parametric study on chemical and enzymatic hydrolysis of alginates from sargassum cristaefolium
DESCRIPTION
A Thesis Research on chemical and enzymatic hydrolysis of alginate, one of carbohydrates found in macroalgae. The hydrolysis study is aimed to determine the yield of reducing sugars and other fermentable carbohydrates which can be used for bioethanol production.TRANSCRIPT
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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
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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 Prof. Denise Ester O. Santiago
Panel Member Panel Member
Date Signed Date Signed
Dr. Jessica F. Simbahan
Panel Member
Date Signed
Ms. Irene G. Pajares Dr. Milagrosa Goss
Co-adviser 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
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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!
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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
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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 bonggang-
bonggang 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.
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Title Page
Acceptance Sheet
i
ii
Acknowledgements
Table of Contents
iii
iv
List of Tables
List of Figures
v
vi
Abstract vii
1
INTRODUCTION
1.1 Significance of the Study
1
1.2 Objectives of the Study 3
1.3 Date and Place of the Study 3
1.4 Scope and Limitations of the Study 4
2
REVIEW OF LITERATURE
2.1 Biofuels
5
2.1.1 Bioethanol 6
2.1.2 Bioethanol Production 8
2.1.2.1 Pretreatment 8
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
2.2.1 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
TABLE OF CONTENTS
2.2 Macroalgae
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2.2.2.1.2 Fucoidan
18
2.2.2.1.3 Cellulose 19
2.2.2.2 Storage Products 19
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
3
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 32
3.5.1 Reducing Sugar Analysis 32
3.5.2 Uronic Acid Analysis 32
3.5.3 Protein Determination 33
4
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
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5
SUMMARY AND CONCLUSION
54
6 RECOMMENDATIONS 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
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LIST OF TABLES
Table # Title Page
2.1
Comparison of Bioethanol against Unleaded Gasoline
7
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
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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
4.16
optimum hydrolysis condition
Enzymatic hydrolysis of Commercial and Extracted Alginate at 45oC
52
and 72hrs
4.17
Comparison of Chemical and Enzymatic Hydrolysis
53
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LIST OF FIGURES
Figure # Title Page
2.1 Mechanism of pre-treatment of lignocellulosic feedstocks 9
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
Reducing Sugar Yield at 80% (v/v) Acid Concentration
36
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4.3 Reducing Sugar Yield at 90% (v/v) Acid Concentration 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% 44
acid concentration
4.9 Comparison of Uronic Acid (UA) and Reducing Sugar (RS) at 80% 45
acid concentration
4.10 Comparison of Uronic Acid (UA) and Reducing Sugar (RS) at 90% 46
acid concentration
4.11 Effect of time and temperature on enzymatic hydrolysis of 48
commercial alginate
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
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ABSTRACT
VIRAY, MICHAEL ANGELO MELO. College of Engineering and Agro-
Industrial 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 (60
oC, 80
oC, and 100
oC) 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, 40
oC and
45oC) 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.
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.
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INTRODUCTION Page 1
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). In addition to that, the very low lignin and high
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INTRODUCTION Page 2
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 L-
guluronic 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.
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INTRODUCTION Page 3
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
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INTRODUCTION Page 4
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 semi-
purified. 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.
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REVIEW OF LITERATURE Page 5
CHAPTER TWO
REVIEW OF LITERATURE
2.1 BIOFUELS
While the science of fuel production from agricultural crops has already been
established, little studies have focused on seaweed resources for renewable energy.
Today, the world is faced with aggravating problems on fuel security and global
warming brought by the rapid growth of industrialization and population. This is mainly
because much of the energy consumption is dependent on non-renewable petroleum-fuels
which are basically derived from fossils. As of 2008, the Philippines oil consumption
reached 11.93 million tons of oil equivalent (MTOE) through which 31.15% are imported
(www.doe.gov.ph).
Aside from the very high fuel demand, attention has also been focused on the
negative impacts on the use of petroleum-fuels in the environment such as global
warming and air pollution.
With these two major problems at hand, researches have been conducted on
finding alternative resources of fuel that will not just aid in fuel scarcity but will also help
in the preservation of the environment. Among the major solutions found today are the
biofuels.
Biofuels are actually fuels derived from biomass materials such as plants, wastes
and other organic materials. And there are three categories mainly: 1) solid fuels, 2)
liquid fuels, and 3) gaseous fuels; and are produced either by biological or
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REVIEW OF LITERATURE Page 6
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.
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REVIEW OF LITERATURE Page 7
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.
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REVIEW OF LITERATURE Page 8
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.
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REVIEW OF LITERATURE Page 9
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
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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
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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
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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
Concentrated Acid
Process
- Low operating temperature
-High sugar yield
-High acid consumption -Equipment corrosion
- High energy consumption for
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 by-
products
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 40oC 50oC and
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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
Compared Variable Dilute-acid hydrolysis Concentrated- acid
hydrolysis
Enzymatic
hydrolysis
Hydrolysis Condition Harsh Relatively mild Mild
Yields of hydrolysis Limited to around
50%
Over 90%
Product inhibition No No Yes
Formation of
inhibitory by- products
Yes
Yes
No
Reaction Time Short Relatively long Long
Energy consumption High Relatively low Low
Reaction Time Short Relatively long Long
Sources: Demirbas, 2005; Taherzadeh and Karimi, 2008
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
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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.
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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
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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 Laminaria, Macrocystis,
Fucus, 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)
Terrestrial
Trees 0.9 2.8
Grasses 1.1 6.8
Marine
microalgae (waste treatment ponds) 4.5
microalgae (laboratory culture) 6.8 13.5
Kelps/ macroalgae (natural beds) 4.9
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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).
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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 L-
fucose 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 D-
xylose, D- galactose and uronic acids (Mackie and Preston, 1974 as cited by Davis et.
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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.
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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
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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)
Mannitol
(%)
Alginic Acid
(%)
Crude Protein
(%)
Crude Fiber
(%)
Sargassumpallidum 5.47 - 12.81 10.7-26.1 6.82-15.83 6.51-6.66
S. kjellmanianum 6.84-13.40 16.3-26.3 17.61-26.50 4.35-14.43
S. thunbergii 1.64-15.48 10.9-26.2 9.97-25.28 3.2-6.27
S. fusiforme 2.45-10.25 11.1-24.5 7.95-12.13 3-4.92
S. hemiphyllum 6.23-11.02 17.3-23.6 9.22-12.4 5.44-5.54
S. horneri 1.62-13.75 25.3-31.0 14.08-17.47 6.26-6.78
S. siliquastrum 9.96-13.68 22.4-25.5 10.58-17.85 5.01-6.74
S. vachellianum 1.3 26.2 12.58 9.3
S. polycystum 1.78 14 15.45 7.79
Sargassumspp. 1.86-10.60 14.1-32.5 4.42-21.46 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).
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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
(grain)
Maize
(kernel)
Sugar
beet
Sugar
cane
Macroal
gae Average world yield (kg ha-1 yr-1 2800 4815 47070 68260 730000
Dry weight of hydrolysable
carbohydrates (kg ha-1 yr-1) 1560 3100 8825 11600 40150
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.
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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.
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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
Brown Algae Specie
Chemical Hydrolysis Enzymatic Hydrolysis
Reference
Condition (Acid
Conc., Temp.,
Time)
Reducing Sugar
(mg/ml)
Condition
(enzyme
loading, Temp.,
Time)
Reducing Sugar
(mg/ml)
Sargassum cristaefolium
3% HCl,
60oC, 300 min
0.2661
0.5 mg/g, o
50 C, 48hrs
2.6278
Reyes
(2009)
Sargassum kushimonte
3% HCl,
60oC,
300min
0.3263
0.5 mg/g o
50 C, 72hrs
2.7372
Rivera
(2009)
Turbinaria ornata
6.67% HCl,
80oC,
255min
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.
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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 Poly-
mannuronic acid (PMA) comparable to the conventional sulphuric acid method.
However, this study did not also indicate the uronic acid yield of the method.
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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.
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CHAPTER THREE
MATERIALS AND METHODS
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.
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MATERIALS AND METHODS Page 28
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 4
oC 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
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MATERIALS AND METHODS Page 29
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, 80
oC and 100
oC) 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
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MATERIALS AND METHODS Page 30
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
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MATERIALS AND METHODS Page 31
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, 40
oC and 45
oC). 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
-
MATERIALS AND METHODS Page 32
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
-
MATERIALS AND METHODS Page 33
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.
-
RESULTS AND DISCUSSION Page 34
CHAPTER FOUR
RESULTS AND DISCUSSION
Macroalgae or more commonly known as seaweed is currently getting attraction
among researchers for its great potential as bioethanol feedstock mainly because of its
high carbohydrate content, absence of lignin content and its non-competing utilization
with food crops.
Alginate, one of its major components (composition could reach to about 40% of
its dry weight), is a polysaccharide consisting of monomeric units of L-guluronic and D-
mannuronic 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, 80
oC, and 100
oC) 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).
-
RESULTS AND DISCUSSION Page 35
Re
du
cin
g Su
gar
(mg/
ml)
Table 4.1 Reducing Sugar Concentration (mg/ml) at different hydrolysis conditions
Reducing Sugar Concentration (mg/ml)
70 % (v/v) HCOOH 80 % (v/v) HCOOH 90 % (v/v) HCOOH
Time (hrs) 60oC 80oC 100oC 60oC 80oC 100oC 60oC 80oC 100oC
1 2.4616 1.0951 0.9594 2.5963 2.2767 1.1607 0.7411 1.0242 1.3312
3 1.7669 0.7086 0.8374 2.3754 2.0700 1.1019 0.6068 0.9834 1.2256
5 1.5230 0.6787 0.7661 1.9337 1.9196 1.0009 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.
3.5000
3.0000
2.5000
2.0000
1.5000
1.0000
0.5000
1hr
3hrs
5hrs
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
-
RESULTS AND DISCUSSION Page 36
Re
du
cin
g Su
gar
(mg/
ml)
R
ed
uci
ng
Suga
r (m
g/m
l)
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
1.5000
1.0000
0.5000
1hr
3hrs
5hrs
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
0.6000
0.4000
0.2000
0.0000
60 80 100
Temperature (oC)
1hr
3hrs
5hrs
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
-
RESULTS AND DISCUSSION Page 37
Number of means 2 3
Critical Range 0.4085 0.4301
Duncan groupinga
Mean (mg/ml)
Time (hrs) A 1.5162 1
A 1.2865 3
A 1.1340 5
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
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.
-
RESULTS AND DISCUSSION Page 38
Level of significance, 0.05
Error degrees of freedom 51
Error mean square 0.33855
Critical Range 0.38949 0.41006
Duncan groupinga
Mean (mg/ml)
Temperature (oC) A 1.6200 60
B 1.2865 80
B 1.0376 100
Level of significance, 0.05
Error degrees of freedom 51
Error mean square 0.24860
Number of means
2
3 Critical Range 0.33376 0.35139
Duncan groupinga
Mean (mg/ml)
Acid Concentration
%(v/v) A 1.8227 80
B 1.1996 70
B 0.9218 90
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 80
oC then remained insignificantly different
after the temperature range.
Table 4.3 3! CRD Analysis for Effect of Temperature on Reducing Sugar
a = values with the same letter are not significantly different
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
a = values with the same letter are not significantly different
-
RESULTS AND DISCUSSION Page 39
Uro
nic
Aci
d (
mg/
ml)
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)
70 % (v/v) HCOOH 80 % (v/v) HCOOH 90 % (v/v) HCOOH
Time (hrs) 60oC 80oC 100oC 60oC 80oC 100oC 60oC 80oC 100oC
1 1.4796 1.1998 0.7402 1.1967 1.3903 1.0355 0.4768 0.7100 0.6539
3 1.2286 0.9070 0.6173 1.0448 1.2702 0.7862 0.4380 0.7029 0.6183
5 1.0648 0.6548 0.5658 0.6374 0.9906 0.7095 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
1.6000
1.4000
1.2000
1.0000
0.8000
0.6000
0.4000
0.2000
0.0000
60 80 100
Temperature (oC)
1hr
3hrs
5hrs
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
-
RESULTS AND DISCUSSION Page 40
Uro
nic
Aci
d (
mg/
ml)
increased, the uronic acid yield decreased. This result corresponded to the trend from
Figure 4.1 regarding the effect of time and temperature on reducing sugar yield. Uronic
acids obtained were in the range 0.5658 mg/ml 1.4796 mg/ml.
Figure 4.5 showed the results of interaction of uronic acid with time and
temperature at 80% (v/v) acid concentration. As seen from Figure 4.5, the uronic acid
also decreased with time just like the trend observed from Figure 4.4 and its
corresponding graph on Figure 4.2. However, a different trend was observed regarding
the effect of temperature on the uronic acid wherein a peak uronic acid yield was
obtained at 80oC. The uronic acid at this condition were found to be in the range from
0.7095 mg/ml to 1.3903 mg/ml. Peak values at 1, 3 and 5 hours were 1.3903 mg/ml,
1.2702 mg/ml and 0.9906 mg/ml respectively.
1.6000
1.4000
1.2000
1.0000
0.8000
0.6000
0.4000
0.2000
0.0000
60 80 100
Temperature (oC)
1hr
3hrs
5hrs
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
-
RESULTS AND DISCUSSION Page 41
Uro
nic
Aci
d (
mg/
ml)
mg/ml with peak values 0.7100 mg/ml, 0.7029 mg/ml and 0.6175 mg/ml for 1, 3 and 5
hours respectively.
0.9000
0.8000
0.7000
0.6000
0.5000
0.4000
0.3000
0.2000
0.1000
0.0000
60 80 100
Temperature (oC)
1hr
3hrs
5hrs
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.
-
RESULTS AND DISCUSSION Page 42
Level of significance, 0.05
Error degrees of freedom 51
Error mean square 0.07949
Critical Range 0.18873 0.19870
a
A 0.9870 1
B 0.8459 3
B 0.6917 5
Error degrees of freedom 51
Error mean square 0.08418
Number of means
2
3 Critical Range 0.19422 0.20447
Duncan groupinga
Mean (mg/ml)
Temperature (oC) A 0.9381 80
B 0.8836 60
B 0.7029 100
Table 4.6 3! CRD Analysis for Effect of Time on Uronic Acid
a = values with the same letter are not significantly different
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 80
oC 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 80
oC.
Table 4.7 3! CRD Analysis for Effect Temperature on Uronic Acid
Level of significance, 0.05
a = values with the same letter are not significantly different
Statistical analysis for the effect of acid concentration on uronic acid was
summarized in Table 4.8. It can be seen from the table that the results were almost in
-
RESULTS AND DISCUSSION Page 43
Level of significance 0.05
Error degrees of freedom 51
Error mean square 0.05734
Number of means
2
3 Critical Range 0.16029 0.16875
Duncan groupinga
Mean
Acid Concentration
% (v/v) A 1.0068 80
A 0.9398 70
B 0.5781 90
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
a = values with the same letter are not significantly different
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.
A B C
Figure 4.7 Degradation of uronic acid
-
RESULTS AND DISCUSSION Page 44
RS/
UA
(m
g/m
l)
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
5% H2SO4, 150oC, 1.5hrs
Conc H2SO4, 160-170oC, 2hrs
6% (from Alginic acid)
10% (from galacturonic acid)
0.4% (from furfural)
Aso (1952)
Feather and Harris(1973)
Aso (1952)
As seen from Table 4.9, harsh hydrolysis conditions are necessary towards the
formation of reductic acid unlike the formation of furfural that is simultaneously formed
upon conversion of polyuronic-acid into its monomeric unit (Feather and Harris, 1973).
To analyse further the results of varying the conditions of hydrolysis, charts
consisting both of the reducing sugar and uronic acid yield at different condition can be
seen from Figures 4.8 to 4.10.
3.0000
2.5000
2.0000
1.5000
1.0000
0.5000
0.0000
60 80 100
Temperature (oC)
1hr-UA
3hrs-UA
5hrs-UA
1hr-RS
3hrs-RS
5hrs-RS
Figure 4.8 Comparison of Uronic Acid (UA) and Reducing Sugar (RS) at 70% acid concentration
-
RESULTS AND DISCUSSION Page 45
RS/
UA
(m
g/m
l)
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 80
oC. 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
2.5000
2.0000
1.5000
1.0000
0.5000
0.0000
60 80 1