Parametric Study On Chemical and Enzymatic Hydrolysis of Alginates From Sargassum Cristaefolium

PARAMETRIC STUDY ON CHEMICAL AND ENZYMATIC HYDROLYSIS
OF ALGINATES FROM Sargassum cristaefolium C.A. Agardh (Phaeophyta)

FOR BIOETHANOL PRODUCTION
MICHAEL ANGELO MELO VIRAY
SUBMITTED TO THE FACULTY OF THE

COLLEGE OF ENGINEERING AND AGRO-INDUSTRIAL TECHNOLOGY
UNIVERSITY OF THE PHILIPPINES, LOS BAOS
IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE
OF
BACHELOR OF SCIENCE IN CHEMICAL ENGINEERING
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.
Dr. Jovita L. Movillon

Panel Member
Prof. Denise Ester O. Santiago

Panel Member
Date Signed
Date Signed
Dr. Jessica F. Simbahan
Panel Member
Date Signed
Ms. Irene G. Pajares

Co-adviser
Dr. Milagrosa Goss

Co-adviser
Date Signed
Date Signed
Prof. Rex B. Demafelis
Adviser
Date Signed
Dr. Jovita L. Movillon

Chair
Department of Chemical Engineering
Date Signed
Dr. Arsenio N. Resurreccion

Dean
College of Engineering and Agro-Industrial Technology
University of the Philippines Los Baos
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.
Viray, Michael Angelo M.
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
1.2 Objectives of the Study
1.3 Date and Place of the Study
1.4 Scope and Limitations of the Study
REVIEW OF LITERATURE
2.1 Biofuels
2.1.1 Bioethanol
2.1.2 Bioethanol Production
2.1.2.1 Pretreatment
2.1.2.2 Hydrolysis
10
2.1.2.2.1 Chemical Hydrolysis
10
2.1.2.2.2 Enzymatic Hydrolysis
12
2.1.2.3 Fermentation
13
2.1.3 Issues and Concerns
13
2.2 Macroalgae
2.2.1 Production and Use
15
2.2.2 Brown Algae
16
2.2.2.1 Cell Wall Structure
17
2.2.2.1.1 Alginic Acid
17
2.2.2.1.2 Fucoidan
18
2.2.2.1.3 Cellulose
19
2.2.2.2 Storage Products
2.2.2.2.1 Mannitol
20
2.2.2.2.2 Laminarin
20
2.2.3 Sargassum spp.
20
2.2.3.1 As Bioethanol Feedstocks
21
2.3 Related Studies on Hydrolysis of Macroalgae
23
2.4 Related Studies on Alginate Hydrolysis
25
MATERIALS AND METHODS

3.1 Feedstock Preparation
27
3.2 Extraction Procedure
27
3.3 Chemical Hydrolysis Procedure
28
3.4 Enzymatic Hydrolysis Procedure
29
3.4.1 Microorganism and Enzyme Production
29
3.4.2 Enzymatic Hydrolysis Proper
31
3.5 Analytical Methods
19
32
3.5.1 Reducing Sugar Analysis
32
3.5.2 Uronic Acid Analysis
32
3.5.3 Protein Determination
33
RESULTS AND DISCUSSION

4.1 Acid Hydrolysis of Commercial Alginate Samples
34
4.1.1 Effect of Parameters on the Reducing Sugar Yield
34
4.1.2 Effect of Parameters on the Uronic Acid Yield
39
4.2 On the Hydrolysis of Alginate Samples
43
4.3 Enzymatic Hydrolysis of Commercial Alginate Samples
47
4.4 Evaluation of Optimum Hydrolysis Condition on Extracted Alginate
51
4.5 Bioethanol Potential of Seaweed Hydrolysates
53
5
6
SUMMARY AND CONCLUSION

RECOMMENDATIONS
54
57
REFERENCES
59
APPENDICES
66
A. Standard Curves
66
B. Raw Data for Reducing Sugar Analysis: Chemical Hydrolysis
70
C. Raw Data for Uronic Acid Analysis: Chemical Hydrolysis
73
D. Raw Data for Enzymatic Hydrolysis
76
E. Evaluation of Chemical and Enzymatic Hydrolysis
78
F. Sample Calculations
80
G. Statistical Analysis
84
H. Material Safety Data Sheets
92
LIST OF TABLES
Table #
Title
Page
2.1
Comparison of Bioethanol against Unleaded Gasoline
2.2
Comparison between Concentrated- and Dilute-Acid Hydrolysis
12
Methods
2.3
Comparison between acid and enzymatic hydrolysis
13
2.4
Terrestrial and Marine Photosynthetic Productivity
16
2.5
Chemical Composition of Various Sargassum species
21
2.6
A comparison between the major bioethanol crops and macroalgae
22
2.7
Chemical and Enzymatic Hydrolysis of Various Brown Macroalgae
24
4.1
Reducing Sugar Concentration (mg/ml) at different hydrolysis
35
conditions
4.2
3! CRD Analysis for Effect of Time on Reducing Sugar
37
4.3
3! CRD Analysis for Effect of Temperature on Reducing Sugar
38
4.4
3! CRD Analysis for Effect of Acid Concentration on Reducing Sugar
38
4.5
Uronic Acid Concentration (mg/ml) at different hydrolysis conditions
39
4.6
3! CRD Analysis for Effect of Time on Uronic Acid
42
4.7
3! CRD Analysis for Effect Temperature on Uronic Acid
42
4.8
3! CRD Analysis for Effect of Acid Concentration on Uronic Acid
43
4.9
Formation of Reductic Acid at Different Conditions
44
4.10
Alginate Lyases from various microorganisms and their optimal
47
temperature
4.11
Enzymatic Activity Determination
47
4.12
Effect of Time and Temperature on Enzymatic Hydrolysis
48
4.13
3! CRD Analysis for Effect of Time on Reducing Sugar Yield
49
4.14
3! CRD Analysis for Effect of Temperature on Reducing Sugar Yield
49
4.15
Chemical hydrolysis of Commercial and Extracted Alginate using the
52
optimum hydrolysis condition

4.16
Enzymatic hydrolysis of Commercial and Extracted Alginate at 45oC
52
and 72hrs
4.17
Comparison of Chemical and Enzymatic Hydrolysis
53
LIST OF FIGURES
Figure #
Title
Page
2.1
Mechanism of pre-treatment of lignocellulosic feedstocks
2.2
Cell wall structures of brown algae
17
2.3
Alginate structural data
18
2.4
Chemical Structure of Cellulose
19
2.5
Pathway for processing brown seaweeds for fuel and other

commercial products
23
3.1
Milled seaweed samples
27
3.2
Gelatinous alginate after precipitation
28
3.3
Chemical Hydrolysis at 80% and 90% acid concentration
29
3.4
Alginate Culture Medium for Enzyme Production
30
3.5
Enzymatic Hydrolysis of Extracted Alginate
31
3.6
Uronic Acid Analyses of Samples
32
4.1
Reducing Sugar Yield at 70% (v/v) Acid Concentration
35
4.2
36
4.3
36
4.4
Uronic Acid Yield at 70% (v/v) acid concentration
39
4.5
Uronic Acid Yield at 80 % (v/v) acid concentration
40
4.6
Uronic Acid Yield at 90% (v/v) acid concentration
41
4.7
Degradation of uronic acid
43
4.8
Comparison of Uronic Acid (UA) and Reducing Sugar (RS) at 70%

acid concentration
44
4.9

acid concentration
45
4.10

acid concentration
46
4.11
Effect of time and temperature on enzymatic hydrolysis of

commercial alginate
48
4.12
Block sites of alginate polymer and alginate lyase reaction
50
4.13
Extracted alginate from raw seaweed material
51
4.14
Chemical Conversion of Uronic Acid to Bioethanol
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
1.1 Significance and Background of the Study

Today, global warming and increasing energy demand brought by rapid growth
of worlds population and industrial developments are driving initiatives for the search
of alternative and renewable resources.
While hydroelectric turbine, photovoltaic cells, geothermal plants, and wind
turbines are generating electricity for commercial and residential uses, liquid biofuels is
the only renewable resource that can be used for transportation which is a major
contributor to global warming (Adams et. al, 2008). Unfortunately, issues on
sustainability of these biofuels are being questioned nowadays due to their contribution
to global warming because of industrial farming methods and their competition for land
and food. As a result, recent researches have diverted on marine biomass like macroalgae
due to their promising advantages.
Macroalgae, commonly known as seaweeds, are vastly cultivated in Asia mainly
for economic purposes. Other than this, seaweeds have also been utilized in the
country as sources of food, phycocolloids (agar, carrageenan and algin), growth
regulators, bioactive compounds and chemicals (Trono, 1999).
As bioethanol feedstocks, they have the advantages over terrestrial plants of fast
growth, removal and conversion of pollutants, and non-requirement of agriculturally
productive land (Ross, 2009).
INTRODUCTION
In addition to that, the very low lignin and high

Page 1
carbohydrate content of seaweeds make it more advantageous because these factors

remove expensive delignification process and produce higher yields involved in the
bioethanol production making it an economically competitive source of biomass.
Several studies have already been conducted regarding the production of ethanol
from seaweeds including those by Horn and Ostgaard (2000a and 2000b) and Adams et.
al (2008). Unfortunately, very little has been done yet in the country.
The alginic acid or alginate (salt compound), the major components of brown
algae, is a polysaccharide containing B-1,4-linked D-mannuronic acid and 1,4-linked Lguluronic acid arranged randomly along the macromolecules (Lewin, 1962) and is very
resistant to hydrolysis by mineral acids. Traditional method commonly employed for the
total liberation of monouronates involves the use of 80% H2SO4. Recent studies have
already developed improved methods for the complete hydrolysis of alginic acid which
resulted in M/G ratios comparable to the traditional method (Anzai et. al, 1990;
Chandia et. al, 2000; Chhatbaret. al, 2009). However, results of these experiments did
not optimize their methods for high monouronate yields.
Thus, this study is conceptualized in order to contribute to the biofuels industry
more specifically to bioethanol production process in the country. This study can provide
significant information regarding the hydrolysis of carbohydrates in seaweeds.
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
1.2 Objectives of the Study

The main objective of this study was to develop methods for the chemical and
enzymatic
hydrolysis of brown macroalgae, Sargassum cristaefolium for bioethanol
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
reducing sugar yield of commercial alginate using enzymatic
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
hydrolysis was conducted at the Thesis Laboratory of the Department of Chemical

Engineering, College of Engineering and Agro-Industrial Technology while enzymatic
hydrolysis was conducted at the Environmental Biotechnology Laboratory (ENVI Lab) of
INTRODUCTION
Page 3
the National Institute for Molecular Biology and Biotechnology (BIOTECH).Analyses of

the samples were done in the ENVI Laboratory.
1.4 Scope and Limitations of Study
This study conducted only the individual effects of time, temperature and acid
concentration on the reducing sugar and uronic acid yield from the acid hydrolysis of
commercial alginate sample. The interactions between these parameters were no longer
investigated. For the enzymatic hydrolysis, only the incubation time and temperature
were only investigated since the enzyme used for the hydrolysis was only semipurified. Other parameters such as substrate concentration, enzyme-substrate ratio and
pH were no longer investigated. In addition the microorganism which was used as
enzyme source was no longer identified. Lastly, the optimization procedure was done
based on the highest yielding conditions for both acid and enzymatic hydrolysis.
INTRODUCTION
Page 4
CHAPTER TWO
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
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.
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.
Page 7
2.1.2 BIOETHANOL PRODUCTION

In early years, bioethanol was produced from sugar feedstocks which are directly
converted into ethanol by the process of fermentation. However, due to the increasing
demand of bioethanol today, alternative sources of bioethanol feedstocks were
considered. In the recent years, researchers have already developed techniques for the
production of bioethanol from polymer-containing feedstocks such as starch, cellullosic
and lignocellulosic biomass. These techniques comprise mainly of two primary processes
which are pre-treatment and hydrolysis or saccharification.
2.1.2.1 PRETREATMENT
Pre-treatment methods refer to the solubilisation and separation of one or more of
the four major components of biomass (hemicellulose, cellulose, lignin, and extractives)
to make the remaining solid biomass more accessible to further chemical or biological
treatment (Demirbas, 2005). It is employed in order to alter the chemical structure of the
carbohydrates (cellulose, hemicellulose, lignin and extractives) present in biomass so that
higher yields of monomeric sugars can be achieved. Structural properties being altered
during the pre-treatment process include the solubility, crystallinity, available surface
area and the pore volume of the carbohydrates. A figure illustrating the mechanism of
pre-treatment process is shown in Figure 2.1.
Page 8
Pre-treatment processes are generally classified into three types namely: a)

physical, b) chemical and c) physico-chemical.
Figure 2.1 Mechanism of pre-treatment of lignocellulosic feedstocks

(Hsu et. al, 1980 as cited by Harmsenet. al, 2010)
Physical pre-treatment processes include milling, grinding, extrusion and

expansion which are generally employed to reduce the size and increase the surface area
available of the feedstocks.In 1976, Millet et. al found that effective break down of
cellulose crystallinity and improvement of its digestibility can be achieved by milling.
Chemical pre-treatment process, on the other hand, involves the use alkali, acids
and cellulose solvents that reacts with the feedstocks carbohydrates and alter their
structural properties. Lastly, physic-chemical pre-treatment process involves the
combination of the physical and chemical pre-treatment. Techniques under this type of
pre-treatment include steam explosion, ammonia fiber explosion (AFEX) and wet
oxidation. AFEX is found to significantly improve the hydrolysis rates of various
herbaceous crops and grasses (Reshamwala et. al, 1995). While among the techniques
Page 9
mentioned, steam explosion is the most widely used physico-chemical pre-treatment

method for the lignocellulosic biomass (McMillan, 1994).
2.1.2.2 HYDROLYSIS
After the pre-treatment process, the next step involves the hydrolysis or
saccharification of the carbohydrates. This process is the breaking down of complex
carbohydrates into their monomeric sugar constituents through the use chemicals or
enzymes. The efficiency of this process is generally affected by the feedstock properties,
the hydrolysis conditions, and the pre-treatment method employed (Moller et. al, 2006).
Andit is generally classified into two types which are the chemical and enzymatic
hydrolysis.
2.1.2.2.1 CHEMICAL HYDROLYSIS
This process, just like the chemical pre-treatment, involves the use of chemicals
such as alkali or acid to break the complex carbohydrates. In the late 19th century,
hydrolysis of complex carbohydrates such as cellulose is commonly employed with an
acid (http://www.plantoils.in/portal/ce/prod/prod.html). Today, the acid hydrolysis is
the most widely used chemical hydrolysis method used to treat lignocellulosic biomass
and it is generally classified as either alkaline, dilute acid or concentrated acid hydrolysis.
2.1.2.2.1.1 ALKALINE HYDROLYSIS
Alkaline hydrolysis is generally applied for delignification purposes rather than
for saccharification purposes. According to Fan and et. al (1987), this method is effective
in increasing the internal surface area of the organic matter, decreasing the crystallinity,
separating structural linkages between lignin and carbohydrates, and disrupting the lignin
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.
While the second stage is conducted under harsher
conditions to recover the 6-carbon sugars.
2.1.2.2.1.3 CONCENTRATED ACID HYDROLYSIS

Concentrated
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
by pumping materials from vessel to vessel (Badger, 2002). The critical
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)
Dilute Acid Process
Low acid consumption

-Short residence time
-
-Operated at high
temperature
-Low sugar yield
-Equipment corrosion
Formation of undesirable byproducts
2.1.2.2.2 ENZYMATIC HYDROLYSIS

This process involves the use of biological catalysts, the enzymes. An effective
pre-treatment, which increases the accessibility of the enzymes to the substrate, is
necessary for the process (Moller et. al, 2006). Depending on the biomass material to be
hydrolysed, either physical or chemical pre-treatment may be employed (Badger, 2002;
Demirbas, 2005).
The use of enzyme is a promising technology for the hydrolysis of complex
carbohydrates because of their highly specific mode of action and their mild operating
conditions. Cellulase for example, an enzyme used to break the B-D-1, 4-glycosydic
bonds in cellulose normally has an operating temperature between 40 oC 50oC and
Page 12
operating pH ranging from 4.0 to 5.0.

However, enzymes have relatively high cost and researches are already being
conducted in order to bring down their price. A comparison between acid hydrolysis and
enzymatic hydrolysis is summarized below.
Table 2.3 Comparison between acid and enzymatic hydrolysis
Dilute-acid hydrolysis Concentrated- acid
hydrolysis
Harsh
Relatively mild
Hydrolysis Condition
Limited to around
Over 90%
Yields of hydrolysis
50%
No
No
Product inhibition
Formation of
Yes
Yes
inhibitory byproducts
Compared Variable
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
is a biological process in which enzymes produced
by microorganisms catalyse energy-yielding reactions that break down complex organic

substrates (Brown, 2003). This happens when the organic substrate such as glucose is
oxidized and electrons are transferred to organic acceptor molecules producing a wide
variety of chemicals of which ethanol is one of the most important. It occurs usually
under anaerobic condition although aerobic processes are still possible.
2.1.3 ISSUES AND CONCERNS
Unfortunately, despite the good promises brought by the biofuels from terrestrial
sources, recent studies have shown that instead of alleviating environmental concerns on
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.
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.
2.2.1 PRODUCTION AND USE

Today, macroalgae are vastly produced among Asian countries mainly as sources
of food, chemicals and livelihood. In the Philippines, macroalgae are a diverse group of
organisms constituting some 820 species divided into 57.6% red algae, 16.3% brown
algae and 26.1% green algae (Trono, 1999). They are widely farmed in the country as
sources of livelihood and food. They are being produced at an average of 45,000 dry tons
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,
bioactive compounds and chemicals (Trono, 1999).

2.2.2 BROWN ALGAE
The Brown Algae, also known as Class Phaeophyceae, has some attractive
characteristics for energy production as compared to other groups: first, mainly for their
high growth rates and yields; second, for their high amounts of hydrolysable
carbohydrates; third, for their large sizes exceeding up to 80m in length; for their absence
of functional parts such as leaves and roots; and lastly, for their ease in harvesting which
can be done without the destruction of the entire plant by clipping (Show, 1981).
Among the species in the group considered are
Fucus,
Laminaria,
Macrocystis,
and Sargassum. Table 2.4 shows a comparison of synthetic productivity of
terrestrial and marine plants.

Table 2.4 Terrestrial and Marine Photosynthetic Productivity (Adapted from Show, 1981)
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)
4.5
6.8 13.5
4.9
Page 16
2.2.2.1 CELL WALL STRUCTURE

The cell wall structure of brown algae, just like the red algae, is composed of at
least two different layers: the innermost layer which is a microfibrillar skeleton that
imparts strength to the wall and the outer layer which consists of amorphous matrix
where the fibrils are embedded. The cell wall of the brown algae consists mainly of three
extracellular polysaccharides: alginic acid, fucoidan, and cellulose. An image of the cell
wall structure of the brown algae is shown below.
Figure 2.2 Cell wall structure of brown algae

(Schiewer and Volesky, 2000 as cited by Davis et. al, 2003)
2.2.2.1.1 ALGINIC ACID
Alginic acid is a slightly water-soluble polysaccharide consisting largely of
calcium and magnesium salts of mixed polymers of D-mannuronic and L-guluronic acids.
They are commonly utilized as sodium salts known as algin (Percival and McDowell,
1967). In brown algae, they serve as the major constituent of the cell wall (Lewin, 1962).
Page 17
The
degradation of this polysaccharide may either be conducted chemically and
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.
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.
Figure 2.4 Chemical Structure of Cellulose (Shleser, 1994)

2.2.2.2 STORAGE PRODUCTS
Carbon in brown algae is stored in two forms either as a monomeric unit or as a
polymeric one. The monomeric unit is a sugar alcohol in form of mannitol while the
polymeric one is in the form of polysaccharide, laminarin.
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)
.
Amounts of mannitol in brown algae usually vary depending on the
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).
The degradation of this polysaccharide is catalysed by an enzyme known as

laminarase or laminarinase which are commonly found in various sources including fungi
and bacteria.
2.2.3 Sargassum spp.
In the Philippines, Sargassum specie appears to have the greatest potential as
bioethanol feedstock. These species are generally large, tall and dark brown or yellowish
in color. They are widely distributed in more than 20 provinces in the country and are
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
2.2.3 AS BIOETHANOL FEEDSTOCKS

The use of macroalgae as feedstocks for energy production is actually not a new
concept at all. In fact, the Pacific giant kelp, Macrocystis pyrifera has already been
utilized as a biomass for methane production under the Marine Biomass Program by
the Agricultural Research Service of the United States Department of Agriculture
(USDA). In addition, experiments regarding the utilization
of
seaweeds have earlier
been performed by the Naval Weapons Center in their Ocean Food and Energy Farm
Project since 1970s (Show, 1981; Benson and Bird, 1987).
Page 21
Unfortunately, little researches have been done on macroalgae for bioethanol

production. As bioethanol feedstocks though, macroalgae are very much advantageous
over terrestrial crops not only in terms of high productivity but also in several aspects
such as the non-utilization of agricultural land, non-requirement of fertilizers during
cultivation, absence of lignin (a component of plant that seals the carbohydrate making it
difficult for bioconversion) and a high amount of carbohydrate that can be converted to
bioethanol. A comparison of the potential of seaweeds for bioethanol production against
the most commonly used terrestrial crops can be seen from Table 2.5.
Table 2.6 A comparison between the major bioethanol crops and macroalgae
Wheat
Maize
Sugar
Sugar
(grain)
(kernel)
beet
cane
Average world yield (kg ha-1 yr-1
2800
4815
47070
68260
Dry
weight
of
hydrolysable
1560
3100
8825
11600
Macroal
gae
730000
40150
carbohydrates (kg ha-1 yr-1)

Potential volume of ethanol (L ha-1 yr-1)
1010
2010
5150
6756
23400
(Adopted from Adams et. al, 2008)

However, despite the high amounts of hydrolysable carbohydrates in seaweeds,
these carbohydrates such as laminarin, mannitol and alginic acids are very complex. In
addition, only a few organisms can convert these to ethanol.
In 2000, Horn and Ostgaard conducted a study regarding the production of
ethanol from mannitol of brown algae using Zymobacter palmae. He found that the
organism was able to produce ethanol from mannitol but was unfortunately incapable of
anaerobic fermentation. In his similar study in which he utilized Pichia angophorae
instead, he found that the organism was capable of ethanolic fermentation of both
mannitol and laminarin. However, he also found that a supply of oxygen is still
necessary.
Page 22
In 2008, a similar study conducted by Adams et. al. pertaining to enzymatic

hydrolysis of laminarin from brown algae to glucose was done in order to easily convert
it to ethanol. However, mannitol and other carbohydrates were not utilized during the
conversion process. An outline for the bioconversion of macroalgae, specifically brown
algae has been presented.
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.
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
Brown Algae Specie
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.
Page 24
2.4 RELATED STUDIES ON ALGINATE HYDROLYSIS

Alginic acid or alginate (salt compound) is a polysaccharide very resistant to
hydrolysis by mineral acids. Thus, in order to completely break down this complex
polysaccharide, harsh hydrolysis conditions are necessary. However, because of these
harsh conditions, destruction particularly of L-guluronic acid usually occurs.
Today, the commonly employed method for complete liberation of mannuronic
and guluronic acid from alginic acid is through the use of 80% H2SO4 (Haug and Larsen,
1962). In a later study conducted by Anzai, Uchida and Nishide (1990), they tried to
improve the previous method and found that reducing the hydrolysis period would
increase the recovery of uronic acid without altering the M/G ratio.
In 2001, Chandia, Matsuhiro and Vasquez also conducted a study for the
hydrolysis of alginic acid using formic acid. They found that the reaction of alginic acid
with 90% formic acid for 6 hours at 100oC followed by treatment with 1.5N formic acid
for 2 hours at 100oC resulted in the total hydrolysis of the alginic acid. The results also
showed an M/G ratio closer to the traditional sulphuric acid method but did not indicate
any information regarding the uronic acid yield.
Recently, a microwave assisted method for the hydrolysis of sodium alginate was
developed to rapidly determine the M/G ratio of the algae (Chhatbar et. al, 2009). The
optimized microwave method employed 0.15M oxalic acid or 0.25M H2SO4 for 4mins
which resulted in M/G ratios and % weight of Poly-guluronic acid (PGA) and Polymannuronic acid (PMA) comparable to the conventional sulphuric acid method.
However, this study did not also indicate the uronic acid yield of the method.
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.
This, though, may also provide a greater innovation for the
hydrolysis of macroalgae for bioethanol production.

Optimum hydrolysis condition indicating the uronic acid yield is necessary as this
will dictate the applicability of the method to the bioethanol production from brown
macroalgae. So, this study was conceptualized in order to contribute to the knowledge of
bioethanol production from macroalgae specifically to the saccharification/ hydrolysis of
this marine biomass. The development of a method for the hydrolysis of marine biomass
(macroalgae) would be beneficial and helpful not only to the growing bioethanol industry
but also to the aggravating issues on fuel security and environmental degradation in the
country.
Page 26
CHAPTER THREE

3.1 FEEDSTOCK PREPARATION
The seaweed used in this experiment, Sargassum cristaefolium, was harvested in
Calatagan, Batangas. The seaweed was washed with tap water to remove salt and other
impurities and was allowed to air-dry. Then, to bring the moisture content to less than
10%, it was oven dried in the Department of Chemical Engineering, College of
Engineering and Agro-Industrial Technology at a temperature not more than 70oC so
as not to denature the samples. The oven dried samples was ground to particle size of
not more than 4mm.
Figure 3.1 Milled seaweed samples

3.2 EXTRACTION PROCEDURE
The method was adopted from the industrial extraction method of alginate
developed by Perez (1970) as cited by Vauchel et. al (2008). The milled seaweed
samples were stored in 2% (w/w) formalin solution to remove polyphenols from the
seaweed.
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.
Figure 3.2 Gelatinous alginate after acid precipitation

3.3 CHEMICAL HYDROLYSIS PROCEDURE
Two grams (2g) of alginate (commercial, extracted, raw seaweed) were mixed
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.
Figure 3.3 Chemical hydrolysis at 80% and 90% acid concentration

3.4 ENZYMATIC HYDROLYSIS PROCEDURE
3.4.1 Microorganism and Enzyme Production
Enzyme production has already been described from an earlier experiment
(Kitamikado et. al, 1992). A pre-selected organism was cultured in a 20-ml liquid
culture medium in 50-ml flasks at 25oC for 2 days. The liquid medium contained the
following (w/v): 1.0% peptone, 0.1% yeast extract, 3.0% NaCl and 0.5% sodium
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.
Figure 3.4 Alginate Culture Medium for Enzyme Production

Page 30
3.4.2 Enzymatic Hydrolysis Proper

The method of Kitamikado et. al (1992) was modified for the enzymatic assay
of commercial sodium alginate. Hydrolysis of alginate samples was conducted in
50ml flasks incubated at varying temperatures (37oC, 40oC and 45oC). A 15-ml of 50
mMTris- HCl buffer (pH 8.0) containing 0.4% sodium alginate and 0.4M NaClwas
added to the 5ml of the enzyme solution containing 20mM CaCl2. The incubation time
was set to 72 hours and samples were obtained every 24 hours for reducing sugar
analysis (See Section 3.5.1).
The optimized enzymatic hydrolysis condition was also employed to the
extracted sodium alginate and raw seaweed powder.
Figure 3.5 Enzymatic Hydrolysis Medium containing

5ml semi-purified enzyme and extracted alginate
Page 31
3.5 ANALYTICAL METHODS

3.5.1 Reducing Sugar Analysis
The method developed by Miller (1972) was employed for the analysis of
reducing sugar in the hydrolysates. Three ml (3ml) of the sample was mixed with 3ml
of DNS solution. The resulting solution was heated in boiling water for 10mins or
when dark brown color has developed. The heated solutions were mixed with 1ml of
Rochelles Salt solution and were let to cool at room temperature. The absorbances
were then measured at 525nm.
3.5.2 Uronic Acid Analysis
The method developed by Filisetti-Cozzi and Carpita (1991) for the analysis of
uronic acids was employed for experiment. A 400uL of sample was placed in tubes.
Then 40uL of 4M sulfamic acid/potassium sulfamate solution (pH 1.6) was added, after
which, was vortex vigorously. A 2.4ml of 75mM sodium tetraborate in sulphuric acid
solutions was then added, after which, was vortex again vigorously. The tubes were
placed in 100oC water bath for 20min then cooled in an ice bath for 10min. An
80uL of m- hydroxydiphenyl solution was added to the tube and absorbances were read
at 525nm.
Figure 3.6 Uronic Acid Analyses of Samples
Page 32
3.5.3 Protein Determination

The method developed by Lowry et. al (1951) was adopted for the determination
of protein content of the enzyme using Bovine-Serum Albumin (BSA) as standard.
Different concentrations (0.1 mg/ml, 0.2 mg/ml, 0.3mg/ml, 0.4 mg/ml and 0.5 mg/ml) of
BSA from the stock solution were prepared. To 1ml of these various concentrations,
5ml of Lowry Reagent 1 was added and was vortex. After 10 minutes, 0.50 ml of Lowry
Reagent 2 was then added and was vortex. After a period of 30 minutes, the
absorbance was read at 750nm. The Lowry Reagents 1 and 2 consisted of the following:
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
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
(time, temperature and
acid
concentration).
Page 34
Table 4.1 Reducing Sugar Concentration (mg/ml) at different hydrolysis conditions

Reducing Sugar Concentration (mg/ml)
Time (hrs)
1
3
5
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
*average of two trials

The relationship between hydrolysis conditions and the yield of reducing sugar
can be seen from Figures 4.1 to 4.3.
Reducing Sugar (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)
Figure 4.1 Reducing sugar yield at 70% (v/v) Acid Concentration

Figure 4.1 showed that at a constant acid concentration of 70% HCOOH, the
reducing sugar yield from the hydrolysis of alginate decreased through time. It can
also be seen from the figure that an almost similar trend was observed with
temperature, which was, as temperature increased the amount of reducing sugar yield
decreased. The amount of reducing sugar obtained from this acid concentration
ranged from 0.6787 mg/ml to 1.4796 mg/ml.
In Figure 4.2, it can be clearly seen that the same trend is observed at 80%
acid concentration. It showed that as time progressed, the reducing sugar recovered
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)
Figure 4.2 Reducing sugar yield at 80% (v/v) acid concentration

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)
Figure 4.3 Reducing sugar yield at 90% (v/v) acid concentration

For Figure 4.3, it showed that at an acid concentration of 90% the amount of
reducing sugar yield from the hydrolysis procedure decreased with time. However, unlike
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
Error degrees of freedom
51
Error mean square
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
a = values with the same letter are not significantly different
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
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
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
Effect of parameters on the Uronic Acid Yield
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
*average of two trials

The relationship of the uronic acid yield on three parameters was summarized in
Figure 4.4 (for treatment at 70% acid concentration), Figure 4.5 (for treatment at 80%
acid concentration) and Figure 4.6 (for treatment at 90% acid concentration).
1.8000
Uronic Acid (mg/ml)
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 Uronic Acid Yield at 70% (v/v) acid concentration
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
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
Uronic Acid (mg/ml)
1.4000
1.2000
1.0000
0.8000
1hr
0.6000
3hrs
0.4000
5hrs
0.2000
0.0000
60
80
100
Temperature (oC)
Figure 4.5 Uronic Acid Yield at 80 % (v/v) acid concentration

Figure 4.6 showed the interaction of uronic acid with time and temperature at
90% (v/v) acid concentration. Like the previous ones, the figure showed that as time
progressed, the uronic acid yield decreased. Regarding the effect of temperature, the
figure showed almost the same trend as Figure 4.5. It also exhibited peak values at
temperature 80oC. However, it did not give the same trend as its corresponding graph
(Figure 4.3).The uronic acid yields were found to be in the range 0.3858 mg/ml 0.7100
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
Uronic Acid (mg/ml)
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)
Figure 4.6 Uronic Acid Yield at 90% (v/v) acid concentration

Comparing the range of uronic acid yield from three different acid concentrations,
it showed that highest average uronic acid yields were obtained at an acid concentration
of 80% (v/v) with an average value of 1.068 mg/ml while the 90% (v/v) acid
concentration yielded the lowest average uronic acid yield with an average value of
0.5781 mg/ml. This result was in accordance with the results obtained regarding the
effect of acid concentration on reducing sugar.
To evaluate whether the effect of the three parameters on the yield of uronic acid
was significant or not, 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 uronic acid was summarized in Table 4.6
It can be seen from Table 4.6 that there was a significant decrease in uronic acid
from 1 hour to 3 hours after which, the uronic acid remained insignificantly different at 5
hours. This result is somewhat different from the results obtained from the effect of time
on reducing sugar yield in which values at different time intervals were not significantly
different from each other.
Page 41
Table 4.6 3! CRD Analysis for Effect of Time on Uronic Acid
Critical Range
A
B
B
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
0.05

Error mean square
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
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
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
4.2 On the hydrolysis of Alginate Samples

Alginate is polysaccharide very much difficult to hydrolyse primarily because the
splitting into monomeric units occurs very slowly and because the condition to effect the
reaction is also the same condition through which the monomeric units degrade into other
products. These monomeric units of the alginate are composed of uronic/ hexuronic acids
and are lost in the hydrolysis due to dehydration and decarboxylation into the forms of
2- furaldehyde/furfural (A), 5-formyl-2-furoic acid (B) and reductic acid (C)(Smidsrod
et. al,1969).
The 2-Furfural and 5-formyl-2-furoic acid were not detectable by DNS whereas
reductic acid was detectable because it is a highly reducing compound.
Figure 4.7 Degradation of uronic acid
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
2% H2SO4, 160oC, 2hrs
6% (from Alginic acid)
Aso (1952)
5% H2SO4, 150oC, 1.5hrs
10% (from galacturonic acid)
Feather and Harris(1973)
0.4% (from furfural)
Aso (1952)
Conc H2SO4, 160-170 C, 2hrs
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
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.
Page 46
4.3 Enzymatic Hydrolysis of Commercial Alginate Samples

In as much as alginate can be degraded chemically, it can also be degraded
biologically through the actions of enzymes known as alginases or alginate lyases. The
hydrolysis of alginate using alginate lyase has already been established. And a lot of
studies have been able to purify alginases from several microorganisms. Table 4.10
showed several microorganisms capable of producing alginases with their corresponding
optimum temperature.
Table 4.10 Alginate Lyases from various microorganisms and their optimal temperature
Microorganism
Pseudoalteromonas citrea
Optimal
Temperature (oC)
References
35 -45
Alekseeva and others (2002)
Marine Bacterium Bacillus
33
Chauchan (1993)
Vibrio sp, YKW -34
40
Fu, Lin and Kim (2007)
26
Hu, Jiang and Hwang (2006)
KMM3297
Marine Bacterium Vibrio sp.

510-64
Vibrio sp. AL 128
40
Kitamikado, Tseng, Yamaguchi and

Nakamura (1992)
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
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
*average to two trials

The relationship of the reducing sugar yield with time and temperature can be
seen from Figure 4.11.
0.3000
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
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
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
15
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)
Page 50
4. 4 Evaluation of Optimum Hydrolysis Conditions on Extracted Alginate

The raw samples of seaweed were extracted for its alginate content as an
evaluation of the hydrolysis procedure developed previously.
The extraction of alginate from the raw seaweed material was employed using the
laboratory-scale industrial extraction method by Vauchel et. al (2008). This method
consisted of four main parts divided into the following procedures: (1) Storage in 2%
(w/w) formalin solution, (2) overnight acidification using 0.50 M H2SO4 at 4oC, (3) Batch
alkaline extraction using 4% (w/v) Na2CO3 solution and (4) Precipitation at pH = 2 and
drying of samples at 35oC. Using this extraction method, an amount of 15.00 grams of
alginate was extracted from the raw material. This value corresponded to 15% of the
dry weight of the sample. The samples were pulverized manually using a mortar and
pestle to obtain particulate sizes approximately equal to that of the commercial alginate
samples.
Figure 4.13 Extracted alginate from raw seaweed material

Optimum hydrolysis condition for this study was defined based on the condition
that yielded the highest amount of reducing sugar. For chemical hydrolysis, the condition
was at 80% (v/v) acid concentration, 60oC for 1hour. On the other hand, the enzymatic
hydrolysis condition was employed at 45oC for 72hrs due to erroneous identification of
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
Average Reducing Sugar

(mg/ml)
Commercial Alginate
2.5963
Average
Uronic Acid
(mg/ml)
1.1967
Extracted Alginate
0.0005
0.0283
*values were average of two trials

Table 4.16 Enzymatic hydrolysis of Commercial and Extracted Alginate at 45oC and 72hrs
Alginate Source
Average Reducing Sugar

(mg/ml)
Commercial Alginate
0.1289
Extracted Alginate
0.9915
*values were average of two trials
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
80% (v/v) acid concentration, 60 C, 1hr
0.0005
Average %
Yield
24.7872
0.0025
As seen in Table 4.17, enzymatic hydrolysis yielded a much greater reducing

sugar in comparison with chemical hydrolysis. This means that enzymatic hydrolysis
could be more favourable for the hydrolysis of alginates from seaweeds towards
bioethanol production.
4.5 Bioethanol Potential of Seaweed Hydrolysates
Using the highest yield of reducing sugar, the bioethanol potential was calculated
based on the reaction presented in Figure 4.14.
Figure 4.14 Chemical Conversion of Uronic Acid to Bioethanol

From the given reaction, 100g grams of uronic acid would yield 39.55grams of
ethanol theoretically. Assuming a fermentation efficiency of 90%, the bioethanol
potential of the highest yield of reducing sugar was 0.3529 g/L which translates to an
ethanol concentration of 0.0447% (v/v). This amount of ethanol concentration was way
too low to be economical. Thus, determination of the real optimum hydrolysis condition
for macroalgae would be necessary.
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,
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
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
ADAMS, J., J. GALLAGHER and I. DONNISON.(2008). Fermentation study on

Saccharina latissima for bioethanol production considering variable pretreatment.J Appl Phycol 21: 569-574.
ANZAI, H, N. UCHIDA and E. NISHIDE.(1990). Determination of D-mannuronic to

L-guluronic ratio in acid hydrolysis of alginate under improved conditions.Nippon
Suisan Gakkaishi 56: 73-81
ASO, K. (1953). Studies on the Autoclaved Products of Polyuronides.Tohoku Journ.

Agri. Res III 2: 359-385
BADGER, P.C. (2002).Trends in new crops and new uses. J. Janick and A. Whipkey
(eds.) ASHS Press, Alexandria, VA. pp. 17 -21
BAYAT-MAKOOI, F., T.M. SINGH and I.S. GOLDSTEIN.(1985). Some factors

influencing the rate of cellulose hydrolysis by concentrated acid.
Biotechno lBioeng Symp 15: 22-37
BROWN, R. C. (2003). Biorenewable resources: engineering new products from

agriculture. Ames, Iowa: Iowa State Press. 286 p.
BENSON, P.H and K.T. BIRD.(1987). Renewable Energy Systems. The Netherlands:
Elsevier Science Publishers B. V.pp 5 12.
CHANDIA, N.P., B. MATSUHIRO and A. E. VASQUEZ. (2001). Alginic acids in

Lessonia trabeculata: characterization by formic acid hydrolysis and FT-IR
spectroscopy. Carb Polym 46: 81-87
CHHATBAR, M., R. MEENA, K. PRASAD, and A.K. SIDDHANTA.(2009).

Microwave assisted rapid method for hydrolysis of sodium alginate for M/G ratio
determination. Carb Polym 76: 650-656.
CRUTZEN, P. J., A.R MOSIER, K.A. SMITH, and W. WINIWARTER . (2008).

N20 release from agro-biofuel production negates global warming reduction by
replacing fossil fuels. Atmos Chem Phys 8:389-395.
DAVIS, T.A., B. VOLESKY and A. MUCCI .(2003). A review of biochemistry of

heavy metal biosorption by brown algae. Water Res 37: 4311-4330.
DEMIRBAS, A. (2005). Bioethanol from cellulosic materials: A renewable motor fuel

from biomass. Energy Sources 27: 327-337.
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,
L.T., M.M. GHARPURAY and Y.H. LEE.(1987). Cellulose

hydrolysis. Biotechnology Monographs Vol 3.Springer-Verlag. NY: Berlin.198 p.
FEATHER, M. S. AND J.F. HARRIS.(1973).

Carbohydrates. Adv. Carb. Chem. 28: 186 213
Dehydration
Reactions of
FILISETTI-COZZI, T.M.C.C and N.C. CARPITA.(1991). Measurement of uronic

acids without interference from neutral sugars. Anal Biochem 197: 157-162.
GOODMAN, L.J. and R.N. LOVE.(1981). Biomass Energy Projects, Planning and
Management. Pergamon Press, Hawaii.184 p.
HARMSEN, P., W. HUIJGEN, L. BERMUDEZ and R. BAKKER.(2010). Literature

review of physical and chemical pretreatment processes for lignocellulosic
biomass. Wageningen UR Food & Biobased Research.49 p.
HAUG, A. and B. LARSEN.(1962). Quantitative determination of the uronic acid

composition of alginates. Act Chem Scand 16: 1908-1918.
HOLTAN, S., Q. ZHANG,W.I. STRAND, and G. SKJAN-BRAEK.(2006).

Characterization of the Hydrolysis Mechanism of Polyalternating Alginate in
Weak Acid and Assignment of the Resulting MG-Oligosaccharidesby NMR
Spectroscopy and ESI-Mass Spectrometry.Biomacromolecules7 (7): 2108-2121
HORN, S., and K. OSTGAARD.(2000). Bioenergy from seaweeds. Graduate

Thesis. Norwegian University of Science and Technology.93 p.
HORN, S., and K. OSTGAARD. (2000a). Ethanol production from seaweed extract.J
Ind Microbiol Biotechnol25: 249-254.
HORN, S., and K. OSTGAARD.(2000b). Production of ethanol from mannitol by

Zymobacter palmae. J Ind Microbiol Biotechnol 24:51-57.
INMAN, M. (2008). Clearing Lands for Biofuels Makes Global Warming Worse.
National Geographic News.
JEIHANIPOUR, A. and M.J. TAHERZADEH.(2009). Ethanol production from

cotton-based waste textiles. Biores Technol 100: 1007-1010.
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.
KITAMIKADO, M., C. TSENG, K. YAMAGUCHI and T. NAKAMURA. (1992).

Two Types of Bacterial Alginate Lyases. Appl Env Microbiol 58 (8): 2474 2478.
LEWIN, R.A. (1962). Physiology and Biochemistry of Algae. NY and London:

Academic Press. 929 p.
LOWRY, O.H., N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL.(1951).

Protein measurement with the Folin-Phenol reagents.J.BiolChem193: 265 275.
McMILLAN, J.D. (1994). Pretreatment of lignocellulosic biomass. Amer Chem Soc

292-324.
MILLER, G.L. (1972).Use of dinitrosalicylic reagent for the determination of reducing

sugar. Anal Chem 31: 426 428.
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.
MONTAO, M., A. RODRIGUEZA and R. BALITAAN.(2006). Ethnobotany of

Sargassum spp. in the Philippines. Coastal Marine Science 30 (1): 222 225.
PATLE, S. and B. LAL.(2007). Ethanol production from hydrolysed agricultural wastes

using mixed culture of Zymomonas mobilis and Candida tropicalis. Biotechnol
Lett 29: 1839-1843.
PERCIVAL, E. and R.H. McDOWELL.(1967). Chemistry and Enzymology of Marine

Algal Polysaccharides. NY and London: Academic Press.219 p.
QUAIN, D. E and C.A BOULTON.(1987). Growth and Metabolism of mannitol by
strains of S. cerevisiae. J Gen Microbiol 133: 1675-1684.
QUIONES, V. R. (2010). Parametric study on Pretreatment and Saccharification of

Turbinaria orrnata(Phaeophyta) for Hydrolysate Production. Undergraduate
Thesis. University of the Philippines Los Baos. Department of Chemical
Engineering. College, Laguna.
RESHAMWALA, S., B.T. SHAWKY, and B.E. DALE.(1995). Ethanol production

from enzymatic hydrolysates of AFEX-treated coastal Bermuda grass and
switchgrass.Appl Biochem Biotechnol 51/52: 43-55.
REYES, K. M. C. (2009). Parametric Study on Pretreatment and Saccharification of

Sargassum cristaefolium for Hydrolysate Preparation in Bioethanol Production.
Undergraduate Thesis.University of the Philippines Los Baos.Department of
Chemical Engineering.College, Laguna.
RIVERA, H.M.D. (2009). Parametric Study on Pretreatment and Saccharification

of Macroalga
(Sargassum kushimonte) for Hydrolysates Production.
Undergraduate Thesis. University of the Philippines Los Baos.Department
of Chemical Engineering.College, Laguna.
ROSS, A. (2009). Marine Biomass Potential and Limitations.University of Leeds.22 p.
SHLESER, R. (1994). Ethanol Production in Hawaii: Processes, Feedstocks, and

Current Economic Feasibility of Fuel Grade Ethanol Production in Hawaii. A
Report Prepared for the State of Hawaii, Department of Business, Economic
Development & Tourism. 87 p.
SHOW, I. T. (1981).Marine Biomass.In Sofer, S. S. & O. R. Zaborsky.Biomass

Conversion Processes for Energy and Fuels. NY and London: Plenum Press. pp
57 76.
SMIDSROD, O., A. HAUG, A. andB. LARSEN.(1966). The Influence of pH on the
Rate of Hydrolysis of Acidic Polysaccharides.Acta. Chem. Scand. 20: 1026-1034.
SMIDSROD, O. ,B. LARSEN, T. PAINTER,andA. HAUG.(1969). The Role of

Intramolecular Autocatalysis in the Acid Hydrolysis of Polysaccharide containing
1,4-Linked Hexuronic Acid. Acta. Chem. Scand. 23:1573-1580.
SMIDSROD, O. and K.I. DRAGET.(1996). Chemistry and physical properties of

alginate. Carbohydrates in Europe 14: 6 -13
TAHERZADEH, M. J and K. KARIMI. (2008). Acid-based hydrolysis process for

ethanol from lignocellulosic material: A review. Bioresources 2(3): 472-479.
TRONO, G. C. Jr. (1999). Diversity of the seaweed flora of the Philippines and its
utilization. Hydrobiologia 398/399: 1-6.
VAN MARIS, J.A., D.A. ABBOTT, E. BELLISSIMI, J. VAN DE BRINK, M.

KUYPER, M.A.H LUTTIK, H.W. WISSELINK, W.A. SCHEFFERS. J.P.
VAN DIJKEN and J.T. PRONK. (2006). Alcoholic fermentation of carbon
sources in biomass hydrolysates by Saccharomyces cerevisiae: current status.
Anton van Leeuwenhoek 90: 391 - 418
VAUCHEL, P., R. KAAS, A. ARHALIASS, R. BARON, andJ. LEGRAND. (2008).

A New Process for Extracting Alginates from Laminaria digitata: Reactive
Extrusion. Food Bioprocess Technol 1: 297-300.
WONG, T.Y., L.A. PRESTON, and N.L. SCHILLER. (2000). ALGINATE

LYASE: Review of Major Sources and Enzyme Characteristics, StructureFunction Analysis, Biological Roles, and Applications. Ann Rev Microbiol 54:
289-340.
YAMASAKI, M., K. OGURA, W. HASHIMOTO, B. MIKAMI, AND K.

MURATA.(2005). A Structural Basis for Depolymerization of Alginate by
Polysaccharide Lyase Family-7.J. Mol. Biol. 352: 11-21
ZUBIA, M., P. CLAUDE and E. DESLANDES. (2008). Alginate, mannitol, phenolic

compounds and biological activities of two-range extending brown algae,
Sargassum mangaravense and Turbinaria ornata (Phaeophyta: Fucales),
from Tahiti (French Polynesia). J Appl Phycol 20:1033-1043
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)
Figure A.1 Standard Curve for Protein Determination
0.45
APPENDIX A2
REDUCING SUGAR STANDARD CURVE
Table A.2 Data for the Standard Curve of Millers DNS Method for ReducingSugar
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
Figure A.2 Standard Curve for Reducing Sugar Determination
0.60
APPENDIX A3
URONIC ACID STANDARD CURVE
Table A.3 Data for the Standard Curve for Uronic Acid Determination
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
Figure A.3 Standard Curve for Uronic Acid Determination
0.60
APPENDIX B
RAW DATA FOR REDUCING
SUGAR ANALYSIS:
CHEMICAL HYDROLYSIS
APPENDIX B
REDUCING SUGAR ANALYSIS FOR CHEMICAL HYDROLYSIS
Table B.1 Raw Data for Reducing Sugar Analysis at 70%AcidConcentration

Acid
Reaction
Temperature
Concentration
Time
(oC)
(%)
(hrs)
1
60
3
5
1
70
80
3
5
1
100
3
5
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
Table B.2 Raw Data for Reducing Sugar Analysis at 80%AcidConcentration

Acid
Reaction
Temperature
Concentration
Time
o
(C)
(%)
(hrs)
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
mg/ml mg/ml Average
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
mg/ml mg/ml Average
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
mg/ml mg/ml Average
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
Table D.1 Raw Data for Enzymatic Hydrolysis at 37oC

Absorbance
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

Absorbance
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

Absorbance
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
TableE.2 Enzymatic Hydrolysis of Extracted Alginate Samplesat45oC and 72 hours
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
F.1 CALCULATION FORREDUCINGSUGAR CONCENTRATION
For the reducing sugar content of hydrolysates at 70% acid concentration,

60oC 1
hour, the absorbance is given to be 0.535:
The value 6 is used to account for the dilution of sample after

heatingfor1hour and the value 1.41 is used to account for the dilution during
neutralization.
F.2 CALCULATION FORURONICACID CONCENTRATION
For the reducing sugar content of hydrolysates at 70% acid concentration,

60oC 1
hour, the absorbance is given to be 0.976:
The value 6 is used to account for the dilution of sample after

heatingfor1hour and the value 1.41 is used to account for the dilution during
neutralization.
F.3. CALCULATION OFAVERAGEPERCENT YIELDS

The initial amount of alginate used for chemical hydrolysis is 20 mg/ml. For
reducing sugar yields 0.0005 mg/ml on both Trials 1 and 2, the percent yield is
calculated
as:
F.4 CALCULATION FORENZYMATICACTIVITY DETERMINATION

For a given sample of 0.5ml of enzyme, the absorbance reading for protein
contentis0.450whiletheabsorbancereadingforreducingsugarcontentis0.094.The
reaction is run for 20minutes.
For the reducing sugar content:
For the protein content:
To determine the specific enzyme activity:
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
TableG1.7 Analysis of Variance for the Effect of Time on Enzymatic Hydrolysis
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
TableG1.8 Analysis of Variance for the Effect of Temperature on Enzymatic Hydrolysis
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
Effect of Acid Concentration

a
0.05
SE
51
0.1175
MSR
2.486E-01
Number of Means
LSR
Critical Range
2
2.84
0.333758287
3
2.99
0.351386365
Mean Value
Level
x1
0.9218
90
x2
1.1996
70
x3
1.8227
80
2v1
0.2778
insignificant
3v2
0.6231
significant
3v1
0.9009
significant
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
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
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

Parametric Study On Chemical and Enzymatic Hydrolysis of Alginates From Sargassum Cristaefolium

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Parametric Study On Chemical and Enzymatic Hydrolysis of Alginates From Sargassum Cristaefolium

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PARAMETRIC STUDY ON CHEMICAL AND ENZYMATIC HYDROLYSIS

OF ALGINATES FROM Sargassum cristaefolium C.A. Agardh (Phaeophyta)

MICHAEL ANGELO MELO VIRAY

SUBMITTED TO THE FACULTY OF THE

BACHELOR OF SCIENCE IN CHEMICAL ENGINEERING

Dr. Jovita L. Movillon

Prof. Denise Ester O. Santiago

Ms. Irene G. Pajares

Dr. Milagrosa Goss

Dr. Jovita L. Movillon

Dr. Arsenio N. Resurreccion

Viray, Michael Angelo M.

1.2 Objectives of the Study

1.3 Date and Place of the Study

1.4 Scope and Limitations of the Study

2.1.2 Bioethanol Production

2.1.2.2.1 Chemical Hydrolysis

2.1.2.2.2 Enzymatic Hydrolysis

2.1.3 Issues and Concerns

2.2.2 Brown Algae

2.2.2.1 Cell Wall Structure

2.2.2.1.1 Alginic Acid

2.2.2.2 Storage Products

2.2.3 Sargassum spp.

2.2.3.1 As Bioethanol Feedstocks

2.3 Related Studies on Hydrolysis of Macroalgae

2.4 Related Studies on Alginate Hydrolysis

MATERIALS AND METHODS

3.2 Extraction Procedure

3.3 Chemical Hydrolysis Procedure

3.4 Enzymatic Hydrolysis Procedure

3.4.1 Microorganism and Enzyme Production

3.4.2 Enzymatic Hydrolysis Proper

3.5 Analytical Methods

3.5.1 Reducing Sugar Analysis

3.5.2 Uronic Acid Analysis

3.5.3 Protein Determination

RESULTS AND DISCUSSION

4.1.1 Effect of Parameters on the Reducing Sugar Yield

4.1.2 Effect of Parameters on the Uronic Acid Yield

4.2 On the Hydrolysis of Alginate Samples

4.3 Enzymatic Hydrolysis of Commercial Alginate Samples

4.4 Evaluation of Optimum Hydrolysis Condition on Extracted Alginate

4.5 Bioethanol Potential of Seaweed Hydrolysates

SUMMARY AND CONCLUSION

B. Raw Data for Reducing Sugar Analysis: Chemical Hydrolysis

C. Raw Data for Uronic Acid Analysis: Chemical Hydrolysis

D. Raw Data for Enzymatic Hydrolysis

E. Evaluation of Chemical and Enzymatic Hydrolysis

H. Material Safety Data Sheets

Comparison of Bioethanol against Unleaded Gasoline

Comparison between Concentrated- and Dilute-Acid Hydrolysis

Comparison between acid and enzymatic hydrolysis

Terrestrial and Marine Photosynthetic Productivity

Chemical Composition of Various Sargassum species

A comparison between the major bioethanol crops and macroalgae

Chemical and Enzymatic Hydrolysis of Various Brown Macroalgae

Reducing Sugar Concentration (mg/ml) at different hydrolysis

3! CRD Analysis for Effect of Time on Reducing Sugar

3! CRD Analysis for Effect of Temperature on Reducing Sugar

3! CRD Analysis for Effect of Acid Concentration on Reducing Sugar

Uronic Acid Concentration (mg/ml) at different hydrolysis conditions

3! CRD Analysis for Effect of Time on Uronic Acid