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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 BAÑOS 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

Dr. Jessica F. Simbahan Panel Member

Date Signed

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 Bos

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 wouldn’t 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. You’re 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 RA’s. 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 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 let’s continue our own lives’ journeys.

journey. So long my friends. Thank you and let’s continue our own lives’ journeys.  Viray,
TABLE OF CONTENTS Title Page   i Acceptance Sheet ii Acknowledgements iii Table of Contents

TABLE OF CONTENTS

Title Page

 

i

Acceptance Sheet

ii

Acknowledgements

iii

Table of Contents

iv

List of Tables

v

List of Figures

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 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

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 19 2.2.2.2.1 Mannitol 20

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

of Optimum Hydrolysis Condition on Extracted Alginate 51 4.5 Bioethanol Potential of Seaweed Hydrolysates 53
5 SUMMARY AND CONCLUSION 54 6 RECOMMENDATIONS 57 REFERENCES 59 APPENDICES 66 A. Standard

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

78 F. Sample Calculations 80 G. Statistical Analysis 84 H. Material Safety Data Sheets 92
Table # LIST OF TABLES Title Page 2.1 Comparison of Bioethanol against Unleaded Gasoline 7

Table #

LIST OF TABLES

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

at Different Conditions 44 4.10 Alginate Lyases from various microorganisms and their optimal 47 temperature
4.11 Enzymatic Activity Determination 4 7 4.12 Effect of Time and Temperature on Enzymatic Hydrolysis

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 45 o C

52

and 72hrs

4.17 Comparison of Chemical and Enzymatic Hydrolysis

53

and Extracted Alginate at 45 o C 52 and 72hrs 4.17 Comparison of Chemical and Enzymatic
LIST OF FIGURES Figure # Title Page 2.1 Mechanism of pre-treatment of lignocellulosic feedstocks 9

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

Sugar Yield at 70% (v/v) Acid Concentration 35 4.2 Reducing Sugar Yield at 80% (v/v) Acid
4.3 Reducing Sugar Yield at 90% (v/v) Acid Concentration 36 4.4 Uronic Acid Yield at

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% acid concentration

44

4.9 Comparison of Uronic Acid (UA) and Reducing Sugar (RS) at 80% acid concentration

45

4.10 Comparison of Uronic Acid (UA) and Reducing Sugar (RS) at 90% 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

4.13 Extracted alginate from raw seaweed material 51 4.14 Chemical Conversion of Uronic Acid to Bioethanol

ABSTRACT

VIRAY, MICHAEL ANGELO MELO. College of Engineering and Agro- Industrial Technology, University of the Philippines Los Baños, 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 o C, 80 o C, and 100 o C) 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 80 o C 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

60

o C, 80% acid concentration and 1hour.

The effect of time (24hrs, 48hrs, and 72hrs) and temperature (37 o C, 40 o C and

o 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 37 o C and 72hours.

45

The optimum conditions were evaluated on the extracted alginate. However, for enzymatic hydrolysis the condition applied was at 45 o C 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

CHAPTER ONE

INTRODUCTION

1.1 Significance and Background of the Study

Today, global warming and increasing energy demand brought by rapid growth

of world’s 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

of agriculturally productive land (Ross, 2009). In addition to that, the very low lignin and high
of agriculturally productive land (Ross, 2009). In addition to that, the very low lignin and high
carbohydrate content of seaweeds make it more advantageous because these factors remove expensive delignification process

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% H 2 SO 4 . 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.

for a more sustainable energy and fuel importation independence of our country in the future. INTRODUCTION
for a more sustainable energy and fuel importation independence of our country in the future. INTRODUCTION
1.2 Objectives of the Study The main objective of this study was to develop methods

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

while enzymatic hydrolysis was conducted at the Environmental Biotechnology Laboratory (ENVI Lab) of INTRODUCTION Page 3
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 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.

procedure was done based on the highest yielding conditions for both acid and enzymatic hydrolysis. INTRODUCTION
procedure was done based on the highest yielding conditions for both acid and enzymatic hydrolysis. INTRODUCTION
CHAPTER TWO REVIEW OF LITERATURE 2.1 BIOFUELS While the science of fuel production from agricultural

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 Philippine’s oil consumption

reached 11.93 million tons of oil equivalent (MTOE) through which 31.15% are imported

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

solid fuels, 2) liquid fuels, and 3) gaseous fuels; and are produced either by biological or
solid fuels, 2) liquid fuels, and 3) gaseous fuels; and are produced either by biological or
thermochemical methods (Goodman and Love, 1981). Among the three categories of biofuels mentioned, liquid biofuels

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.

others. Among the major producers of bioethanol are Brazil, United States, China and India. REVIEW OF
others. Among the major producers of bioethanol are Brazil, United States, China and India. REVIEW OF
As a transport fuel, bioethanol has been blended to gasoline which would account for 5%

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

Table 2.1 Comparison of Bioethanol against Unleaded Gasoline Sourc e: www.doe.gov.ph Most of the feedstocks used

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.

in the Philippines and some recent researches on cellulosic and lignocellulosic feedstocks. REVIEW OF LITERATURE Page
in the Philippines and some recent researches on cellulosic and lignocellulosic feedstocks. REVIEW OF LITERATURE Page
2.1.2 BIOETHANOL PRODUCTION In early years, bioethanol was produced from sugar feedstocks which are directly

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.

A figure illustrating the mechanism of pre-treatment process is shown in Figure 2.1. REVIEW OF LITERATURE
A figure illustrating the mechanism of pre-treatment process is shown in Figure 2.1. REVIEW OF LITERATURE
Pre-treatment processes are generally classified into three types namely: a) physical, b) chemical and c)

Pre-treatment processes are generally classified into three types namely: a)

physical, b) chemical and c) physico-chemical.

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

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

various herbaceous crops and grasses (Reshamwala et. al, 1995). While among the techniques REVIEW OF LITERATURE
various herbaceous crops and grasses (Reshamwala et. al, 1995). While among the techniques REVIEW OF LITERATURE
mentioned, steam explosion is the most widely used physico-chemical pre-treatment method for the lignocellulosic biomass

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 19 th 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

separating structural linkages between lignin and carbohydrates, and disrupting the lignin REVIEW OF LITERATURE Page 10
separating structural linkages between lignin and carbohydrates, and disrupting the lignin REVIEW OF LITERATURE Page 10
structure. However, in two separate studies conducted by Patle and Lal (2007) and Jeihanipour and

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

those created by pumping materials from vessel to vessel (Badger, 2002). The critical REVIEW OF LITERATURE
those created by pumping materials from vessel to vessel (Badger, 2002). The critical REVIEW OF LITERATURE
factors needed to make this process economically viable are to optimize sugar recovery and cost

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 40 o C 50 o C and

in cellulose normally has an operating temperature between 40 o C – 50 o C and
in cellulose normally has an operating temperature between 40 o C – 50 o C and
operating pH ranging from 4.0 to 5.0. However, enzymes have relatively high cost and researches

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

 

Limited to around

     

Yields of hydrolysis

 

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

sources, recent studies have shown that instead of alleviating environmental concerns on REVIEW OF LITERATURE Page
sources, recent studies have shown that instead of alleviating environmental concerns on REVIEW OF LITERATURE Page
fuel use, it is actually the one causing major problems on global warming due to

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.

biodiesel on palm, 86 years; and peatland rainforest also for biodiesel on palm, 423 years. REVIEW
biodiesel on palm, 86 years; and peatland rainforest also for biodiesel on palm, 423 years. REVIEW
Aside from the environmental concerns reported by Inman on the use of biofuels from terrestrial

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

as sources of livelihood and food. They are being produced at an average of 45,000 dry
as sources of livelihood and food. They are being produced at an average of 45,000 dry
costing to around $48,000 from 1990 to 1995. In addition to that, exports of dried

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)

Photosynthetic Productivity (Adapted from Show, 1981) Vegetation Type Production (kg/m 2 -yr) Terrestrial

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

4.5 microalgae (laboratory culture) 6.8 – 13.5 Kelps/ macroalgae (natural beds) 4.9 REVIEW OF LITERATURE Page
4.5 microalgae (laboratory culture) 6.8 – 13.5 Kelps/ macroalgae (natural beds) 4.9 REVIEW OF LITERATURE Page
2.2.2.1 CELL WALL STRUCTURE The cell wall structure of brown algae, just like the red

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.

the cell wall structure of the brown algae is shown below. Figure 2.2 Cell wall structure

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).

1967). In brown algae, they serve as the major constituent of the cell wall (Lewin, 1962).
1967). In brown algae, they serve as the major constituent of the cell wall (Lewin, 1962).
The degradation of this polysaccharide may either be conducted chemically and enzymatically. Alginate lyase, the

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,

microorganisms (Wong et. al, 2000).

and

a

wide

range

of

The figure below shows the chemical constituent and overall structure of an

alginic acid.

constituent and overall structure of an alginic acid. Figure 2.3Alginate structural data: (a) alginate monomers (M

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.

of D- xylose, D- galactose and uronic acids (Mackie and Preston, 1974 as cited by Davis
of D- xylose, D- galactose and uronic acids (Mackie and Preston, 1974 as cited by Davis
al, 2003). In addition to acid hydrolysis, fucoidan may also be degraded using enzymes known

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.

1962).Figure 2.4 shows the chemical structure of cellulose. Figure 2.4 Chemical Structure of Cellulose (Shleser, 1994)

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.

in form of mannitol while the polymeric one is in the form of polysaccharide, laminarin. REVIEW
in form of mannitol while the polymeric one is in the form of polysaccharide, laminarin. REVIEW
2.2.2.2.1 MANNITOL Mannitol is a 6-Carbon sugar alcohol/ polyol/ hexitol universally found in brown algae.

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

 

.

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

in color. They are widely distributed in more than 20 provinces in the country and are
in color. They are widely distributed in more than 20 provinces in the country and are
commonly found all over the rocky, wave exposed or sheltered areas (Montano et. al, 2006).

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).

Ocean Food and Energy Farm Project since 1970 ’ s (Show, 1981; Benson and Bird, 1987).
Ocean Food and Energy Farm Project since 1970 ’ s (Show, 1981; Benson and Bird, 1987).
Unfortunately, little researches have been done on macroalgae for bioethanol production. As bioethanol feedstocks though,

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

Macroal

(grain)

(kernel)

beet

cane

gae

Average world yield (kg ha-1 yr-1

2800

4815

47070

68260

730000

Dry

weight

of

hydrolysable

1560

3100

8825

11600

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.

mannitol and laminarin. However, he also found that a supply of oxygen is still necessary. REVIEW
mannitol and laminarin. However, he also found that a supply of oxygen is still necessary. REVIEW
In 2008, a similar study conducted by Adams et. al. pertaining to enzymatic hydrolysis of

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.

of macroalgae, specifically brown algae has been presented. Figure 2.5 Pathway for processing brown seaweeds for

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.

provide a greater innovation for cleaner and greener bioethanol industry for our country. REVIEW OF LITERATURE
provide a greater innovation for cleaner and greener bioethanol industry for our country. REVIEW OF LITERATURE
In 2009, two parallel studies were conducted regarding the hydrolysis of two species of brown

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 Quiñones

(2010).

A

summary of the results of their chemical and enzymatic hydrolysis is shown in Table 2.7.

Table 2.7 Chemical and Enzymatic Hydrolysis of Various Brown Macroalgae

 

Chemical Hydrolysis

Enzymatic Hydrolysis

 
   

Condition

 

Brown Algae Specie

Condition (Acid

Conc., Temp.,

Time)

Reducing Sugar

(mg/ml)

(enzyme

loading, Temp.,

Time)

Reducing Sugar

(mg/ml)

Reference

Sargassum cristaefolium

3% HCl, 60 o C, 300 min

0.2661

0.5

mg/g,

2.6278

Reyes

50

o

C, 48hrs

(2009)

 

3% HCl,

     

Rivera

Sargassum kushimonte

60

o C,

0.3263

0.5

50

mg/g

o

C, 72hrs

2.7372

(2009)

 

300

i

       
 

6.67% HCl,

     

Quiñones

(2010)

Turbinaria ornata

80

o C,

0.7014

0.5

mg/g,

o

1.6333

255min

50

C, 72hrs

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.

analysis and no detailed analysis was done on the exact amounts of these carbohydrates. REVIEW OF
analysis and no detailed analysis was done on the exact amounts of these carbohydrates. REVIEW OF
2.4 RELATED STUDIES ON ALGINATE HYDROLYSIS Alginic acid or alginate (salt compound) is a polysaccharide

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% H 2 SO 4 (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 100 o C followed by treatment with 1.5N formic acid

for 2 hours at 100 o C 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 H 2 SO 4 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.

acid method. However, this study did not also indicate the uronic acid yield of the method.
acid method. However, this study did not also indicate the uronic acid yield of the method.
On the other hand, although researches have already been conducted regarding the isolation of alginases

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.

to the aggravating issues on fuel security and environmental degradation in the country. REVIEW OF LITERATURE
to the aggravating issues on fuel security and environmental degradation in the country. REVIEW OF LITERATURE
CHAPTER THREE MATERIALS AND METHODS 3.1 FEEDSTOCK PREPARATION The seaweed used in this experiment, Sargassum

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 70 o C so

as not to denature the samples.

not more than 4mm.

The oven dried samples was ground to particle size of

4mm. The oven dried samples was ground to particle size of Figure 3.1 Milled seaweed samples

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.

samples were stored in 2% (w/w) formalin solution to remove polyphenols from the seaweed. MATERIALS AND
samples were stored in 2% (w/w) formalin solution to remove polyphenols from the seaweed. MATERIALS AND
Prior to extraction, several steps were done. First, the stored seaweed was washed with distilled

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 H 2 SO 4 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) Na 2 CO 3 solution was added.

The

resulting

mixture

was

magnetically

stirred

for

1hour

after

which

it

was

centrifuged (10,000 x g) for 10mins at 10 o C. The supernatant was stored at 4 o C prior to

precipitation. The gelatinous precipitate that formed was pressed manually to remove the

liquid. Then it was dried in an oven at 35 o C. The dried alginate was pulverized using

a mortar and pestle.

The dried alginate was pulverized using a mortar and pestle. Figure 3.2 Gelatinous alginate after acid

Figure 3.2 Gelatinous alginate after acid precipitation

3.3 CHEMICAL HYDROLYSIS PROCEDURE

Two grams (2g) of alginate (commercial, extracted, raw seaweed) were mixed

PROCEDURE Two grams (2g) of alginate (commercial, extracted, raw seaweed) were mixed MATERIALS AND METHODS Page
PROCEDURE Two grams (2g) of alginate (commercial, extracted, raw seaweed) were mixed MATERIALS AND METHODS Page
with 100 ml of formic acid in varying concentrations (70%, 80%, and 90%). It was

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 (60 o C, 80 o C and 100 o C) 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 100 o C for 2 hours. After the period, 5ml samples were obtained

and placed in a vial. Samples were stored at 4 o C prior to analysis.

a vial. Samples were stored at 4 o C prior to analysis. Figure 3.3 Chemical hydrolysis

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 25 o C 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

contained the following (w/v): 1.0% peptone, 0.1% yeast extract, 3.0% NaCl and 0.5% sodium MATERIALS AND
contained the following (w/v): 1.0% peptone, 0.1% yeast extract, 3.0% NaCl and 0.5% sodium MATERIALS AND
alginate (pH 7.8). After cultivation, the culture supernatant was obtained by centrifugation (10,000 x g)

alginate

(pH

7.8).

After

cultivation,

the

culture

supernatant

was

obtained

by

centrifugation (10,000 x g) at 4 o C 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, (NH 4 ) 2 SO 4 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 4 o C 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.

or L-guluronic acid per min under the above conditions. Figure 3.4 Alginate Culture Medium for Enzyme

Figure 3.4 Alginate Culture Medium for Enzyme Production

min under the above conditions. Figure 3.4 Alginate Culture Medium for Enzyme Production MATERIALS AND METHODS
min under the above conditions. Figure 3.4 Alginate Culture Medium for Enzyme Production MATERIALS AND METHODS
3.4.2 Enzymatic Hydrolysis Proper The method of Kitamikado et. al (1992) was modified for the

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 (37 o C, 40 o C and 45 o C). 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 CaCl 2 . 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.

to the extracted sodium alginate and raw seaweed powder. Figure 3.5 Enzymatic Hydrolysis Medium containing 5ml

Figure 3.5 Enzymatic Hydrolysis Medium containing 5ml semi-purified enzyme and extracted alginate

3.5 Enzymatic Hydrolysis Medium containing 5ml semi-purified enzyme and extracted alginate MATERIALS AND METHODS Page 31
3.5 Enzymatic Hydrolysis Medium containing 5ml semi-purified enzyme and extracted alginate MATERIALS AND METHODS Page 31
3.5 ANALYTICAL METHODS 3.5.1 Reducing Sugar Analysis The method developed by Miller (1972) was employed

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

Rochelle’s 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 100 o C 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.

was added to the tube and absorbances were read at 525nm. Figure 3.6 Uronic Acid Analyses

Figure 3.6 Uronic Acid Analyses of Samples

to the tube and absorbances were read at 525nm. Figure 3.6 Uronic Acid Analyses of Samples
to the tube and absorbances were read at 525nm. Figure 3.6 Uronic Acid Analyses of Samples
3.5.3 Protein Determination The method developed by Lowry et. al (1951) was adopted for the

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.

ml Folin-Ciocalteau Phenol Reagent was diluted with distilled water to 25 ml solution. MATERIALS AND METHODS
ml Folin-Ciocalteau Phenol Reagent was diluted with distilled water to 25 ml solution. MATERIALS AND METHODS
CHAPTER FOUR RESULTS AND DISCUSSION Macroalgae or more commonly known as seaweed is currently getting

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 (60 o C, 80 o C, and 100 o C) 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).

at different hydrolysis conditions (time, temperature and acid concentration). RESULTS AND DISCUSSION Page 34
at different hydrolysis conditions (time, temperature and acid concentration). RESULTS AND DISCUSSION Page 34
Table 4.1 Reducing Sugar Concentration (mg/ml) at different hydrolysis conditions   Reducing Sugar Concentration

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)

60

o C

80

o C

100

o C

60

o C

80

o C

100

o C

60

o C

80

o C

100

o C

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
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
1hr
1.5000
3hrs
1.0000
5hrs
0.5000
0.0000
60
80
100
Temperature ( o C)
Reducing Sugar (mg/ml)

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

80% acid concentration. It showed that as time progressed, the reducing sugar recovered RESULTS AND DISCUSSION
80% acid concentration. It showed that as time progressed, the reducing sugar recovered RESULTS AND DISCUSSION
from hydrolysis decreased. The same was true for temperature, which was, as temperature increased the
from hydrolysis decreased. The same was true for temperature, which was, as temperature increased the
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 ( o C)
Reducing Sugar (mg/ml)

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 Reducing Sugar (mg/ml)
1.6000
1.4000
1.2000
1.0000
0.8000
0.6000
0.4000
0.2000
0.0000
60
80
100
Reducing Sugar (mg/ml)

Temperature ( o C)

1hr1.0000 0.8000 0.6000 0.4000 0.2000 0.0000 60 80 100 Reducing Sugar (mg/ml) Temper at ure (

3hrs1.0000 0.8000 0.6000 0.4000 0.2000 0.0000 60 80 100 Reducing Sugar (mg/ml) Temper at ure (

5hrs1.0000 0.8000 0.6000 0.4000 0.2000 0.0000 60 80 100 Reducing Sugar (mg/ml) Temper at ure (

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

reducing sugar yield from the hydrolysis procedure decreased with time. However, unlike RESULTS AND DISCUSSION Page
reducing sugar yield from the hydrolysis procedure decreased with time. However, unlike RESULTS AND DISCUSSION Page
the previous two figures, the figure showed a different trend with regards to the temperature.

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

Level of significance,

Level of significance,  0.05

0.05

Error degrees of freedom

51

Error mean square

0.37244

Number of means Critical Range 2 3 0.4085 0.4301 A 1.5162 1 A 1.2865 3
Number of means
Critical Range
2
3
0.4085
0.4301
A
1.5162
1
A
1.2865
3
A
1.1340
5

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.

of alginate would have no significant effect on the amount of reducing sugar produced. RESULTS AND
of alginate would have no significant effect on the amount of reducing sugar produced. RESULTS AND
Table 4.3 shows the 3! CRD analysis of data for the effect of temperature on

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 60 o C to 80 o C then remained insignificantly different

after the temperature range.

Table 4.3 3! CRD Analysis for Effect of Temperature on Reducing Sugar

Level of significance,  Error degrees of freedom 0.05 51 Error mean square 0.33855 Critical
Level of significance, 
Error degrees of freedom
0.05
51
Error mean square
0.33855
Critical Range
0.38949
0.41006
A
1.6200
60
B
1.2865
80
B
1.0376
100

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

Level of significance,  Error degrees of freedom Error mean square 0.05 51 0.24860 Critical
Level of significance, 
Error degrees of freedom
Error mean square
0.05
51
0.24860
Critical Range
0.33376
0.35139
Duncan grouping a
Mean (mg/ml)
Acid Concentration
A
1.8227
80
B
1.1996
70
B
0.9218
90

a = values with the same letter are not significantly different

1.1996 70 B 0.9218 90 a = values with the same letter are not significantly different
1.1996 70 B 0.9218 90 a = values with the same letter are not significantly different
4.1.2 Effect of parameters on the Uronic Acid Yield In addition to the reducing sugar

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)

60

o C

80

o C

100

o C

60

o C

80

o C

100

o C

60

o C

80

o C

100

o C

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
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
1hr
0.8000
3hrs
0.6000
5hrs
0.4000
0.2000
0.0000
60
80
100
Temperature ( o C)
Uronic Acid (mg/ml)

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

of uronic acid decreased with time. In addition, Figure 4.4 showed that as temperature RESULTS AND
of uronic acid decreased with time. In addition, Figure 4.4 showed that as temperature RESULTS AND
increased, the uronic acid yield decreased. This result corresponded to the trend from Figure 4.1

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 80 o C. 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 Uronic Acid (mg/ml)
1.6000
1.4000
1.2000
1.0000
0.8000
0.6000
0.4000
0.2000
0.0000
60
80
100
Uronic Acid (mg/ml)

Temperature ( o C)

1hr1.0000 0.8000 0.6000 0.4000 0.2000 0.0000 60 80 100 Uronic Acid (mg/ml) Temper at ure (

3hrs1.2000 1.0000 0.8000 0.6000 0.4000 0.2000 0.0000 60 80 100 Uronic Acid (mg/ml) Temper at ure

5hrs1.2000 1.0000 0.8000 0.6000 0.4000 0.2000 0.0000 60 80 100 Uronic Acid (mg/ml) Temper at ure

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 80 o C. 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

4.3).The uronic acid yields were found to be in the range 0.3858 mg/ml – 0.7100 RESULTS
4.3).The uronic acid yields were found to be in the range 0.3858 mg/ml – 0.7100 RESULTS
mg/ml with peak values 0.7100 mg/ml, 0.7029 mg/ml and 0.6175 mg/ml for 1, 3 and
mg/ml with peak values 0.7100 mg/ml, 0.7029 mg/ml and 0.6175 mg/ml for 1, 3 and
mg/ml with peak values 0.7100 mg/ml, 0.7029 mg/ml and 0.6175 mg/ml for 1, 3 and 5
hours respectively.
0.9000
0.8000
0.7000
0.6000
0.5000
1hr
0.4000
3hrs
0.3000
5hrs
0.2000
0.1000
0.0000
60
80
100
Temperature ( o C)
Uronic Acid (mg/ml)

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.

values at different time intervals were not significantly different from each other. RESULTS AND DISCUSSION Page
values at different time intervals were not significantly different from each other. RESULTS AND DISCUSSION Page
Table 4.6 3! CRD Analysis for Effect of Time on Uronic Acid Level of significance,

Table 4.6 3! CRD Analysis for Effect of Time on Uronic Acid

Level of significance, Error degrees of freedom Error mean square 0.05 51 0.07949 Critical Range
Level of significance,
Error degrees of freedom
Error mean square
0.05
51
0.07949
Critical Range
0.18873
0.19870
A
0.9870
1
B
0.8459
3
B
0.6917
5

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 60 o C to 80 o C after which a significant decrease occurred until it reached

100 o C. This showed a different result as compared with the effect of temperature on

reducing sugar in which there was a significant decrease from 60 o C to 80 o C.

Table 4.7 3! CRD Analysis for Effect Temperature on Uronic Acid

Level of significance,  0.05
Level of significance,  0.05

Level of significance,

Level of significance,  0.05

0.05

Level of significance,  0.05
Error degrees of freedom Error mean square 51 0.08418 Critical Range 0.19422 0.20447 A 0.9381
Error degrees of freedom
Error mean square
51
0.08418
Critical Range