.8000
0.6000
3hrs
0.4000
5hrs
0.2000
0.0000
60
80
100
Temperature (oC)
Figure 4.4 showed that at an acid concentration of 70% (v/v), the amount of
uronic acid decreased with time. In addition, Figure 4.4 showed that as temperature
RESULTS AND DISCUSSION
Page 39
increased, the uronic acid yield decreased. This result corresponded to the trend from
Figure 4.1 regarding the effect of time and temperature on reducing sugar yield. Uronic
acids obtained were in the range 0.5658 mg/ml 1.4796 mg/ml.
Figure 4.5 showed the results of interaction of uronic acid with time and
temperature at 80% (v/v) acid concentration. As seen from Figure 4.5, the uronic acid
also decreased with time just like the trend observed from Figure 4.4 and its
corresponding graph on Figure 4.2. However, a different trend was observed regarding
the effect of temperature on the uronic acid wherein a peak uronic acid yield was
obtained at 80oC. The uronic acid at this condition were found to be in the range from
0.7095 mg/ml to 1.3903 mg/ml. Peak values at 1, 3 and 5 hours were 1.3903 mg/ml,
1.2702 mg/ml and 0.9906 mg/ml respectively.
1.6000
1.4000
1.2000
1.0000
0.8000
1hr
0.6000
3hrs
0.4000
5hrs
0.2000
0.0000
60
80
100
Temperature (oC)
Page 40
mg/ml with peak values 0.7100 mg/ml, 0.7029 mg/ml and 0.6175 mg/ml for 1, 3 and 5
hours respectively.
0.9000
0.8000
0.7000
0.6000
0.5000
1hr
0.4000
0.3000
3hrs
0.2000
5hrs
0.1000
0.0000
60
80
100
Temperature (oC)
Page 41
Critical Range
A
B
B
Level of significance,
Error degrees of freedom
Error mean square
0.05
51
0.07949
0.18873
0.19870
0.9870
0.8459
0.6917
1
3
5
For the effect of temperature on the uronic acid, Table 4.7 showed the statistical
analysis of the data. It showed that there was a significant increase of uronic acid from
temperature 60oC to 80oC after which a significant decrease occurred until it reached
100oC. This showed a different result as compared with the effect of temperature on
reducing sugar in which there was a significant decrease from 60oC to 80oC.
Table 4.7 3! CRD Analysis for Effect Temperature on Uronic Acid
Level of significance,
0.05
51
0.08418
Number
means
CriticalofRange
2
0.19422
3
0.20447
Duncan A
groupinga
B
B
0.9381
Mean
(mg/ml)
0.8836
0.7029
80
Temperature
(oC)
60
100
Statistical analysis for the effect of acid concentration on uronic acid was
summarized in Table 4.8. It can be seen from the table that the results were almost in
RESULTS AND DISCUSSION
Page 42
correspondence with the results from Table 4.4. It showed that there was a significant
decrease of uronic acid from 80% to 90%. However, the uronic acid yield was
statistically not different from 70% to 80%.
Table 4.8 3! CRD Analysis for Effect of Acid Concentration on Uronic Acid
0.05
Level of significance
51
Error degrees of freedom
0.05734
Error mean square
Number
means
CriticalofRange
2
0.16029
3
0.16875
Duncan groupinga
A
A
B
Mean
1.0068
0.9398
0.5781
Acid Concentration
% 80
(v/v)
70
90
Page 43
It has been stated by Smidsrod et. al(1969) that the dehydration and
decarboxylation reactions occur consecutively rather than simultaneously. In addition to
that, the distribution of the products will largely depend on the pH (corresponding to the
acid concentration) and temperature conditions of the reaction. Table 4.9 showed
hydrolysis condition resulting into formation of reductic acid.
Table 4.9 Formation of Reductic Acid at Different Conditions
Conditions
Yield
References
Aso (1952)
Aso (1952)
As seen from Table 4.9, harsh hydrolysis conditions are necessary towards the
formation of reductic acid unlike the formation of furfural that is simultaneously formed
upon conversion of polyuronic-acid into its monomeric unit (Feather and Harris, 1973).
To analyse further the results of varying the conditions of hydrolysis, charts
consisting both of the reducing sugar and uronic acid yield at different condition can be
seen from Figures 4.8 to 4.10.
3.0000
RS/ UA (mg/ml)
2.5000
2.0000
1hr-UA
3hrs-UA
1.5000
5hrs-UA
1.0000
1hr-RS
3hrs-RS
0.5000
5hrs-RS
0.0000
60
80
100
Temperature (oC)
Figure 4.8 Comparison of Uronic Acid (UA) and Reducing Sugar (RS) at 70% acid concentration
Page 44
It can be seen from Figure 4.8 that the uronic acid yield followed exactly the
same trend as the reducing sugar yield. However, it should be noted from the results of
our statistical analysis that time has no significant effect on the reducing sugar yield
but is only affected when the temperature changes from 60oC to 80oC. Since the
increase in temperature causes a harsher condition of hydrolysis at 70%
acid
concentration, it is possible that uronic acids have been degraded into other
products such as furfural, reductic acid and 5-formyl-2-furoic acid. However since
the reducing sugar also decreased through the temperature, it is possible that the rate
of degradation of uronic acid towards furfural and 5-formyl-2-furoic acid is greater
than the rate of degradation towards reductic acid because the two compounds are not
detected by DNS.
3.0000
RS/ UA (mg/ml)
2.5000
2.0000
1hr-UA
3hrs-UA
1.5000
5hrs-UA
1.0000
1hr-RS
3hrs-RS
0.5000
5hrs-RS
0.0000
60
80
100
Temperature (oC)
Figure 4.9 Comparison of Uronic acid (UA) and Reducing Sugar (RS) at 80% acid concentration
For Figure 4.9, it can be seen that the reducing sugar still decreased when the
temperature increased from 60oC to 80oC however the uronic acid increased with the
change in temperature. Possible reason for this is that the change in the acid
concentration might have increased the rate of hydrolysis of alginate into uronic acid.
However, since the conditions are much worse, it might have followed the same scenario
as the previous one. It could have been that the rate at which uronic acid degrades into
RESULTS AND DISCUSSION
Page 45
furfural or 5-formyl-2-furoic acid is still greater than the rate of degradation towards
reductic acid.
For Figure 4.10, this graph shows a totally different trend as compared from the
previous figures. The reducing sugar increased with the temperature. However for uronic
acid, the increase stopped after reaching 80oC. Most possible explanation for this
phenomenon is that the high acid concentration might have caused the rate of hydrolysis
of alginate to increase but when temperature reached 100oC, degradation has become
more severe. In addition to that, since this is at a very much harsher condition from the
previous two, the rate of degradation might have increase towards the formation of
reductic acid instead of 2-furfural or 5-formyl-2-furoic acid. It is also possible that
furfural have been further decarboxylated into reductic acid from the severe hydrolysis
condition since it is a precursor of reductic acid (Feather and Harris, 1973).
1.4000
RS/ UA (mg/ml)
1.2000
1.0000
1hr-UA
0.8000
3hrs-UA
0.6000
5hrs-UA
1hr-RS
0.4000
3hrs-RS
0.2000
5hrs-RS
0.0000
60
80
100
Temperature (oC)
Figure 4.10 Comparison of Uronic Acid (UA) and Reducing Sugar (RS) at 90% acid concentration
The effect of acid concentration with regard to the increasing rate of hydrolysis of
alginate mentioned before satisfied the findings of Smidsrod et. al (1969 and 1966) and
Holtan et. al (2006). The increasing rate of hydrolysis is caused by the un-dissociated
carboxyl group of the polymer that provoke an intramolecular autocatalysis of the
reaction.
RESULTS AND DISCUSSION
Page 46
Microorganism
Pseudoalteromonas citrea
Optimal
Temperature (oC)
References
35 -45
33
Chauchan (1993)
40
26
KMM3297
40
For this study, an isolated marine bacterium of BIOTECH, UPLB from mangrove
waters was tested for its enzymatic activity on commercial alginate samples. The strain
was pre-selected based on the diameter of clearing on cetylpyridinimchloride. In the
evaluation of enzymatic hydrolysis of commercial alginate samples, the enzymes were
semi-purified first using ammonium sulphate (NH4)2SO4 precipitation in order to have a
stable and consistent enzymatic activity. The result of the determination of activity of the
semi-purified enzyme was summarized below.
Table 4.11 Enzymatic Activity Determination
Enzyme Volume (ml) Protein (mg) Reducing Sugar (mg) Sp. Acitivity
1.0275
0.0500
0.0125
Semi
0.5000
Page 47
Parameters tested for the semi-purified enzyme were only temperature and
incubation with response only limited to reducing sugar due to difficulties in the analysis
of uronic acid. Table 4.12 summarized the effect of time and temperature on the reducing
sugar yield from the enzymatic hydrolysis.
Table 4.12 Effect of Time and Temperature on Enzymatic Hydrolysis
Reducing Sugar (mg/ml)
Time(hrs)
37oC
40oC
45oC
24.0000
0.0517
0.0389
0.0367
48.0000
0.1403
0.0887
0.0805
72.0000
0.2317
0.1963
0.1289
0.2500
0.2000
0.1500
37C
40C
0.1000
45C
0.0500
0.0000
24.00
-0.0500
48.00
72.00
Time (hrs)
Figure 4.11 Effect of time and temperature on enzymatic hydrolysis of commercial alginate
RESULTS AND DISCUSSION
Page 48
It can be seen through the figure that as time increased the amount of
reducing sugar also increased. However, as temperature increased the reducing sugar
decreased. A 3! CRD analysis on whether time has a significant effect on the reducing
sugar yield was seen on Table 4.13
Table 4.13 3! CRD Analysis for Effect of Time on Reducing Sugar Yield
Level of significance,
Error degrees of freedom
Error mean square
0.05
15
0.00103
CriticalofRange
Number
means
0.02278
2
0.02388
3
Duncan groupinga
A
B
C
Mean (mg/ml)
0.1856
0.1032
0.0424
Time (hrs)
72
48
24
As seen from Table 4.13, time significantly affected the amount of reducing
sugar being produced from enzymatic hydrolysis. It showed that as time increased, the
reducing sugar and the per cent conversion of alginate increased.
For the temperature, the summary of 3! CRD analysis is presented in Table 4.14
Table 4.14 3! CRD Analysis for Effect of Temperature on Reducing Sugar Yield
0.05
Level of significance,
15
Error degrees of freedom
0.00446
Error mean square
Number
means
CriticalofRange
2
0.04743
3
0.04973
Duncan A
groupinga
B
B
0.1412
Mean
(mg/ml)
0.1080
0.0820
37
Temperature
(oC)
40
45
Page 49
As seen Table 4.14, there was a significant effect on the amount of reducing sugar
yield from 37oC to 40oC after which it became insignificantly different until it reached
45oC.
The results of the enzymatic hydrolysis of alginate samples were quite satisfactory
as the data was in agreement with the findings from several studies about the properties
of alginases from microorganisms. As seen from the effect of temperature, the enzyme
produced higher reducing sugar from 37oC as compared to higher temperatures.
Comparing it with the typical optimum values of alginases from several microorganisms
from Table 4.10 showed that the semi-purified enzymes highest yielding temperature
lies within the range thus following the general property of alginases.
Although the mechanism through which alginases catalyses the degradation of
alginates is not yet fully elucidated, it has been the general finding that the enzymes
degrade alginate through a beta-elimination mechanism that release unsaturated
saccharides with C=C double at their non-reducing terminal urinate residues (Wong,
Preston and Chiller, 2000; Yamasaki, Ogura, Hashimoto, Mikami and Murata, 2005).
The figure below showed the cleavage sites for alginate lyase reactions.
Figure 4.12 Block sites of alginate polymer and alginate lyase reaction. (a) MM; (b) GG; (c) MG
block sites. Vertical and horizontal arrows indicate cleavage sites for alginate lyase reaction and
reaction schemes. (Yamasaki et. al, 2005)
RESULTS AND DISCUSSION
Page 50
Page 51
optimum condition using the raw enzyme. Table 4.15 and 4.16 showed the optimum
chemical and enzymatic hydrolyses their corresponding reducing sugar/ uronic acid yield.
Table 4.15 Chemical hydrolysis of Commercial and Extracted Alginate using the optimum
hydrolysis condition
Alginate Source
Commercial Alginate
2.5963
Average
Uronic Acid
(mg/ml)
1.1967
Extracted Alginate
0.0005
0.0283
Alginate Source
Commercial Alginate
0.1289
Extracted Alginate
0.9915
Based on the values presented in Table 4.15, it showed that hydrolysis using
commercial alginate yielded higher values both for uronic acid and reducing sugar.
However, in Table 4.16, extracted alginate yielded higher value of reducing sugar in
comparison with the commercial alginate. The most probable explanation for these
results was the nature of the commercial and extracted alginates. Considering that the
reducing sugar decreased when chemical hydrolysis was conducted on extracted alginate
and it increased when enzymatic hydrolysis was employed, it is possible that the
extracted alginate has a more readily hydrolysable characteristic than commercial
alginate. This means that milder condition would be enough to hydrolyse the extracted
alginate. Since the chemical hydrolysis employed a harsher condition for extracted
alginate in relation to its nature, it is possible that the extracted alginate severely
degraded into 2-furfural or 5-formyl-2-furoic acid and not reductic acid. It can be be
explained through the reducing sugar and uronic acid values given in Table 4.15. Another
explanation for that is through the yields of enzymatic hydrolysis. The enzyme did not
Page 52
have much difficulty hydrolysing the extracted alginate resulting into much higher yield
as compared to commercial alginate.
To compare the yields of both chemical and enzymatic hydrolysis, Table 4.17 is
presented below. The percent yield was calculated based on the initial amount of alginate
to be hydrolysed which were 4mg/ml for enzymatic and 20mg/ml for chemical.
Table 4.17 Comparison of Chemical and Enzymatic Hydrolysis
Hydrolysis Condition
Semi-purified enzyme, 45oC, 72 hrs
Average
Reducing Sugar
(mg/ml)
0.9915
0.0005
Average %
Yield
24.7872
0.0025
Page 53
CHAPTER FIVE
SUMMARY AND CONCLUSIONS
This study was conceptualized in order to develop a hydrolysis procedure for the
alginate component of seaweeds for bioethanol production. Two types of hydrolysis were
considered namely the chemical and the enzymatic hydrolysis of alginates.
The parametric study for chemical hydrolysis was conducted using commercial
alginate in order to determine the effect of temperature (60oC, 80oC, and 100oC), acid
concentration (70%, 80% and 90%) and time (1hr, 3hrs, and 5hrs) on the reducing sugar
and uronic acid yield of the hydrolysis. It was found out that both the temperature and
acid concentration has a significant effect on the reducing sugar yield. Result showed that
the reducing sugar yield had a peak value at 60oC and 80% acid concentration and further
increase in those values results in the decrease of yield. However, unlike the two
parameters, time does not have a significant effect on reducing sugar yield.
For the uronic acid, it was found out that all three parameters affect the yield.
Results showed that the uronic acid yield decreases with time. Meanwhile for
temperature and acid concentration, it was found out that further increase of temperature
from 80oC results in the decrease of uronic acid yield and an increase in the acid
concentration from 80% also cause a decrease in the uronic acid yield.
Possible explanation for the observed effects of parameters was mainly due to the
degradation of uronic acid formed after being hydrolysed from the alginate. Uronic acids,
SUMMARY AND CONCLUSIONS
Page 54
after being released from the alginate polysaccharide undergo decarboxylation into 2furfuraldehyde, 5-formyl-2furoic acid and reductic acid at the hydrolysis condition. The
increase in the rate formation of 2-furfuraldehyde and 5-formyl-2-furoic acid which are
compounds not detectable by DNS are the reason for the sudden decrease in reducing
sugar and uronic acid yield after 80oC and 80% acid concentration. Meanwhile the
formation of reductic acid causes the DNS reading for reducing sugar to increase and the
uronic acid reading to decrease. Based on the results of the experiment, optimum
condition for chemical hydrolysis was found to be at 60 oC, 80% acid concentration for
1hour which yielded a reducing sugar and uronic acid of 2.5963 mg/ml and 1.1967 mg/ml
respectively.
For the enzymatic hydrolysis, the parametric study was conducted using
commercial alginate to determine the effect of time and temperature on the reducing
sugar yield. It was found out that both time and temperature had a significant effect on
the reducing sugar yield. Results showed that reducing sugar yield increases through time
while it decreases with temperature. The results of the experiment were in agreement
with results of various studies conducted regarding the properties of the alginate lyases.
Optimum condition was found to be at 37oC and 72 hours which yielded a reducing sugar
of 0.2317 mg/ml.
Optimum conditions for both chemical and enzymatic hydrolysis were evaluated
using the extracted alginate from the seaweed samples. However, optimum condition for
the enzymatic hydrolysis was not conducted at the above conditions. Instead it was
conducted at 45oC and 72 hours. Results showed reducing sugar yields of 0.0005 mg/ml
and 0.9915 mg/ml for chemical and enzymatic hydrolysis respectively. As seen from the
SUMMARY AND CONCLUSIONS
Page 55
values enzymatic hydrolysis yielded the highest reducing sugar as compared to the
chemical hydrolysis. It was also found out that the reducing sugar yield obtained from
enzymatic hydrolysis of extracted alginate was higher compared to the commercial
alginate implying an efficient hydrolysis by the enzyme. However, acid hydrolysis of the
extracted alginate showed a very low reducing sugar yield as compared to the
commercial alginate.
The highest reducing sugar yield among all treatments was used for the
determination of bioethanol potential of the seaweed using stoichiometric relationship. It
was found out that bioethanol yield is very low such that distillation of the hydrolysates
for ethanol purification is no longer feasible.
Page 56
CHAPTER SIX
RECOMMENDATIONS
Only the individual effects of time, acid concentration and temperature on the
reducing sugar and uronic acid yield for the chemical hydrolysis and the individual
effects of time and temperature for enzymatic hydrolysis were studied for this
experiment. However, it can be clearly seen from the results of the experiment that
parametric interaction are present in the hydrolysis of alginate. Thus, it is highly
recommended to study whether the interactions between the parameters cause a
significant effect on the reducing sugar and uronic acid yield.
In addition to that, this study only evaluated the optimum conditions based on the
highest amount of reducing sugar yield obtained from the various treatments. Use of
software such as Design Expert for the determination of optimum condition might
provide significant insights and more meaningful results for the hydrolysis of alginate as
this present complete statistical analysis of regarding the effects of each parameters and
the existing interaction among them.
Since it was found that chemical hydrolysis of alginate leads to significant losses
of uronic acid upon degradation; it is therefore recommended to conduct a thorough study
for the enzymatic hydrolysis instead. This not only leads to higher yields of reducing
sugar and uronic acid but also prevents the formation of inhibitory compounds that might
hinder fermentation during the bioethanol production. In addition to that purified enzyme
could also provide more meaningful results for the production of hydrolysates from
RECOMMENDATIONS
Page 57
alginate. Moreover, other parameters which might also affect the yield of hydrolysates
could also be studied such as pH, enzyme loading and substrate concentration.
The extraction of alginate from the seaweed material was not properly conducted
due to the insufficient capacity of the stirring equipment. It is therefore recommended to
use a stirrer which will be able to handle the extraction procedure to obtain higher yields
of alginate.
Lastly, hydrolysates could also be evaluated for bioethanol production using
ethanologenic microorganisms like Zymomonas and some yeast strains such as Pichiaand
Kluyveromyces. These strains of microorganisms were suggested since it has already
been found out that Saccharomyces cerevisiae could not utilize uronic acid for bioethanol
production (van Maris et. al, 2006).
RECOMMENDATIONS
Page 58
REFERENCES
BADGER, P.C. (2002).Trends in new crops and new uses. J. Janick and A. Whipkey
(eds.) ASHS Press, Alexandria, VA. pp. 17 -21
BENSON, P.H and K.T. BIRD.(1987). Renewable Energy Systems. The Netherlands:
Elsevier Science Publishers B. V.pp 5 12.
DIEN, B.S., M.A. COTTA and T. W. JEFFRIES. (2003). Bacteria engineered for fuel
ethanol production: current status. Appl Microbiol Biotechnol 63: 258-266.
FAN,
Dehydration
Reactions of
GOODMAN, L.J. and R.N. LOVE.(1981). Biomass Energy Projects, Planning and
Management. Pergamon Press, Hawaii.184 p.
HORN, S., and K. OSTGAARD. (2000a). Ethanol production from seaweed extract.J
Ind Microbiol Biotechnol25: 249-254.
INMAN, M. (2008). Clearing Lands for Biofuels Makes Global Warming Worse.
National Geographic News.
JI, M. H. and Y.X. ZHANG.(1962). Studies on the chemical composition of the Chinese
Economic Brown Seaweeds. Oceanolet Limnol Sin 4(3-4): 161-168.
MILLET, M.A., A.J. BAKER and L.D. SATTER.(1976). Physical and chemical pre
treatment for enhancing cellulose saccharification. Biotechnol. And Bioeng.
Symp 6: 125 153.
MOLLER, R., S. HAKE and M. PAULY. (2006). EPOBIO project: Cell wall
saccharification. CPL Press, UK. pp 9 41.
TRONO, G. C. Jr. (1999). Diversity of the seaweed flora of the Philippines and its
utilization. Hydrobiologia 398/399: 1-6.
http://www.doe.gov.ph/AF/Bioethanol.htm
http://en.wikipedia.org/wiki/Mannitol
APPENDIX A
STANDARD CURVES
APPENDIXA1
PROTEIN STANDARD CURVE
Table A.1 Data for the Standard Curve for Lowrys Method of Protein Determination
Concentration, mg/ml
Trial 1
Trial 2
0.00
0.00
0.10
0.10
0.20
0.20
0.30
0.30
0.40
0.40
Absorbance
Trial 1
Trial 2
0.0000
0.0000
0.2610
0.2820
0.4510
0.4530
0.6900
0.6890
0.8820
0.8970
Average
0.0000
0.2715
0.4520
0.6895
0.8895
1.0000
Absorbance
0.8000
y=2.197x+
R=
0.6000
0.4000
0.2000
0.0000
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
Concentration(mg/ml)
0.45
APPENDIX A2
REDUCING SUGAR STANDARD CURVE
Table A.2 Data for the Standard Curve of Millers DNS Method for ReducingSugar
Concentration, mg/ml
Absorbance
Trial 1
Trial 2
Trial 1
Trial 2
Average
0.00
0.00
0.0000
0.0000
0.0000
0.20
0.20
0.3470
0.3450
0.3460
0.30
0.30
0.5280
0.5480
0.5380
0.40
0.40
0.7310
0.7130
0.7220
0.50
0.50
0.9210
0.9190
0.9200
1.0000
0.8000
y=1.8386x-0.0096
R=0.9992
Absorbance
0.6000
0.4000
0.2000
0.0000
0.00
-0.2000
0.10
0.20
0.30
0.40
0.50
Concentration(mg/ml)
0.60
APPENDIX A3
URONIC ACID STANDARD CURVE
Table A.3 Data for the Standard Curve for Uronic Acid Determination
Concentration, mg/ml
Absorbance
Trial 1
Trial 2
Trial 1
Trial 2
Average
0.00
0.00
0.0000
0.0000
0.0000
0.10
0.10
0.5150
0.5150
0.5150
0.30
0.30
1.7800
1.7760
1.7780
0.40
0.40
2.2910
2.2870
2.2890
0.50
0.50
2.7080
2.7100
2.7090
3.0000
2.5000
Abosrobance
2.0000
y=5.5776x+
R=
1.5000
1.0000
0.5000
0.0000
0.00
0.10
0.20
0.30
0.40
0.50
Concentration(mg/ml)
0.60
APPENDIX B
RAW DATA FOR REDUCING
SUGAR ANALYSIS:
CHEMICAL HYDROLYSIS
APPENDIX B
REDUCING SUGAR ANALYSIS FOR CHEMICAL HYDROLYSIS
RS
Trial1
(Abs)
0.535
0.382
0.332
0.238
0.154
0.148
0.211
0.182
0.165
mg/ml
2.462
1.758
1.528
1.095
0.709
0.681
0.971
0.837
0.759
RS
Trial2
(Abs)
0.535
0.386
0.330
0.238
0.154
0.147
0.206
0.182
0.168
mg/ml
2.462
1.776
1.518
1.095
0.709
0.676
0.948
0.837
0.773
Average
(mg/ml)
2.462
1.767
1.523
1.095
0.709
0.679
0.959
0.837
0.766
60
80
80
100
RS
Trial1
(Abs)
mg/ml
RS
Trial2
(Abs)
mg/ml
0.543
2.552
0.562
2.641
2.596
0.509
2.392
0.502
2.359
2.375
0.411
1.931
0.412
1.936
1.934
0.484
2.274
0.485
2.279
2.277
0.443
2.082
0.438
2.058
2.070
0.408
1.917
0.409
1.922
1.920
0.249
1.170
0.245
1.151
1.161
0.235
1.104
0.234
1.100
1.102
0.205
0.963
0.208
0.977
0.970
Average
(mg/ml)
Table B.3 Raw Data Reducing Sugar Analysis at 90% Acid Concentration
Acid
Reaction
Temperature
Concentration
Time
o
(C)
(%)
(hrs)
60
90
80
100
RS
Trial1
(Abs)
mg/ml
RS
Trial2
(Abs)
mg/ml
0.154
0.739
0.155
0.744
Average
(mg/ml)
0.741
0.126
0.604
0.127
0.609
0.607
0.120
0.576
0.120
0.576
0.576
0.213
1.022
0.214
1.027
1.024
0.205
0.983
0.205
0.983
0.983
0.170
0.815
0.173
0.830
0.823
0.277
1.329
0.278
1.334
1.331
0.258
1.238
0.253
1.214
1.226
0.207
0.993
0.204
0.979
0.986
APPENDIX C
RAW DATA FOR URONIC
ACID ANALYSIS:
CHEMICAL HYDROLYSIS
APPENDIX C
URONIC ACID ANALYSIS FOR CHEMICAL HYDROLYSIS
Table C.1 Raw Data for Uronic Acid Analysis at 70%AcidConcentration
Acid
Reaction
Temperature
Concentration
Time
o
( C)
(%)
(hrs)
60
70
80
100
UA
Trial1
Abs
UA
Trial2
Abs
0.976
0.975
1.480
1.479
1.480
0.811
0.809
1.230
1.227
1.229
0.702
0.702
1.065
1.065
1.065
0.793
0.789
1.203
1.197
1.200
0.599
0.597
0.909
0.906
0.907
0.4318
0.4316
0.655
0.655
0.655
0.494
0.482
0.749
0.731
0.740
0.409
0.405
0.620
0.614
0.617
0.374
0.372
0.567
0.564
0.566
Table C.2 Raw Data for Uronic Acid Analysis at 80%Acid Concentration
Acid
Reaction
Temperature
Concentration
Time
o
(C)
(%)
(hrs)
60
80
80
100
UA
Trial1
Abs
UA
Trial2
Abs
0.772
0.773
1.196
1.197
1.197
0.675
0.674
1.046
1.044
1.045
0.412
0.411
0.638
0.637
0.637
0.898
0.897
1.391
1.390
1.390
0.815
0.825
1.262
1.278
1.270
0.638
0.641
0.988
0.993
0.991
0.669
0.668
1.036
1.035
1.036
0.509
0.506
0.788
0.784
0.786
0.456
0.46
0.706
0.713
0.709
Table C.3 Raw Data for Uronic Acid Analysis at 90%Acid Concentration
Acid
Reaction
Temperature
Concentration
Time
(oC)
(%)
(hrs)
60
90
80
100
UA
Trial1
Abs
UA
Trial2
Abs
0.301
0.302
0.476
0.478
0.477
0.276
0.278
0.436
0.440
0.438
0.243
0.245
0.384
0.387
0.386
0.448
0.45
0.708
0.712
0.710
0.446
0.443
0.705
0.701
0.703
0.387
0.394
0.612
0.623
0.618
0.414
0.413
0.655
0.653
0.654
0.392
0.39
0.620
0.617
0.618
0.382
0.376
0.604
0.595
0.599
APPENDIX D
RAW DATA FOR
ENZYMATIC HYDROLYSIS
APPENDIX D
ENZYMATIC HYDROLYSIS OF COMMERCIAL ALGINATES
Reducing Sugar(mg/ml)
Ave
Reducing
Sugar
(mg/ml)
Time(hrs)
T1
T2
T1
T2
24.00
0.0950
0.0950
0.0517
0.0517
0.0517
48.00
0.1300
0.1280
0.1414
0.1392
0.1403
72.00
0.2100
0.2160
0.2284
0.2350
0.2317
Reducing Sugar(mg/ml)
Average
Reducing
Sugar
(mg/ml)
Time(hrs)
T1
T2
T1
T2
24.00
0.0720
0.0710
0.0392
0.0386
0.0389
48.00
0.0810
0.0820
0.0881
0.0892
0.0887
72.00
0.1830
0.1780
0.1991
0.1936
0.1963
Reducing Sugar(mg/ml)
Average
Reducing
Sugar
(mg/ml)
Time(hrs)
T1
T2
T1
T2
24.00
0.0670
0.0680
0.0364
0.0370
0.0367
48.00
0.0750
0.0730
0.0816
0.0794
0.0805
72.00
0.1180
0.1190
0.1284
0.1294
0.1289
APPENDIX E
EVALUATION OF
CHEMICAL AND
ENZYMATIC HYDROLYSIS
APPENDIX E
EVALUATION OFCHEMICAL AND ENZYMATIC HYDROLYSIS
TableE.1 Chemical Hydrolysis of Extracted Alginate Samples at OptimumCondition
Analysis
Absorbance
Concentration (mg/ml)
Average
%Yield
Trial1
Trial2
Trial1
Trial2
Reducing
Sugar
Analysis
0.001
0.001
0.0005
0.0005
0.0025
Uronic Acid
Analysis
0.160
0.156
0.0287
0.0280
0.2833
Analysis
Reducing
Sugar
Analysis
Absorbance
Concentration (mg/ml)
Trial1
Trial2
Trial1
Trial2
0.001
0.001
0.0005
0.0005
Average
%Yield
24.7872
APPENDIX F
SAMPLE CALCULATIONS
APPENDIX G
STATISTICAL ANALYSIS
APPENDIX G1
STATISTICAL ANALYSIS: Analysis of Variance (ANOVA)
Table G1.1 Analysis of Variance for the Effect of Time on Reducing Sugar
Source of Variation
df
SS
MS
Between Treatments
Error (within
treatments)
1.3466
0.6733
1.8078
51
18.9943
0.3724
Total
53
Decision
Accept
Table G1.2 Analysis of Variance for the Effect of Time on Uronic Acid
Source of Variation
df
SS
MS
Between Treatments
Error (within
treatments)
0.7850
0.3925
4.9380
51
4.0540
0.0795
Total
53
Decision
Reject
Table G1.3 Analysis of Variance for the Effect of Temperature on Reducing Sugar
Sourceof Variation
df
SS
MS
Between Treatments
Error (within
treatments)
3.0746
1.5373
4.5408
51
17.2663
0.3386
Total
53
Decision
Reject
Table G1.4 Analysis of Variance for the Effect of Temperature on Uronic Acid
Source of Variation
df
SS
MS
Between Treatments
Error (within
treatments)
0.5459
0.2729
3.2423
51
4.2931
0.0842
Total
53
Decision
Reject
TableG1.5 Analysis of Variance for the Effect of Acid Concentration on Reducing Sugar
Source of Variation
df
SS
MS
Between Treatments
Error (within
treatments)
7.6623
3.8312
15.4110
51
12.6786
0.2486
Total
53
Decision
Reject
TableG1.6 Analysis of Variance for the Effect of Acid Concentration on Uronic Acid
Source of Variation
df
SS
MS
Between Treatments
Error (within
treatments)
1.9148
0.9574
16.6984
51
2.9241
0.0573
Total
53
Decision
Reject
Source of Variation
Between Treatments
Error (within
treatments)
Total
df
2
SS
0.0620
MS
0.0310
F
30.1540
15
17
0.0154
0.0010
Decision
Reject
Source of Variation
Between Treatments
Error (within
treatments)
Total
df
2
SS
0.0106
MS
0.0053
F
1.1850
15
17
0.0669
0.0045
Decision
Accept
Where: df= degrees of freedom, F = test statistic, MS= mean squares, SS= sums of squares
Note: A decision of Reject signifies that at least 2of the treatment means are significantly
different. An Accept decision signifies that treatment means are not significantly
different.
APPENDIX G2
STATISTICAL ANALYSIS: Duncans Multiple Range Test (DMRT)
Table G2.1 3! CRD Analysis for the Effect of Time on Reducing Sugar
Effect of Time
a
0.05
SE
51
MSR
3.724E-01
Number of Means
LSR
2
2.84
3
2.99
Mean Value
x1
1.1306
x2
1.2865
x3
1.5162
2v1
0.1559
3v2
0.2297
3v1
0.3856
0.1438
Critical Range
0.408515995
0.430092544
Level
5
3
1
insignificant
insignificant
insignificant
Table G2.2 3! CRD Analysis for the Effect of Temperature on Reducing Sugar
Effect of Temperature
a
0.05
SE
51
MSR
3.386E-01
Number of Means
LSR
2
2.84
3
2.99
Mean Value
x1
1.0376
x2
1.2865
x3
1.6200
2v1
0.2489
3v2
0.3335
3v1
0.5824
0.1371
Critical Range
0.389490065
0.410061723
Level
100
80
60
insignificant
insignificant
significant
TableG2.3 3! CRD Analysis for the Effect of Acid Concentration on Reducing Sugar
Table G2.4 3! CRD Analysis for the Effect of Time on Uronic Acid
Effect of Time
a
0.05
SE
51
MSR
7.949E-02
Number of Means
LSR
Critical Range
2.84
0.188727989
2.99
0.198696016
Mean Value
0.0665
Level
x1
0.6917
x2
0.8459
x3
0.9870
2v1
0.1542
insignificant
3v2
0.1410
insignificant
3v1
0.2952
significant
Table G2.5 3! CRD Analysis for the Effect of Temperature on Uronic Acid
Effect of Temperature
a
0.05
SE
51
0.0684
MSR
8.418E-02
Number of Means
LSR
Critical Range
2
2.84
0.194215395
3
2.99
0.20447325
Mean Value
Level
x1
0.7029
100
x2
0.8836
60
x3
0.9381
80
2v1
0.1807
insignificant
3v2
0.0545
insignificant
3v1
0.2352
significant
Table G2.6 3! CRD Analysis for the Effect of Acid Concentration on Uronic Acid
Effect of Acid Concentration
a
0.05
SE
51
0.0564
MSR
5.734E-02
Number of Means
LSR
Critical Range
2
2.84
0.16028635
3
2.99
0.168752178
Mean Value
Level
x1
0.5781
90
x2
0.9398
70
x3
1.0068
80
2v1
0.3617
significant
3v2
0.0670
insignificant
3v1
0.4287
significant
Table G2.7 3! CRD Analysis for the Effect Time on Enzymatic Hydrolysis
Effect of Time
a
0.05
SE
15
MSR
1.028E-03
Number of Means
LSR
2
3.014
3
3.16
Mean Value
x1
0.0424
x2
0.1032
x3
0.1856
2v1
0.0607
3v2
0.0825
3v1
0.1432
0.0076
Critical Range
0.022779853
0.023883323
Level
24
48
72
significant
significant
significant
Table G2.8 3! CRD Analysis for the Effect Temperature on Enzymatic Hydrolysis
Effect of Temperature
a
0.05
SE
15
MSR
4.458E-03
Number of Means
LSR
2
3.014
3
3.16
Mean Value
x1
0.0820
x2
0.1080
x3
0.1412
2v1
0.0259
3v2
0.0333
3v1
0.0592
0.0157
Critical Range
0.047432088
0.049729727
Level
45
40
37
insignificant
insignificant
significant
Where: a =significance level, SE = error degrees of freedom, MSR = error mean square,
LSR =least significant studentized range
APPENDIX H
MATERIAL SAFETY
DATA SHEETS