CLS-12A091

SCHOOL OF CHEMICAL AND LIFE SCIENCES
Diploma in Chemical Engineering
Academic Year 2012/2013
CP512Y Final Year Project
CLS-12A091
Evaluation Study on using seaweed as liquid fertilizer & bio-sorbent
Project Done by:
YULYA 1015272
DHARMEN HARISH RUPAWALA 1015959
LIM WEI JIE 1016158
In partial fulfillment of the requirements for the Diploma in Chemical Engineering
January 2013
Project Supervised by:
MS JESSY YAU

i
MODULE: CP512Y Final YearProject
DCHE091/Academic Year 2012/13 School of Chemical & Life Sciences
Acknowledgements
We would like to express our appreciation to the following people who have assisted
in the completion of this Final Year Project.
Firstly, our heartfelt gratitude goes out to our supervisor, Ms Jessy Yau. With her
consistent guidance and support, intertwined with our co-operation, we were able to
accomplish much more than what was expected during the span of this project.
Following which, we are thankful for Ms Teo Meng Choo (Technical Support Officer
of W314), Ms Serene Chiam (Technical Support Officer of W312) and Ms Wong Sue
Ting (Technical Support Officer of W318). They played an important role in our
project as they provided a brilliant work space to keep our equipment and providing
materials efficiently and sourcing for vendors in spite of their busy schedules.
We express our gratefulness to Ms Liew Poi Hoon (Technical Support Officer of
T11B406) and Ms Chiang Lee Keau (Technical Support Officer of T11B407) for
allowing us to make use of the Inductively Coupled Plasma Optical Emission
Spectrometry (ICP-OES) machine for our testing in T11B406.
Last but definitely not least, we thank Mr Andrew Kon and his Final Year Project
group consisting of Ng Pey Ling, Chee Kai Li and Ivan Cheng Jia Xing for giving us
much needed assistance and lending us their testing equipment.
Without any of their help, the completion of this project would not have been
possible. (This project was financially supported by Singapore Polytechnic.)

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Abstract
The objective of this Final Year Project was to conduct an evaluation study on using
Sargassum Sp. Seaweed as a potential source of liquid fertilizer on flowering plants
and heavy metal bio-sorbent for various sustainable development purposes. Apart
from that, a small scale feasibility study on using seaweed as a budding source of bio-
fuel was carried out.
After obtaining the seaweed from Sentosa, it was manually squeezed to obtain the
seaweed sap. The seaweed sap was used as the liquid fertilizer in our experimentation.
After extraction of seaweed sap, an elemental test was conducted using a HACH
DR890 Colorimeter and HACH DR2010 Spectrometer to analyze the N:P:K ratio of
the raw seaweed sap. From the testing, the N:P:K ratio of the seaweed sap was found
to be 1.091: 0.003 : 0.128.
To study the effectiveness of our liquid fertilizer on flowering plants, we selected an
evergreen flowering plant, Cuphea Hyssopifolia, which we applied our liquid
fertilizer to. The fertilizer was applied at 6 different volumes for a consecutive 8
weeks. After our study, a balanced NPK fertilizer was proved to be effective for
flowering plants.
The solid residue, which was left behind after extraction of the seaweed sap, was used
for 2 purposes, namely as a bio-sorbent and source of biofuel.
For bio-sorbent, we looked into the absorption of heavy metal ions. In our study, two
main factors that affect absorption efficiency were examined. These factors include
time and pH. From our experimentations, a pH of 4.5 and adsorption time of 5.5 hours
were deduced to be the optimum conditions in a batch adsorption system.
For biofuel, experimentations using the soxhlet extractor and conventional soaking
were carried out to test for the extraction of lipid oil from the solid seaweed residue.
After our experimentations, it was concluded that only a minimal amount of lipid oil
could be extracted. Therefore, further studies and experimentations have to be done to
determine the feasibility of seaweed as a source of biofuel.

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Table of Contents
Acknowledgements..........................................................................................................................................i
Abstract................................................................................................................................................................. ii
Table of Contents............................................................................................................................................iii
List of Illustrations.........................................................................................................................................vi
List of Tables...................................................................................................................................................... x
Chapter 1: Introduction...............................................................................................................................1
1.1 Project Aim......................................................................................................................................1
1.2 Project Objectives........................................................................................................................1
1.3 Project Scope..................................................................................................................................2
Chapter 2: Literature Review...................................................................................................................3
2.1 Introduction to Seaweed.........................................................................................................3
2.1.1 Introduction to the Genus of Sargassum.............................................................4
2.1.2 Morphology............................................................................................................................5
2.2 Introduction to Fertilizer........................................................................................................7
2.2.1 Organic Fertilizer and Inorganic Fertilizer ........................................................7
2.2.2 Essential Nutrients in Fertilizers and Their Roles in Plants....................8
2.2.3 N-P-K ratio of Fertilizers ............................................................................................12
2.3 Introduction to Flowering Plants....................................................................................14
2.3.1 Physical Characteristics and Behavior...............................................................14
2.3.2 Introduction to Cuphea Hyssopifolia..................................................................15
2.4 Introduction to Biosorption...............................................................................................17
2.4.1 Passive Accumulation Processes...........................................................................19
2.4.2 Characteristics of Brown Algae..............................................................................21
2.4.3 Biochemistry of Heavy Metal Removal in Brown Seaweed..................23
2.4.4 Biochemistry of Biosorption in Sargassum Sp..............................................25
2.5 Introduction to Biofuel..........................................................................................................26
2.5.1 World’s Energy Consumption and Energy Sources...................................26
2.5.2 Increasing Need for Alternative Energy and Fuel Sources....................28
2.5.3 Algal Biofuel........................................................................................................................33
2.5.4 Extraction and Conversion of Biofuel.................................................................37
2.5.5 Challenges of Extracting Biofuel from Seaweed..........................................40
2.6 Sustainable Development...............................................................................................41
Chapter 3: Design of Experiment........................................................................................................42
3.1 Extraction of Seaweed Sap..................................................................................................42
3.1.1 List of Materials and Apparatusfor Extraction of Seaweed Sap.........43

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3.1.2 Experimental Procedures..........................................................................................44
3.2 Elemental Testing of Seaweed Sap extracted..........................................................48
3.2.1 Testing Procedures........................................................................................................50
3.3 Testing on Effectiveness of Seaweed Liquid Fertilizer......................................66
3.3.1 List of Materials and Apparatus for Testing on Effectiveness of
Seaweed Liquid Fertilizer.............................................................................................................68
3.3.2 Experimental Procedures..........................................................................................70
3.4 Testing on Effectiveness of Seaweed Waste as Bio-sorbent..........................73
3.4.1 List of Materials and Apparatus for Bio-sorbent Testing.......................78
3.4.2 Experimental Procedures for Using Seaweed as a Bio-sorbent in a
Batch System........................................................................................................................................80
3.4.3 Experimental Procedures for Using Activated Carbon as an
Adsorbent in a Batch System......................................................................................................82
3.4.4 Experimental Procedures for Preparation of Heavy Metal Ions
Standards................................................................................................................................................83
3.4.5 Experimental Procedures of Operating the ICP-OES................................84
3.4.6 Experiment Procedures for Controlled pH Biosorption.........................86
3.5 Extraction of Lipid Oil from Seaweed Waste...........................................................88
3.5.1 List of Materials and Apparatus.............................................................................89
3.5.2 Experimental Procedures (Soxhlet Extractor)..............................................91
3.5.3 Experimental Procedures (Conventional Soaking)....................................95
Chapter 4: Results and Discussion.....................................................................................................96
4.1 Elemental Testing Results and Discussion................................................................96
4.1.1 Elemental Testing of Seaweed extract...............................................................96
4.1.2 Elemental Test Results of Enhancer.................................................................100
4.1.3 NPK rating of our enhanced seaweed fertilizer ........................................104
4.1.4 Amount of fertilizer to apply to plants............................................................106
4.2 Analysis of Effectiveness of Seaweed Liquid Fertilizer..................................109
4.2.1 Analysis on Yellow Leaves Growth...................................................................109
4.2.2 Analysis on Appearance Rating...........................................................................123
4.2.3 Discussion on effectiveness of seaweed liquid fertilizer .....................137
4.3 Analysis of Seaweed as a Bio-sorbent.......................................................................138
4.3.1 Pre-treatment of Bio-sorbent...............................................................................138
4.3.2 Results Obtained from Varying Adsorption Time under
Uncontrolled pH..............................................................................................................................140
4.3.3 Results Obtained from Varying pH under Controlled Adsorption
Time 143

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4.3.4 Results Obtained from pH Optimization Analysis under Controlled
Adsorption Time.............................................................................................................................147
4.3.5 Results Obtained from Time Optimization Analysis under the
Optimized pH Value.......................................................................................................................149
4.3.6 Results Obtained through Comparison with Activated Carbon......152
4.4 Analysis on feasibility of producing biofuel from seaweed waste..........155
Chapter 5: Recommendation..............................................................................................................156
5.1 Future works on seaweed fertilizer ...........................................................................156
5.1.1 Aeroponic System........................................................................................................156
5.1.2 Comparison between Aeroponics and Hydroponics..............................157
5.1.3 Substitution of Nutrient Solution with Seaweed Fertilizer................158
5.2 Futureworks for bio-sorbent..........................................................................................159
5.3 Future works on biofuel....................................................................................................162
5.3.1 Improvement on extraction yield......................................................................162
5.3.2 Analysis and processing of raw lipid oil........................................................162
Chapter 6: Conclusion.............................................................................................................................163
References.....................................................................................................................................................165
Appendix A: Information of Commercial Fertilizer Enhancer.......................................170
Appendix B: Commercial Seaweed Fertilizer Information..............................................171
Appendix C: Fertilizer Calculation Model...................................................................................172
Appendix D: Data of number of yellow leaves growing on plants..............................174
Appendix E: Data of appearance rating of plants..................................................................177
Appendix F: Rubric used for the appearance rating scale of 1 to 5............................180
Appendix G: Calculation to Preparation Heavy Metal Ions Solution.........................181
Appendix H: Heavy Metal Ions Standards Calibration CurveIron...............................183
Appendix I: Activated Carbon used for bio-sorbent comparison................................186
Appendix J: Solubility of Metal Hydroxides as a Function of pH..................................187
Appendix K: Half Reactions for Metal Precipitation............................................................188
Appendix L: Gantt Chart........................................................................................................................189

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List of Illustrations
Figure
Number
Figure Title Page
2.1.1 Plant body of seaweed 3
2.1.2 (From left to right) Phaeophyta, Rhodophyta and Chlorophyta 4
2.1.3 Sargassum sp. (seaweed) provided by Sentosa Authority 4
2.1.4
(a) Picture of Sargassum sp. collected from Sentosa shores;
(b) Picture of Sargassum sp.
5
2.2.1 Classification of Essential Nutrients for Plants 9
2.2.2 Label stating the percentage of N-P-K in the Fertilizer 12
2.3.1 Reproductive structures of Flowering Plants 14
2.3.2 Cuphea Hyssopifolia 15
2.4.1 Schematic diagram of adsorption vs. ion exchange 20
2.4.2 Cell wall structure of brown algae 22
2.4.3 Molecular Formula of β(14)-linked unbranchedglucan 23
2.4.4 Molecular Formula of fucoidan 23
2.4.5
Molecular formulae of the M-block (left) and the G-block
(right)
24
2.4.6
Covalently bonded M- and G- blocks forming the alginate
polymer
24
2.5.1
Line graph of world total final consumption from 1971 to
2010 by fuel
26
2.5.2 Pie chart of fuel shares of final consumption 27
2.5.3
Line graph of crude oil production from 1971 to 2011 by
region
28
2.5.4
Bar graph of recorded & projected world energy consumption
from 1990 to 2035
29
2.5.5 Estimate summary of energy statistic 29
2.5.6
The average atmospheric carbon dioxide concentration over
the last millennium compared with the average temperature
changes in the northern hemisphere
30
2.5.7
The effect of anthropogenic emissions on atmospheric
concentrations of carbon dioxide
31
2.5.8
Percent reduction in emissions of pollutant in 100% biodiesel
(B100) and 20% biodiesel blend (B20)
32
2.5.9 Cell wall of brown seaweed 37
2.5.10 Structural formula of triglycerides 37
2.6.1 Constituting portions of Sustainable Development 41

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3.1.1
List of materials, apparatus and their uses in extraction of
seaweed sap
43
3.1.2
A conductivity probe used to measure conductivity of tap
water
44
3.1.3 Seaweed is washed with tap water in basin 44
3.1.4 Seaweed is cut into smaller pieces 45
3.1.5 Seaweed is weighed 46
3.1.6 Seaweed is blender 46
3.1.7 Seaweed sap is filtered through cheesecloth 47
3.2.1 DRB 200 Reactor 50
3.2.2 A reagent blank is prepared 50
3.2.3 Organic-free water is added to the vial 50
3.2.4 A sample is prepared 51
3.2.5 Sample is added to the vial 51
3.2.6 Vials are placed in the Reactor for heating 51
3.2.7 Hot vials are allowed to cool 52
3.2.8 “PRGM” button on HACH DR/890 Colorimeter 52
3.2.9
“6”, “9” and “ENTER” buttons on HACH DR/890
Colorimeter
52
3.2.10 Total Nitrogen Reagent A is added 53
3.2.11 Total Nitrogen Reagent B is added 53
3.2.12 Sample or reagent blank is added to the vial 53
3.2.13 Vial is inverted slowly to mix 54
3.2.14
“TIMER” and “ENTER” buttons on HACH DR/890
Colorimeter
54
3.2.15 COD/TNT Adapter is inserted into the cell holder 54
3.2.16 Vial containing the reagent blank is placed into the adapter 55
3.2.17 “ZERO” button on HACH DR/890 Colorimeter 55
3.2.18 Sample vial is wiped 55
3.2.19 Vial containing the sample is placed into the adapter 56
3.2.20 “READ” button on HACH DR/890 Colorimeter 56
3.2.21 “PRGM” button on HACH DR/890 Colorimeter 57
3.2.22
“8”, “2” and “ENTER” buttons on HACH DR/890
Colorimeter
57
3.2.23 COD/TNT Adapter is inserted into the cell holder 57
3.2.24 Sample is added to the vial 58
3.2.25 Sample vial is wiped 58

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3.2.26 Sample vial is placed into the adapter 58
3.2.27 Sample vial is covered 59
3.2.28 “ZERO” button on HACH DR/890 Colorimeter 59
3.2.29 PhosVer 3 Phosphate is added 59
3.2.30 Vial is shook 60
3.2.31
“TIMER” and “ENTER” buttons on HACH DR/890
Colorimeter
60
3.2.32 Sample vial is placed in the adapter 60
3.2.33 Sample vial is covered 61
3.2.34 “READ” button on HACH DR/890 Colorimeter 61
3.2.35 Mixing cylinder is filled with sample 62
3.2.36
Potassium 1 Reagent and Potassium 2 Reagent are added;
Mixing cylinder is inverted to mix
62
3.2.37 Potassium 3 Reagent is added; Mixing cylinder is shook 63
3.2.38 Three-minute reaction period is started 63
3.2.39 Prepared sample is poured into sample cell 63
3.2.40 Second sample cell is filled with the blank sample 64
3.2.41 “ZERO” button on HACH DR/2010 Spectrophotometer 64
3.2.42 Sample cell is wiped and placed into the cell holder 64
3.2.43 “READ” button on HACH DR 2010 Spectrophotometer 65
3.3.1
List of Materials, Apparatus and their use in Testing of
Effectiveness of Seaweed Liquid Fertilizer
68-69
3.3.2 Pots of Cuphea arranged in rows 70
3.3.3 First row of pots is labeled 70
3.3.4 Fertilizer enhancer is pounded into powdered form 71
3.3.5 Seaweed fertilizer is applied onto the plants’ roots 72
3.3.6 Number of yellow leaves are counted and recorded 72
3.4.1
List of Materials, Apparatus and their use in bio-sorbent
testing
78-79
3.5.1
List of Materials, Apparatus and their use in lipid oil
extraction
89-90
3.5.2 Seaweed residue 91
3.5.3 Seaweed residue is placed inside soxhlet chamber 92
3.5.4 Water bath is used to supply heat to soxhlet extractor 92
3.5.5 A simple distillation column 93
3.5.6 Lipid Oil is collected as residue 94
3.5.7 Lipid Oil is transferred into measuring cylinder 94

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4.1.1 1/Concentration of Nitrogen against Dilution Factor graph 97
4.1.2 1/Concentration of Phosphorous against Dilution Factor graph 98
4.1.3 1/Concentration of Potassium against Dilution Factor graph 99
4.1.4 1/Concentration of Nitrogen against Dilution Factor graph 101
4.1.5 1/Concentration of Phosphorous against Dilution Factor graph 102
4.1.6 1/Concentration of Potassium against Dilution Factor graph 103
4.3.1 Waste Seaweed Residue used for Bio-sorption 138
4.3.2
Percentage of Heavy Metal Ions Adsorption vs. Time under an
uncontrolled pH
141
4.3.3
Acidic and Basic Set-up to study Adsorption capabilities.
From left, Set-up of pH 13, pH 11, pH 9, pH 7, pH 5, pH 3
and pH 1.
143
4.3.4
Bar Chart comparing Percentage Adsorption with varying
acidic pH
146
4.3.5
Comparison of Percentage Adsorption with narrowed down
pH range
148
4.3.6 Study of Optimal Time of Bio-sorption under a controlled pH 151
4.3.7
Comparison of Adsorption Efficiency between Activated
Carbon & Seaweed Bio-sorbent
153
4.4.1 Lipid oil floating on water 155
5.1.1 Aeroponic System 156
5.3.1 PFD of batch bio-sorption design 160
5.2.2
Implementation of a packed bed column designed for bio-
sorption and desorption
161

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List of Tables
Table
Number
Figure Title Page
2.2.1 Sixteen Essential Plant Nutrients 8
2.2.2 Roles of Nutrients in Plants and Their Deficiency Symptoms 10
2.4.1
Three common algae divisions which possess cell walls and
the prominent characteristics
21
2.5.1 Data of crude oil production in 2011 28
2.5.2
Typical oil content and fatty acid compositions (% by wt of
total lipids) of plant and animal oils
33
2.5.3
Typical oil content and fatty acid content in oils from
microbial sources
34
2.5.4
Production of oil from plant sources, land area, and percent
cropping area required to displace transportation fuel in the
United States in biodiesel equivalents
35
2.5.5 Overcoming barriers to algae biofuels: Technology goals 40
3.2.1 Test Elements and Test Equipment 48-49
3.3.1 Volumes of enhanced seaweed fertilizer applied respectively 67
3.4.1 Effluent Discharge Limits according to NEA Guidelines 75
4.1.1 Concentrations of elements in our raw seaweed extract 96
4.1.2 NPK rating of our raw seaweed extract 96
4.1.3 Nitrogen Concentration Testing 97
4.1.4 Nitrogen Content 97
4.1.5 Phosphorous Concentration Testing 98
4.1.6 Phosphorous Content 98
4.1.7 Potassium Concentration Testing 99
4.1.8 Potassium Content 99
4.1.9
Concentrations of elements by dissolving 12g of enhancer in
100ml of DI water
100
4.1.10 Actual NPK rating of the enhancer 100
4.1.11 Nitrogen Concentration Testing 101
4.1.12 Nitrogen Content 101
4.1.13 Phosphorous Concentration Testing 102
4.1.14 Phosphorous Content 102
4.1.15 Potassium Concentration Testing 103
4.1.16 Potassium Content 103
4.1.17 Calculation on elemental weight in enhanced sap 104
4.1.18 NPK rating of enhanced seaweed fertilizer 105

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4.1.19
Ideal volume of fertilizer to apply to a hectare of land based
on nitrogen priority
106
4.1.20
on phosphorous priority
106
4.1.21
on potassium priority
106
4.1.22 Ideal volume of fertilizer to apply based on nitrogen priority 107
4.1.23
Ideal volume of fertilizer to apply based on phosphorous
priority
107
4.1.24 Ideal volume of fertilizer to apply based on potassium priority 107
4.3.1
Conductivity reading of water used in cleaning seaweed after
several washes
138
4.3.2 Concentration of Heavy Metal Remaining w.r.t time 140
4.3.3
Percentage Adsorption of Heavy Metal Ions w.r.t time under
uncontrolled pH
141
4.3.4 Visual Observation of the 7 selected pH values 144
4.3.5 Effect of Acidic and Basic Conditions in Bio-sorption 144
4.3.6
Heavy Metal Ions Concentration remaining with varying
acidic pH
145
4.3.7
Percentage Adsorption of Heavy Metals with varying acidic
pH
146
4.3.8 Optimization of pH Results 147
4.3.9 Adsorption Percentage of Optimization of pH 148
4.3.10
Concentration values of Heavy Metals at a fixed pH of 4.5
w.r.t time 149
4.3.11 Percentage Adsorption of the ideal pH value 150
4.3.12
Concentration values of Heavy Metals Remaining using
Carbon Adsorbent
152
4.3.13
Concentration values of Heavy Metals Remaining using
Seaweed Biosorbent
152
4.3.14
Percentage Adsorption of Heavy Metals using Carbon
Adsorbent
153
4.3.15
Percentage Adsorption of Heavy Metals using Seaweed Bio-
sorbent
153
4.4.1 Results on lipid oil extracted 155
5.1.1 Comparison between Aeroponics and Hydroponics 157

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Chapter 1: Introduction
In this chapter, the project aim, project objectives and project scope are stated.
1.1 Project Aim
The aim of this final year project is to evaluate potential uses of seaweed as fertilizer
for flowering plants and bio-sorbent. We are also exploring the feasibility of
producing biofuel out of the seaweed. By doing so, we are practicing sustainable
development as complied with responsible care concept, which is widely practiced by
chemical engineers and strongly promoted by Singapore Chemical Industry Council
(SCIC).
1.2 Project Objectives
In order to achieve the project aim, project objectives were set to guide us through the
project. The project objectives are as follows:
 Enhance the formulation of seaweed fertilizer used for both flowering and non-
flowering plants.
 Conduct experiments to test for the effectiveness of seaweed extract used as
fertilizer for flowering plants.
 Allow seaweed to serve as a potent, cheap and reliable source of adsorption
material.
 Conduct experiments to test for the effectiveness of seaweed residue used as bio-
sorbent.
 Maximize the potential of seaweed residue by means of producing biofuel.
 Conduct experiments to test for the feasibility of seaweed residue used as a
biofuel source.

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1.3 Project Scope
Seaweed is a very versatile product, which is widely used for many applications such
as food products, agricultural purposes, cosmetic products and even wastewater
treatment.
In this project, the seaweed collected from Sentosa Authority is Sargassum sp., which
is no applicable to all the applications stated. Nevertheless, this project focuses on
three main aspects of the seaweed, namely as organic liquid fertilizer for flowering
plants, bio-sorbent and source of biofuel.
ProjectScope
Seaweed as Fertilizer
Seaweed as Bio-sorbent
Seaweed as Biofuel

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Chapter 2: Literature Review
In this chapter, our literature study on focuses 4 main subjects: seaweed, fertilizer,
bio-sorbent and biofuel are elaborated in this section. A brief introduction to
flowering plants and sustainable development is also being explored in our literature
study to further aid our process through this project.
2.1 Introduction to Seaweed
Seaweed is classified under algae, which refers to plants where the body is
distinguishable into leaves, stem and roots. Marine algae are a term used to describe
seaweeds since it is found in the sea.
The plant body of seaweed is simple; the whole body of the plant is called thallus,
which consists of the holdfast, main axis and fronds.
Figure 2.1.1: Plant body of seaweed(Dhargalkar & Kavlekar 2004)
Fronds
Holdfast
Main axis

4
2.1.1 Introduction to the Genus of Sargassum
Seaweed is further classified into three broad categories based on pigmentation:
brown, red and green. These broad groups are referred to as Phaeophyta, Rhodophyta
and Chlorophyta respectively.
Figure 2.1.2: (From left to right) Phaeophyta, Rhodophyta and Chlorophyta
Phaeophyta varies in coloration from olive-yellow to deep brown; it is commonly
known as brown seaweed. Brown seaweeds are generally larger as compared to green
and red seaweed.
Sargassum sp, which is the raw material provided to us by Sentosa Authority for this
project, is classified under the Phaeophyta category due to its brown appearance.
Sargassum is a genus of marine algae, commonly found in marine water.
Figure 2.1.3: Sargassum sp. (seaweed) provided by Sentosa Authority

5
The scientific classification of Sargassum sp. is as follows:
Domain : Eukaryota
Kingdom : Chromalyeolata
Phylum : Heterokontophyta
Class : Phaeophyceae
Order : Fucales
Family : Sargassaceae
Genus : Sargassum
2.1.2 Morphology
In this section, the morphology of Sargassum sp. is discussed.
Figure 2.1.4: (a) Picture of Sargassum sp. collected from Sentosa shores;
(b) Picture of Sargassum sp. (Teo & Wee 1983)
The tissues of Sargassum sp. are differentiated into holdfast, main axis and fronds.
The holdfasts are very strong fibrous affairs, which sprout a growth of flat leaves. The
holdfast helps the main axis to remain attached to the substratum.

6
Branches arise in alternate fashion on primary axis. The axillary branch system
develops into fronds and flattened structure, called receptacles. The fronds are broad-
linear, 1 to 8cm long, 1 to 2cm wide, dense and frilly. On both surfaces of the fronds,
small pores, known as ostioles, are present. On the other hand, the receptacles bear
reproductive organs in special flask-shaped structures, which are known as
conceptacles.
Furthermore, Sargassum sp. has gas-filled bladders (vesicles). The vesicles increase
the plants’ buoyancy. The vesicles help in respiration and keep the fronds afloat for
purpose of photosynthesis. The vesicles are spherical, 3 to 7mm long, 2 to 5mm
across.

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2.2 Introduction to Fertilizer
Fertilizer is added to the soil, plant, or other growing medium to supply the essential
macronutrients and micronutrients to growing plants. The use of fertilizers has
increased crop yields and reduced hunger more than any other single agricultural
practice. Nevertheless, fertilizer must be applied correctly, in the right amount and
with regard to the surrounding ecosystem. Over-application of fertilizer can cause
toxic symptoms, whereas too little application of fertilizer can cause deficiency
symptoms.
In addition to supplying nutrients to crops to increase yields, fertilizers can pollute the
environment and result in marked changes in soil characteristics, some are beneficial
while some are not. These secondary influences play an important role in the choice
of fertilizer.
Fertilizers are generally categorized into two types, namely organic fertilizers and
inorganic fertilizers. This will be further elaborated in the next section.
2.2.1 Organic Fertilizer and Inorganic Fertilizer
Organic fertilizers are supplied to the soil by applying crop residues, animal manures
or naturally occurring minerals. Organic fertilizers are especially beneficial in adding
organic matter to help improve the ability of the soil to hold moisture and nutrients,
foster growth of soil organisms and promote healthier root development of the
plant.Moreover, the nutrients in organic fertilizer are released only when broken down
by soil microbes. Hence, they are less susceptible to mineral leaching, provides long-
term nutrition and steady plant growth. Unlike chemical fertilizers, using manure as a
soil additive also reduces its impact as a pollutant. Examples of organic fertilizer are
animal manures, poultry manure, blood, bone meal and seaweed.
Inorganic fertilizers are often referred as chemical fertilizers or synthetic fertilizers.
They usually consist of macronutrients, which include nitrogen (N), phosphorous (P)
and potassium (K). Of these three elements, nitrogen promotes the growth of leaves
and vegetation, phosphorous promotes root and shoot growth and potassium promotes

8
flowering, fruiting and general hardiness. The chemical fertilizers act fast, whereby
the nutrients are released too quickly, resulting in mineral leaching. Moreover, they
are rich in nutrients and can be easily over applied that “burn” roots or create toxic
concentration of salts. Therefore, continuous use of chemical fertilizer may destroy
nitrogen-fixing bacteria in the soil, affecting the plant growth. Besides creating harm
to the plants, chemical fertilizers also pose negative impacts on the environment.
Over-application and misuse of chemical fertilizers has resulted in the nutrients
infiltrating ground and surface water in some areas. Nitrates in ground water have
become a serious issue with human health, while phosphorous has been associated
with surface water problems. Examples of chemical fertilizers are urea, ammonium
and phosphates.
2.2.2 Essential Nutrients in Fertilizers and Their Roles in Plants
Nutrients are defined as chemical elements needed by plants for normal growth and
development. There are sixteen chemical elements that are considered to be the
essential plant nutrients. As shown in Table 2.2.1, these essential nutrients are divided
into mineral and non-mineral elements. Carbon, hydrogen, oxygen and nitrogen are
the non-mineral nutrients; the remaining twelve are mineral nutrients.
Non-mineral Primary Secondary Mineral Micronutrients
Name Symbol Name Symbol Name Symbol Name Symbol
Carbon C Nitrogen N Calcium Ca Boron B
Hydrogen H Phosphorous P Magnesium Mg Chlorine Cl
Oxygen O Potassium K Sulfur S Copper Cu
Iron Fe
Manganese Mn
Molybdenum Mo
Zinc Zn
Table 2.2.1: Sixteen Essential Plant Nutrients(Parker 2004)
Non-mineral nutrients, such as hydrogen and oxygen are supplied to plants from
carbon dioxide and water through photosynthesis. The mineral nutrients are supplied
by the soil through nutrient uptake processes. The sugars produced by the
photosynthetic process account for most of the increase in plant growth.

9
The twelve mineral nutrients and nitrogen are further classified into three groups,
depending upon the amount of each used by plants. The three groups are mainly the
primary or macronutrients, the secondary nutrients and the minor or micronutrients.
The following diagram shows the classification of the nutrients.
Figure 2.2.1: Classification of Essential Nutrients for Plants
Primary or macronutrients are nutrients used in the largest amounts, which include
nitrogen, phosphorous and potassium. Secondary nutrients are nutrients used in
intermediate amounts by plants, which include calcium, magnesium and sulfur. The
other seven nutrients are required by plants in small or micro amounts and hence, are
called micronutrients. Nevertheless, all nutrients are equally essential for plant growth
and metabolism.
The roles of the various plant nutrients and their deficiency symptoms are indicated in
Table2.2.2in the following page.

10
Nutrient Function Deficiency Symptoms
Nitrogen Promotes rapid growth; chlorophyll
formation; synthesis of amino acids
and proteins.
Stunted growth; yellow lower
leaves; spindly stalks; pale green
color.
Phosphorous Stimulates root growth; aids seed
formation; used in photosynthesis
and respiration.
Purplish color in lower leaves and
stems; dead spots on leaves and
fruits.
Potassium Increases vigor, disease resistance,
stalk strength and seed quality.
Scorching or browning of leaf
margins on lower leaves; weak
stalks.
Calcium Constituent of cell walls; aids cell
division.
Deformed or dead terminal leaves;
pale green color.
Magnesium Component of chlorophyll, enzymes
and vitamins; aids nutrient uptake.
Interveinal yellowing (chlorosis) of
lower leaves.
Sulfur Essential in amino acids, vitamins;
gives green color.
Yellow upper leaves; stunted
growth.
Boron Important to flowering, fruiting and
cell division.
Terminal buds die; thick, brittle
upper leaves with curling.
Copper Component of enzymes; chlorophyll
synthesis and respiration.
Terminal buds and leaves; blue-
green color.
Chlorine Not well defined; aids in root and
shoot growth.
Wilting; chlorotic leaves.
Iron Catalyst in chlorophyll formation;
component of enzymes.
Interveinal chlorosis of upper leaves.
Manganese Chlorophyll synthesis. Dark green leaf veins; interveinal
chlorosis.
Molybdenum Aids nitrogen fixation and protein
synthesis.
Similar to nitrogen.
Zinc Needed for auxin and starch
formation.
Interveinal chlorosis of upper leaves.
Carbon Component of most plant
compounds.
Hydrogen Component of most plant
compounds.
Oxygen Component of most plant
compounds.
Table 2.2.2: Roles of Nutrients in Plants and Their Deficiency Symptoms(Parker 2004)
The roles of essential macronutrients, such as nitrogen, phosphorous and potassium in
fertilizers and their deficiency symptoms will be further elaborated.

11
Nitrogen
Nitrogen is a part of chlorophyll, which gives plants their green color. When plants do
not receive enough nitrogen, their leaves lose their normal green color and turn
yellow. Tips of lower or bottom leaves turn yellow first.
Nitrogen-deficient plants usually grow slowly with spindly stalks and stems.
However, over-application of nitrogen will lead to a lush green overgrowth with
increased susceptibility to frosts and then collapse. Proper fertilization is very
important to prevent these symptoms in order to grow healthy plants.
Nitrogen can be very mobile in the soil and is subjected to many physical, chemical
and biological processes. Therefore, significant losses of the nutrient can occur. The
main pathways through which nitrogen not used by plants is lost from the soil are
leaching, erosion, denitrification and volatilization.
Phosphorous
Phosphorous is required mainly for root growth, photosynthesis and respiration.
Phosphorous-deficient plants usually have purplish lower leaves and stems and dead
spots on leaves and fruits.
Unlike nitrogen, phosphorous is very immobile in the soil and moves primarily as soil
particles are moved. It is lost from the soil through plant removal and soil erosion.
Due to its immobility and the relatively high need for phosphorous by young plants,
an adequate supply of phosphorous must be near the plant’s root system early in
growth.

12
Potassium
Potassium is second only to nitrogen in amounts used by plants. It is required by
plants mainly to increase vigor, disease resistance and stalk strength.
Potassium-deficient plants commonly experience scorching or browning along leaf
margins (edges) of lower or bottom leaves.
Although potassium is relatively immobile in the soil, some movement or leaching
loss of potassium occurs in soils containing large amounts of sand. Potassium
deficiencies are most likely to occur in plants growing in sandy soils.
2.2.3 N-P-K ratio of Fertilizers
A complete fertilizer contains the three primary nutrients: nitrogen, phosphorous and
potassium. Each bag of fertilizer must carry a label stating the analysis of its contents,
regardless of the source of the nutrients. This analysis is represented by three figures
prominently displayed on the bag; for example, 18-24-6.
Figure 2.2.2: Label stating the percentage of N-P-K in the Fertilizer(Lowe’s 2013)

13
The first figure is the percentage by weight/weightnitrogen, the second figure
represents phosphorous and the third figure is potassium. Other nutrients would be
listed by percentage by weight/weightelsewhere on the label. Any remaining
percentage consists of filler, which most often is dolomitic limestone or gypsum. This
applies for all solid fertilizer. Liquid fertilizers usually have their NPK rating in
percentage by weight/volume.
Although the label indicates percent nitrogen (N), phosphorous (P) and potassium
(K), only N is in the elemental form. Phosphorous is in the form of phosphate (P2O5)
and K is potash (K2O). In order to obtain the percentage of elemental P and K,
multiply the percent P by a correction factor of 0.44 (the percent of P in P2O5) and the
percent K by a correction factor of 0.83 (the percent of K in K2O) respectively.
While the label on the bag states the percentage of each primary nutrient, it may or
may not indicate the compounds used to make up the fertilizer. For example, the
nitrogen in the fertilizer might be supplied as urea, ammonium nitrate, ammonium
sulfate, calcium nitrate, or a combination of these. The formulation is important
because it informs the user of what compounds are used and their chemical form. By
knowing the formulation, a grower can manipulate plant growth by manipulating the
type of fertilizer applied to the plants. For example, nitrogen in the nitrate form
promotes a slower, less succulent growth than ammonium.
Fertilizer recommendations for plant growth are often expressed as a ratio, in pounds
per acre or kg per hectare. A ratio differs from an analysis because it expresses the
amount of one nutrient in relation to the other. For example, the ratio of nutrients in a
5-10-5 fertilizer is 1-2-1. Thus, if the recommendation was for a 1:2:1 fertilizer, a
product labeled 5-10-5, 10-20-10, or 15-30-15 would be equally acceptable as long as
the rate of application was adjusted accordingly. Moreover, for fertilizer
recommendations, the three figures (N-P-K) represent elemental nitrogen,
phosphorous and potassium respectively.
Each plant or crop has its own nutritional requirement. While most of these are very
similar, some plants have particular needs or sensitivities. For many crops, fertility
recommendations are available.

14
2.3 Introduction to Flowering Plants
Flowering plants, also known as angiosperms, are the largest grouping within the
plant kingdom (Kingdom Plantae or Viridiplantae) in terms of the numbers of
described species. Approximately 260,000 species of flowering plant have been
named so far, constituting nearly 90% of all known species of plants. There are about
450 families of flowering plants, displaying extremely diverse life histories and
ecological adaptions. In addition to dominating plant biodiversity, angiosperms are
the dominant photosynthetic organisms (primary producers) in most terrestrial
ecosystems.
2.3.1 Physical Characteristics and Behavior
Flowering plants are anatomically distinguished from other plant groups by several
development and anatomical features. They produce flowers, which are very short
branches bearing a series of closely spaced leaves modified to facilitate pollination
(sepals and petals) or to bear the organs involved in sexual production (stamens and
pistils), as shown in Figure 2- below.
Figure 2.3.1: Reproductive structures of Flowering Plants (Woods K. & Caley K.J. 2012)

15
Developing seeds are completely enclosed in an ovary derived from a portion of the
pistil. Ovary tissues mature to form a fruit that is generally involved in protecting the
seed and facilitating its dispersal. Seeds at some point in their development contain a
distinctive tissue, the triploid endosperm, which serves as a nutritional reserve for the
developing embryo.
2.3.2 Introduction to Cuphea Hyssopifolia
Cuphea hyssopifolia, also commonly known as Mexican false heather, is a compact
bushy spreading evergreen shrub, with height ranging from 30 cm to 45 cm. As
shown on Figure 2- below, C. hyssopifolia has small, lance-shaped dark green leaves
and small clusters of light purple flowers 1 cm across.
Figure 2.3.2: Cuphea Hyssopifolia
C. hyssopifolia produces quaint, small and trumpet-shaped flowers with six spreading
lavender petals and green calyx tubes. Its flowers appear singly in the leaf axils along
stems crowded with lance-shaped glossy green leaves. Although C. hyssopifolia is
heather-like in appearance, it is not a member of the heather family, hence the
common name of false heather. Its flowers are attractive to hummingbirds and
butterflies.

16
C. hyssopifolia is easily grown in average, medium and well-drained soils in full sun.
It may be grown from seed indoors for 8 to 10 weeks before maturity. C. hyssopifolia
requires regular watering, weekly or more depending on the period of exposure to
sunlight. Regular watering during the first growing season allows the plant to
establish a deep, extensive root system. Ideal fertilizer dosage for C. hyssopifolia in
NPK rating is 10:10:10.

17
2.4 Introduction to Biosorption
Biosorption is a term used to describe a physicochemical process that enables the
accumulation and concentration of containments such as heavy metals ions from an
aqueous solution through the use of biomass. Biosorption is a process which involves
a mixture of active and passive transport mechanisms. First the metal ion diffuses
itself on to the surface of the cell wall. After which, it binds onto the surface of the
cell wall that displays some affinity with the heavy metal ions. During this process, a
few passive accumulation processes get involved. This includes adsorption, chelation,
complexation, coordination and ion-exchange. Some of these terms will be further
discussed in later pages. All these passive processes are generally classified using an
umbrella term adsorption, as it helps to simplify our understanding of the mechanisms
behind the binding that occurs in algae. The processes described above are both fast
and reversible. A slower phase may be followed after the fast phase. In this phase,
metals are found to become permanently bound onto the cell wall of the seaweed.
This involves a few different mechanisms such as covalent bonding, redox reactions
and crystallization on the surface of the cell wall.
More often than not, the term biosorption is used interchangeably with
bioaccumulation. However, bioaccumulation varies drastically as it is used to describe
the accumulation of heavy metals due to a metabolic activity of the living biomass.
(Haytoglu et al. 2001). Therefore, it is crucial for us to understand this difference as it
helps in understanding the basic concept for the mechanisms of biosorption in
seaweed.
As stated in the earlier paragraph, biosorption is a process which occurs in dead
biomass. The use of dead biomass poses to be advantageous, as high concentrations of
heavy metals found in waters do not affect the cell. In addition, dead biomass is not
affected by the toxicity in the environment.
In the current years, much research and development is being employed to cater to the
studies of biosorption. These studies are looking into how biomass can be tied down
to the heavy metal industry where tons and tons of metal waste effluent is being
disposed off inappropriately. In addition, studies are looking into recovering precious
heavy metals from the production waste.

18
All varieties of biomass, ranging from yeast right down to bacteria, have been
employed to facilitate the study of biomass as the next big thing in heavy metal
recovery. Among all the biomass studied, brown algae biomass has demonstrated to
be the most reliable and effective in the extraction of heavy metal ions from an
aqueous solution.
The family of these brown algae was found to be with a common name of Sargassum
Sp. (As discussed in the earlier pages) In knowing our algae species, there is a greater
necessity to understand and appreciate the basic cell structure and biochemistry
underlying the biosorption mechanism. In addition, how brown algae compares to
other families of algae.

19
2.4.1 Passive Accumulation Processes
As described above, there are various passive accumulation processes which are
involved in the biosorption. They include adsorption, ion-exchange, chelation and
coordination.
Adsorption
Adsorption is the process of accumulating various substances that are in a solution on
a suitable interface. (Metcalf & Eddy, 2003) It is a mass transfer operation whereby, a
component in the liquid phase is transferred to the solid phase. In an adsorption
process, 2 common terms are used namely;Adsorbate and adsorbent. Adsorbate is the
component which is extracted from the liquid phase at the interface. On the other
hand, adsorbent is the solid phase, in which the adsorbate is accumulated upon. In
our study, we would only be focusing on solid-liquid adsorption.
Ion-Exchange
Ion-exchange is the mechanisms which is primarily responsible for the results
obtained through biosorption. It involves the reversible exchange of ions between an
aqueous solution and a solid ion-exchange matrix. This process is a unique case of
adsorption whereby a chemical reaction between free-moving ions in a liquid phase
and ions in an insoluble solid phase occurs. Mobile ions of the opposing charge will
exchange with ions of the same charge and attach themselves onto the external
surface of the solid. The diagram below describes the difference between adsorption
and ion exchange.

20
Figure 2.4.1: Schematic diagram of adsorption vs. ion exchange (Bakir 2010)
Ion exchange comes into the picture of biosorption as it helps us to understand and
explain the phenomenon of what is observe from heavy metal extraction experiments.
Complexation
Complexation is a chemical reaction which happens between a metal ion and a
molecule or ionic entity known as a ligand. This ligand contains at least a single atom
with an unshared pair of electrons. (McGraw-Hill, 2003).
Chelation
Chelation is a process which combines a metal ion to a chemical compound to
ultimately form a heterocyclic ring. The ring contains coordinate1
bonds which hold
the metal ion to at least 2 other nonmetal ions. (The American Heritage Science
Dictionary, 2005).
1
Coordinate bonds:Covalent bonds in which both electrons come from the same atom. These bonds
are also known as dipolar bonds.

21
2.4.2 Characteristics of Brown Algae
Algae can be defined as a wide group of autotrophic2
organisms. This diverse group
consists of unicellular to multicellular species. Most of the algae are microscopic in
size, thus most of them are classified as microorganisms. However, some forms of
algae take on a macroscopic structure. In light of the massive diversification, there are
several characteristics which are used to group varies classes of algae together. Table
1.1 below demonstrates the classification of algae which possess cell walls.
Table 2.4.1: Three common algae divisions which possess cell walls and the prominent
characteristics(T Davis, B Volesky and A Mucci, 2003)
In biosorption, the mechanism behind it is associated with the chemistry and the
presence of a cell wall. Where algae are concerned, biosorption is mainly accredited
to the properties of its cell wall. Electrostatic forces of attraction and complexationare
some of the cell wall properties which play the role of biosorption in most instances.
As seen from Table 2.3.1, all 3 algal groups (Chlorophyta, Phaeophyta and
Rhodophyta) are made up of an inner cell wall fibrillar skeleton and an outer cell wall
amorphous embedding matrix. This fibrillar skeleton is most commonly made up of
cellulose, while the amorphous embedding matrix is saturated with polysaccharides.
The polysaccharides include; xylans, pectin, mannans, algnic acids and fucinic acid.
2
Autotrophic: Self-sustaining and self-nourishing organisms which have the capabilities of making
their own food with the use of inorganic materials, for example, carbon dioxide.

22
The external structure of these diatoms3
is still preserved after death. Due to this
reason, the cell wall structure of seaweed is indeed responsible for the structural
integrity of the cell.
Focusing directly on brown algae, Phaeophyta, the amorphous matrix is mainly
constituted by alginic acid, followed by sulfated polysaccharides, fucoidan, in smaller
quantities.The diagram below aids our understanding in viewing the cell wall
structure of brown algae.
Figure 2.4.2: Cell wall structure of brown algae (T Davis, B Volesky and A Mucci, 2003)
The Phaeophyta and Rhodophyta families have the topmost amount of amorphous
embedding matrix polysaccharides. Polysaccharides have a well-known ability to
bind heavy metals to it. Thus,with this great volume of polysaccharides, it allows the
seaweed to be excellent heavy metal binders.
3
Diatoms: A large group of algae

23
2.4.3 Biochemistry of Heavy Metal Removal in Brown Seaweed
As mentioned previously, the cell wall is made up of 2 regions. The inner cell wall is
responsible for providing the rigidity to the cell wall. On the other hand the outer cell
wall is an amorphous embedding matrix. The rigid inner layer is primarily made up of
uncharged cellulose polymer. In brown algae specifically, the polymer found is a β
(14) - linked unbranched glucan. The molecular formula of this particular type of
glucan is displayed in the diagram below.
Figure 2.4.3: Molecular Formula of β(14)-linked unbranchedglucan
(T Davis, B Volesky and A Mucci, 2003)
Extracellular Polysaccharides
There are 2 main polysaccharides available on the extracellular surface of a brown
algae cell. They consist of fucoidan and alginate acid.
Fucoidan is a branched sulfated polysaccharide ester. L-fucose 4-sulfate is the major
building block of this polysaccharide. The average molecular weight of this substance
is 20,000. They constitute to about 5-20% of the dry weight in the Seaweed. The
structure of a fucoidan is shown in the diagram below.
Figure 2.4.4: Molecular Formula of fucoidan (T Davis, B Volesky and A Mucci, 2003)

24
Alginic acid is present in all types of brown algae. This compound is found on the
embedding matrix and the skeleton of the cell. Alginate acid makes up about 10-40%
of the dry weight of the seaweed. However, the amount present is highly dependent
on seasons and depth that they are grown at.
Alginic acid is also known as alginate. This name is given to a group of linear
copolymer which consist of a β-1- 4,-linked D-mannuronic acid (M-blocks) and α-1-
4,-linked –glucuronic acid (G-blocks). The molecular formulae of the M- and G-
blocks are displayed in the diagram below.
Figure2.4.5: Molecular formulae of the M-block (left) and the G-block (right)
These 2 monomers are covalently bonded in irregular patterns. Their arrangement in
the cell wall is unpredictable and the sequence of arrangement of the M- and G-
blocks forming the alginate are found to play a big role in the physical properties and
reactivity of the alginate. The fraction of the M-blocks and G-blocks vary from one
genus of brown algae to another. All these variations do demonstrate a difference in
affinity of heavy metals in the algae. The diagram below illustrates the how the blocks
are bonded together via covalent bonds which forms the alginate.
Figure 2.4.6: Covalently bonded M- and G- blocks forming the alginate polymer

25
2.4.4 Biochemistry of Biosorption in Sargassum Sp.
In Sargassum Sp., the alginate group is the most available acidic functional group
present in their cells. Where adsorption capabilities are concerned, this function group
is directly related to the binding sites which are available on the embedding matrix.
This alginate matrix makes up about 40% of a brown algae’s dry weight.
Apart from the carboxylic group, the second most present acidic functional group in
brown algae is found to be sulfonic acid in fucoidan. This group is typically a runner
up in terms of adsorption capabilities in the seaweed cell. When biosorption is
conducted below pH 2, they start playing a stronger role in adsorption.
Another group present in Sargassum Sp. which contributes to adsorption would be the
hydroxyl groups. They are present in all polysaccharides however are less bountiful
and are only found to become negatively charged when the pH exceed 10. Thus,
where metal affinity is concerned, their role is greatly hindered at low pH.
In light of Sargassum Sp., it naturally fits into our fundamental knowledge which
states that alginate plays one of the greatest roles in heavy metal binding. However, it
is important to note that ion-exchange used here doesn’t account for all the binding
which occurs throughout the algae. Instead, it is just as a hyponym used to describe
experimental observations from scientist. The accurate mechanism behind the binding
may range from physical right down to chemical bonding. Therefore, in the case of
the brown algae, the entire mechanism behind it can be viewed as an extracellular
occurrence on the surface of the cell wall.

26
2.5 Introduction to Biofuel
Biofuel can be obtained through physical extraction or chemical conversion of
biomasses. Common biofuels produced are bio-alcohols, biodiesel, vegetable oils and
biogas. In fact, fossil fuels like crude oil and natural gas are fuels formed by anaerobic
decomposition of ancient biomasses over the millions of years, similar to the
formation of biofuels. However, the main difference between biofuels and fossil fuels
is that biofuels are renewable source of energy while fossil fuels are not, since fossil
fuels take too long to produce.
Common biomasses used for biofuels production are listed in ascending order:
corn/maize, rapeseed, sugarcane, palm oil, jatropha, soybean, cottonseed, sunflower,
wheat and switchgrass.
2.5.1 World’s Energy Consumption and Energy Sources
Figure 2.5.1: Line graph of world total final consumption from 1971 to 2010 by fuel
(International Energy Agency 2012)

27
Figure 2.5.2: Pie chart of fuel shares of final consumption (International Energy Agency 2012)
In the year 2010, the world’s total energy consumption is estimated to be 8677 Mtoe;
equivalent to approximately 100.9 trillion kWh. Out of the total consumption, only
12.7% of the energy sources come from biofuels and waste.

28
2.5.2 Increasing Needfor Alternative Energy and Fuel Sources
Depletion natural resources
Figure 2.5.3: Line graph of crude oil production from 1971 to 2011 by region (International
Energy Agency 2012)
Producers Mt
% of
world
total
Saudi Arabia
Russian Federation
United States
Islamic Rep. of Iran
People’s Rep. of China
Canada
United Arab Emirates
Venezuela
Mexico
Nigeria
517
510
346
215
203
169
149
148
144
139
12.9
12.7
8.6
5.4
5.1
4.2
3.7
3.7
3.6
3.5
Rest of the world 1471 36.6
World 4011 100
Table 2.5.1: Data of crude oil production in 2011 (International Energy Agency 2012)
Crude oil production hit 4011 Mt in the year 2011; equivalent to approximately 26
billion barrels of crude oil (varies with density of crude oil). Crude oil consumption is
estimated to be approximately 34 billion barrels per year (CIA - The World Factbook,
2012). With current proven oil reserves of approximately 1.48 trillion barrels,
assuming that crude oil consumption remains at 34 billion barrels per year, that leaves
us with approximately 40 years left before oil reserves run out.

29
However, our world does not work as ideal we like it to be. With projected growth in
the world’s population and expending markets, especially in China and India, the
world’s energy consumption will definitely rise with the increasing demand. This puts
us more vulnerable to extinction of our natural resources even if more reserves are
available than proven. Alternative energy sources need to emerge and overtake fossil
fuels as our primary energy source to feed our needs.
Figure 2.5.4: Bar graph of recorded & projected world energy consumption from 1990 to 2035
(U.S. Energy Information Administration 2012)
Figure 2.5.5: Estimate summary of energy statistic (Worldometers 2012, Accessedon 28/12/12)

30
Climate changes
Figure 2.5.6: The average atmospheric carbon dioxide concentration over the last millennium
compared with the average temperature changes in the northern hemisphere (Hodgson 2010)
With reference from the above graph, we can see a general trend that as atmospheric
concentration of CO2rises, the temperature change in the world’s temperature will rise
as well. Atmospheric concentration of CO2 will continue to rise as CO2 emissions
continue to rise.

31
Figure 2.5.7: The effect of anthropogenic emissions on atmospheric concentrations of carbon
dioxide (Hodgson 2010)
Studies predicted that the world’s average temperature will rise by about 0.2°C per
decade and sea levels will rise by about 17cm in the next century due to global
warming caused by increasing greenhouse effect. Results of global warming could
range from inconvenience to disaster. Extreme weather conditions, melting of the
polar ice caps and glaciers, and disruption or extinction of flora and fauna are but a
few of the results of global warming. Below are the likely consequences of different
temperature rises as quoted from The Report by Sir Nicholas Stern in 2006:
1°C: Smaller mountain glaciers disappear in the Andes, threatening the water
supply of 50 million people. More than 300,000 extra people die from increase in
climate-related diseases in tropical regions.
2°C: Water scarcity increases in southern Africa and the Mediterranean.
Significant decline in food production in Africa, where malaria affects up to 60
million people.Up to 10 million extra people affected by coastal flooding each year.
3°C: Serious droughts in southern Europe occur once every ten years.
Between 1 and 4 billion people suffer water shortages and a similar number suffer
from floods. Many millions of people are at risk of malnutrition, as agriculture yields
at higher latitudes reach peak output. More than 100 million people are affected by
the risk of coastal flooding.

32
4°C: Sub-Saharan Africa and the southern Mediterranean suffer between 30
and 50% decrease in availability of water. Agricultural yields decline by 15-35% in
Africa. Crops fail in entire regions. Up to 80 million extra people are exposed to
malaria.
5°C: There is a possible disappearance of the large glaciers in the Himalayas,
affecting the supply of 25% of the population of China and hundreds of millions more
in India. Ocean acidity increases with threat of total collapse in the global fisheries
industry. Sea levels rise inexorably, inundating vast regions of Asia and about half of
the world’s major cities, including London, New York and Tokyo.
Alternative sources of energy like solar or wind energy that does not require
combustion or like biofuel are carbon-neutral sources of energy have to emerge and
overtake environment-destroying fossil fuels as our primary energy sources in hope to
savage our already damaged environment.
*TH is total hydrocarbons,CO2 is carbon dioxide, CO is carbon monoxide, PM is particulate matter, NOx is nitrogen oxides,
PAH is polycyclic hydrocarbons, and NPAH is nitrated PAHs.
Figure 2.5.8: Percent reduction in emissions of pollutant in 100% biodiesel (B100) and 20%
biodiesel blend (B20) (Drapcho, Nghiem & Walker 2008)

33
2.5.3 Algal Biofuel
Algal biofuels are considered second generation biofuels, whereby second generation
biofuels are classified as biofuels products of refinement of agriculture and forestry
waste. Similar to first generation biofuels, algal biofuels can be obtained from
physical extraction or chemical conversion of waste algae.
Why algal biofuel?
Higher oil content
Microalgae have been proven to be a potential leading source of energy since they
have relatively high oil content compared to other biofuel feed stocks. However, no
intensive research has been conducted on macroalgae since they have lower oil
content compared to microalgae.
Table 2.5.2: Typical oil content and fatty acid compositions (% by wt of total lipids) of plant and
animal oils (Drapcho, Nghiem & Walker 2008)

34
Table 2.5.3: Typical oil content and fatty acid content in oils from microbial sources (Drapcho,
Nghiem & Walker 2008)

35
Table 2.5.4: Production of oil from plant sources, land area, and percent cropping area required
to displace transportation fuel in the United States in biodiesel equivalents (Drapcho, Nghiem &
Walker 2008)
However, with the data available above, even with lower oil content in macroalgae
compared to microalgae, macroalgae still have a potential to be a viable source of
energy since microalgae have such high theoretical yields. The solid residues of the
seaweed after seaweed fertilizer extraction could be put to good use in such case.

36
Higher viability of seaweed
Seaweeds are generally harvested from open sea or seaweed farms while other biofuel
feed stocks are grown on arable land or high-tech farms like hydroponics farms and
aeroponics farms. This puts seaweed at a greater advantage to conventional biofuel
feed stocks since they are able to grow on outside arable land, or even in the sea,
hence allowing greater land area for other expansions such as food crops, natural
reserves and infrastructures, enabling large scales cultivation without fear of shortage
of lands.
On top of advantages on land usage, seaweeds can grow in sea water while
conventional biofuel feed stocks can only grow in fresh water. With water scarcity in
various area of the world, seaweeds prove to be a better choice of biofuel feed stock.
Food vs. oil dilemma
A general concern with biofuel is that it uses edible, staple food as its feed stocks.
This raises an ethical issue since millions still go hungry around the world. A
common proverb depicts this issue, “The rich get richer and the poor get poorer.”
Fuels are used to power the rich while the poor starve. Extracting algal oil from
Sargassum sp. remove the food vs. oil dilemma since Sargassum sp. isnot considered
a food source.

37
2.5.4 Extraction and Conversion of Biofuel
Figure 2.5.9: Cell wall of brown seaweed (Bakir 2010)
Extraction of biofuel from seaweed is to isolate the phospholipids from the
cell wall of the seaweed. Extracted phospholipids are in the form of triglycerides.
*C is carbon, H, is hydrogen, O is oxygen, R is carbon functional group
Figure 2.5.10: Structural formula of triglycerides (Doyle & Bell 2011)

38
Methods of extraction
1) Cold press method
Seaweeds are milled under high pressure to “squeeze” the phospholipids out of
the cell wall.
Advantage(s): Low cost, simple equipment used
Disadvantage(s): Low yield, difficult to clean
2) Solvent extraction method
Seaweeds are treated with solvents (hexane, ethanol, etc.) to dissolve the
phospholipids and hence, allowing the phospholipids to secrete out of the cell
wall.
Advantage(s): Simple equipment used, high yield, solvent can be used as
reactants for phospholipids conversion.
Disadvantage(s): Extra energy needed to separate solvent from phospholipids,
solvents might be harmful to health or environment.
3) Pyrolysis
Seaweeds are chemically decomposed by heating into oil. Reaction can occur in
both the presence and absence of oxygen and other reagents. Different operating
parameters generate different fuel.
Advantage(s): Very fast, high yield.
Disadvantage(s): Needs significant dehydration of seaweed for pyrolysis to work.

39
4) Supercritical Processing
Similar to solvent extraction method, supercritical processing uses supercritical
fluids, usually carbon dioxide, to extract phospholipidsout of the seaweeds
through dissolving and secreting.
Advantage(s): High purity products, high yield, no need for dewatering of
seaweed.
Disadvantage(s): Complicated method, require precise conditions for it to work.
Methods of conversion of extracted phospholipids
1) Chemical transesterification
As mentioned previously, the phospholipids existing in the cell walls are
triglycerides. They need to be treated before they become useful products.
Chemical transesterification treats triglycerides by reacting them with alcohol to
from fatty acid esters, which are used as biodiesel.
TAG + 3R’OH ↔ 3R’COOR + C3H5(OH)3
2) Catalytic Cracking
Catalytic cracking breaks down long carbon chains into simpler molecules. These
products are then further refined into fuels like gasoline, kerosene, etc.
Triglycerides Alcohol Fatty acidesters Glycerol

40
2.5.5 Challenges of Extracting Biofuel from Seaweed
As good as the statistics shows, there is always limitations hindering full efficiency of
the production of something. Below are the few challenges the world has to overcome
to have high efficiency in obtaining biofuel from seaweed.
OVERCOMING BARRIES TO ALGAE BIOFUELS: TECHNOLOGY GOALS
PROCESS
STEP
R&D CHALLENGES
FEEDSTOCK
Algal
Biology
• Sample strains from a wide variety of environments for maximum diversity
• Develop small-scale, high-throughput screening technology
• Develop open-access database and collection of existingstrains with detailed characterization
• Investigate genetics and biochemical pathways for production of fuel precursors
• Improve on strains for desired criteria by gene manipulation techniques or breeding
Algal
Cultivation
• Investigate multiple approaches (i.e., open, close, hybrid, and coastal/off-shore systems;
phototropic, heterotrophic, andmixotrophic growth)
• Achieve robust and stable cultures at a commercial scale
• Optimize system for algal productivity of fuel precursors (e.g., lipids)
• Sustainably and cost-effectively manage the use of land, water, and nutrients
• Identify and address environmental risks and impacts
Harvesting
and
Dewatering
• Investigate multiple harvestingapproaches (e.g., sedimentation, flocculation, dissolved air
floatation, filtration, centrifugation, andmechanizedseaweed harvesting)
• Minimize process energy intensity
• Lower capital and operatingcosts
• Assess each technology option in terms of overall system compatibility andsustainability
CONVERSION
Extraction
and
Fractionation
• Investigate multiple approaches (e.g., sonication, microwave, solvent systems, supercritical
fluid, subcritical water, selective extraction, andsecretion)
• Achieve high yield of desired intermediates; preserve co-products
• Minimize process energy intensity
• Investigate recycling mechanisms to minimize waste
• Address scaling challenges, such as operational temperature, pressure, carryingcapacity, side
reactions, and separations
Fuel
Conversion
• Investigate multiple approaches to liquid transportation fuels (e.g., direct fuel production,
thermochemical/catalytic conversion, biochemical conversion, andanaerobic digestion)
• Improve catalytic specificity, activity, anddurability
• Reduce contaminants andreaction inhibitors
• Minimize process energy intensity and emissions over the life cycle
• Achieve high conversion rates under scale-up conditions
Co-products
• Identify and evaluate the co-production of value-added chemicals, energy, and materials from
algal remnants (e.g., bio gas, animal/fish feeds, fertilizers, industrial enzymes, bioplastics, and
surfactants)
• Optimize co-product extraction andrecovery
• Conduct market analyses, including quality and safety trials to meet applicable standards
INFRASTRUCTURE
Distribution
and
Utilization
• Characterize algal biomass, intermediates, biofuel, and bioproducts under different storage and
transport scenarios for contamination, weather impacts, stability, andend-product variability
• Optimize distribution for energy and costs in the context of facility siting
• Comply with all regulatory and customer requirements for utilization (e.g., engine
performance andmaterial compatibility)
Resources
and Siting
• Assess and characterize land, climate, water, energy, and nutrient resource requirements for
siting of microalgae (heterotrophic& photoautotrophic) andmacroalgae production systems
• Integrate with wastewater treatment treatment and/or CO2 emitter industries (in the case of
heterotrophic approach)
• Address salt balance, energy balance, water & nutrient reuse, and thermal management
PURSING STRATEGIC R&D: TECHNO-ECONMIC MODELING AND ANALYSIS
Given the multiple technology andsystem options andtheir interdependency, anintegratedtechno-economic modeling
and analysis spanningthe entire algae to biofuels supply chain is crucial in guiding research efforts alongselect
pathways that offer the most opportunity to practically enable a viable and sustainable algae-based biofuels and co-
products industry.
Table 2.5.5: Overcoming barriers to algae biofuels: Technology goals (Doyle & Bell 2011)

41
2.6 Sustainable Development
Sustainable development is a widely discussed topic in today’s day and age.
Resources are depleting, prices are rising; carbon footprint is at an all-time high.
Everyone seems to be asking the same question, “At the rate of how things are
proceeding, who knows what is install for our future generations.”Therefore, to
answer all these questions the Brundtland Commission came up with the term
‘Sustainable Development’. Sustainable development can be defined as the
development that meets the needs of the present without the compromising the ability
of future generations to meet their own needs (Brundtland Report, 1987). Sustainable
development comprises of 2 major portions:
• The concept of needs, whereby the needs of the world’s poor should be given
utmost priority (Brundtland Report, 1987).
• The idea of limitations, enforced by the state of technology and social
organization on the environment’s ability to meet the present and future needs
(Brundtland Report, 1987).
All in all, sustainable development helps us to view the world as a system. When a
change is made to any part of the world, another portion of the world will indefinitely
be affected either directly or indirectly.
Sustainable development can be categorized into 3 parts: Social sustainability,
environmental sustainability and economical sustainability. When social and
environment sustainability is met, it is said to be bearable. When social and economic
sustainability is met it is said to be equitable. When economic and environment
sustainability are met it is said to be a viable system. A sustainable system is only
achieved when a system is said to socially, environmentally and economically
sustainable.
Figure 1.1: Constituting portions of Sustainable Development (Johann Dréo, 2006)

42
Chapter 3: Designof Experiment
In this chapter, the main experimental procedures involved in this project would be
discussed:
3.1 Extraction of Seaweed Sap
Extraction of seaweed sap involves obtaining the seaweed liquid extract and seaweed
solid waste. In this section, the materials and apparatus used are listed and the
procedures carried out for the extraction of seaweed sap are elaborated.

43
3.1.1 List of Materials and Apparatusfor Extraction of Seaweed Sap
Figure 3.1.1: List of materials, apparatus and their usesin extraction of seaweed sap

44
3.1.2 Experimental Procedures
Washing and Cutting of Seaweed collected
1. Use a conductivity probe to measure the conductivity of tap water, and record the
value.
Figure 3.1.2: A conductivity probe used to measure conductivity of tap water
2. Wash a portion of the seaweed with tap water in a basin to further remove sand
and salt particles from the seaweed surface for 10 minutes.
Figure 3.1.3: Seaweed is washed with tap water in basin
3. Use the conductivity probe to measure the conductivity of tap water again, after it
is used to remove impurities from the seaweed surface. If the conductivity of tap
water measured is higher than that recorded earlier, it implies that salt particles
have been removed from the seaweed surface.

45
4. Cut the washed seaweed into smaller pieces using a pair of scissors.
Figure 3.1.4: Seaweed is cut into smaller pieces
5. Repeat steps 2 to 4 for all the batches of seaweed.

46
Extraction of Seaweed Sap
1. Set up the blender.
2. Weigh a small portion of seaweed.
Figure 3.1.5: Seaweed is weighed
3. Place the seaweed into the blender and blend it for 2 to 3 minutes.
Figure 3.1.6: Seaweed is blender

47
4. Wrap the blended seaweed in a piece of cheesecloth.
5. Squeeze/Filter the seaweed sap through the cheesecloth in a container.
Figure 3.1.7: Seaweed sap is filtered through cheesecloth
6. Measure the volume of seaweed liquid extract collected using a measuring
cylinder.
7. Repeat steps 2 to 6 for all the batches of seaweed.
8. Record the total volume of seaweed liquid extract collected.

48
3.2 Elemental Testing of Seaweed Sap extracted
A sample of seaweed liquid extract was used for the elemental testing of nitrogen,
phosphorous and potassium using three testing equipment, namely the HACH DRB
200 Reactor, HACH DR/890 Colorimeter and HACH DR/2010 Spectrophotometer.
The test elements and their respective testing equipment are as follows.
Test Element Testing Equipment
Nitrogen HACH DRB 200 Reactor
HACH DR/890 Colorimeter

49
Phosphorous HACH DR/890 Colorimeter
Potassium HACH DR/2010 Spectrophotometer
Table 3.2.1: Test Elements and Test Equipment

50
3.2.1 Testing Procedures
Elemental Testing of Nitrogen
1. Turn on the DRB 200 Reactor. Heat to 103 – 106 °C (optimum temperature is
105°C).
Figure 3.2.1: DRB 200 Reactor
2. Prepare a reagent blank: Using a funnel, add the contents of one Total Nitrogen
Persulfate Reagent Power Pillow to one HR Total Nitrogen Hydroxide Digestion
Vial. (Note: Wipe off any reagent that gets on the lid or the tube threads.)
Figure 3.2.2: A reagent blank is prepared
3. Add 0.5 mL of organic-free water to the vial. Cap the vial and shake vigorously
for about 30 seconds. Process this reagent blank exactly the same as the sample,
including digestion and color finish. (Note: Alternate water must be free of all
nitrogen-containing species.)
Figure 3.2.3: Organic-free water is added to the vial

51
4. Prepare a sample: Using a funnel, add the contents of one Total Nitrogen
Persulfate Reagent Powder Pillow to one HR Total Nitrogen Hydroxide Digestion
Vial. (Note: Wipe off any reagent that gets on the lid or the tube threads.)
Figure 3.2.4: A sample is prepared
5. Add 0.5 mL of sample to the vial. Cap the vial and shake vigorously for about 30
seconds. (Note: The persulfate reagent may not dissolve completely after
shaking.)
Figure 3.2.5: Sample is added to the vial
6. Place the vials in the Reactor. Heat for 30 minutes.
Figure 3.2.6: Vials are placed in the Reactor for heating

52
7. Using finger cots or gloves, remove the hot vials from the reactor and allow to
cool to room temperature. (Note: It is very important to remove the vials from the
Reactor after exactly 30 minutes.)
Figure 3.2.7: Hot vials are allowed to cool
8. Enter the stored program number for Test ‘N Tube HR Total Nitrogen. Press:
PRGM. The display will show: PRGM ?
Figure 3.2.8: “PRGM” button on HACH DR/890 Colorimeter
9. Press: 69 ENTER. The display will show mg/L, N and the ZEROicon. (Note:
For alternate forms (NH3, NO3), press the CONC key.)
Figure 3.2.9: “6”, “9” and “ENTER” buttons on HACH DR/890 Colorimeter

53
10. Add the contents of one Total Nitrogen Reagent A Powder Pillow to the vial
containing the digested blank or sample. Cap the vial and shake vigorously for 15
seconds. Press: TIMER ENTERafter shaking. A three-minute reaction period
will begin.
Figure 3.2.10: Total Nitrogen Reagent A is added
11. After the timer beeps, add one Total Nitrogen Reagent B Powder Pillow to the
vial. Cap the vial and shake for 15 seconds. The display will show: 02:00 Timer
2. Press: ENTERafter shaking. A two-minute reaction period will begin. (Note:
The reagent will not completely dissolve. The solution will begin to turn yellow.)
Figure 3.2.11: Total Nitrogen Reagent B is added
12. After the timer beeps, remove the cap from one Total Nitrogen Reagent C Vial.
Add 2 mL of digested, treated sample (or reagent blank) to the vial. The vial will
be warm.
Figure 3.2.12: Sample or reagent blank is added to the vial

54
13. Cap and invert slowly 10 times to mix. The vial will be warm. (Note: Proper
mixing is important for complete recovery. Hold the vial vertical with the cap up.
Invert the vial and wait for all of the solution to flow to the cap end. Pause. Return
the vial to the upright position and wait for all of the solution to flow to the vial
bottom. This is one inversion (10 inversions = 30 seconds).)
Figure 3.2.13: Vial is inverted slowly to mix
14. The display will show: 05:00 Timer 3. Press: ENTER. A five-minute reaction
period will begin. Do not invert the vial again. (Note: The yellow color will
intensify.)
Figure 3.2.14: “TIMER” and “ENTER” buttons on HACH DR/890 Colorimeter
15. Insert the COD/TNT Adapter into the cell holder by rotating the adapter until it
drops into place. Then push down to fully insert it. (Note: For increased
performance, a diffuser band covers the light path holes on the adapter. Do not
remove the diffuser band.)
Figure 3.2.15: COD/TNT Adapter is inserted into the cell holder

55
16. When the timer beeps, wipe the outside of the Total Nitrogen Reagent C vial
containing the reagent blank. Place the vial into the adapter with the Hach logo
facing the front of the instrument. Push straight down on the top of the vial until it
seats solidly into the adapter. Tightly cover the vial with the instrument cap.
(Note: Do not move the vial from side to side during insertion, as this can cause
errors. Wipe with a damp towel, followed by a dry one, to remove fingerprints or
other marks.)
Figure 3.2.16: Vial containing the reagent blank is placed into the adapter
17. Press: ZERO. The cursor will move to the right, then the display will show: 0
mg/L N.
Figure 3.2.17: “ZERO” button on HACH DR/890 Colorimeter
18. Wipe the Total Nitrogen Reagent C vial containing the sample. (Note: Wipe with
a damp towel, followed by a dry one, to remove fingerprints or other marks.)
Figure 3.2.18: Sample vial is wiped

56
19. Place the vial into the adapter with the Hach logo facing the front of the
instrument. Push straight down on the top of the vial until it seats solidly into the
adapter. Tightly cover the vial with the instrument cap. (Note: Do not move the
vial from side to side during insertion, as this can cause errors. Multiple samples
may be read after zeroing on one reagent blank.)
Figure 3.2.19: Vial containing the sample is placed into the adapter
20. Press READ. The cursor will move to the right, then the result in mg/L nitrogen
(N) will be displayed. (Note: If the display flashes Limit, dilute the sample and
repeat the digestion and colorimetric finish. The digestion must be repeated for
accurate results; diluting and repeating the color finish does not yield complete
results. Multiply the result by the dilution factor.)
Figure 3.2.20: “READ” button on HACH DR/890 Colorimeter

57
Elemental Testing of Phosphorous
1. Enter the stored program number for reactive phosphorous (PO4
3-
), Test ‘N Tube.
Press: PRGM. The display will show: PRGM ?.
Figure 3.2.21: “PRGM” button on HACH DR/890 Colorimeter
2. Press: 82 ENTER. The display will show mg/L, PO4 and the ZEROicon. (Note:
For alternate forms (P, P2O5), press the CONC key.)
Figure 3.2.22: “8”, “2” and “ENTER” buttons on HACH DR/890 Colorimeter
3. Insert the COD/TNT Adapter into the cell holder by rotating the adapter until it
drops into place. Then push down to fully insert it. (Note: A diffuser band covers
the light path holes of the adapter to give increased performance. The band
should not be removed.
Figure 3.2.23: COD/TNT Adapter is inserted into the cell holder

58
4. Use a TenSette Pipet to add 5.0 mL of sample to a Reactive Phosphorous Test ‘N
Tube Dilution Vial. Cap and mix.
Figure 3.2.24: Sample is added to the vial
5. Clean the outside of the vial with a towel. (Note: Wiping with a damp towel,
followed by a dry one, will remove fingerprints or other marks.)
Figure 3.2.25: Sample vial is wiped
6. Place the sample vial into the adapter. Push straight down on the top of the vial
until it seats solidly into the adapter. (Note: Do not move the vial from side to side
as this can cause errors.)
Figure 3.2.26: Sample vial is placed into the adapter

59
7. Tightly cover the sample vial with the instrument cap.
Figure 3.2.27: Sample vial is covered
8. Press: ZERO. The cursor will move to the right, then the display will show: 0.00
mg/L PO4. (Note: For multiple samples, zero only on the first sample. Read the
remaining samples after adding the PhosVer 3 Reagent.)
Figure 3.2.28: “ZERO” button on HACH DR/890 Colorimeter
9. Using a funnel, add the contents of one PhosVer 3 Phosphate Powder Pillow to
the vial.
Figure 3.2.29: PhosVer 3 Phosphate is added

60
10. Cap the vial tightly and shake for 10 – 15 seconds. (Note: The powder will not
completely dissolve.)
Figure 3.2.30: Vial is shook
11. Press: TIMER ENTER. A 2-minute reaction time will begin. (Note: Read
samples between 2 and 8 minutes after the addition of the PhosVer 3 reagent. A
blue color will develop if phosphate is present.)
Figure 3.2.31: “TIMER” and “ENTER” buttons on HACH DR/890 Colorimeter
12. Immediately after the timer beeps, place the sample vial in the adapter. Push
straight down on the top of the vial until it seats solidly into the adapter. (Note: Do
not move the vial from side to side as this can cause errors.)
Figure 3.2.32: Sample vial is placed in the adapter

61
13. Tightly cover the vial with the instrument cap.
Figure 3.2.33: Sample vial is covered
14. Press: READ. The cursor will move to the right, then the result in mg/L
phosphate (PO4
3-
) will be displayed.
Figure 3.2.34: “READ” button on HACH DR/890 Colorimeter

62
Elemental testing of Potassium
1. This procedure requires a user-entered calibration prior to sample measurement.
See the User Calibration section to set up and calibrate a program for potassium.
2. Enter the stored user program number for Potassium (K). Press: 9 ? ? (954)
ENTER. The display will show: Dial to 650 nm.
3. Rotate the wavelength dial until the small display shows: 650 nm. When the
correct wavelength is dialed in, the display will quickly show: Zero Sample then:
mg/L K.
4. Fill a graduated mixing cylinder with 25 mL of sample. (Note: Filter highly
colored or turbid samples. Use filtered sample here and in Step 9.)
Figure 3.2.35: Mixing cylinder is filled with sample
5. Add the contents of one Potassium 1 Reagent Pillow. Add the contents of one
Potassium 2 Reagent Pillow. Stopper. Invert several times to mix.
Figure 3.2.36: Potassium 1 Reagent and Potassium 2 Reagent are added; Mixing cylinder is
inverted to mix

63
6. Add the contents of one Potassium 3 Reagent Pillow after the solution clears.
Stopper. Shake for 30 seconds. (Note: A white turbidity will form if potassium is
present.)
Figure 3.2.37: Potassium 3 Reagent is added; Mixing cylinder is shook
7. Press: SHIFT TIMER. A three-minute reaction period will begin.
Figure 3.2.38: Three-minute reaction period is started
8. Pour the solution from the cylinder into a sample cell (the prepared sample).
Figure 3.2.39: Prepared sample is poured into sample cell

64
9. When the timer beeps, the display will show: mg/L K. Fill the second sample cell
(the blank) with 10 mL of sample. Place it into the cell holder.
Figure 3.2.40: Second sample cell is filledwith the blank sample
10. Press: ZERO. The display will show: Zeroing…. Then: 0.0 mg/L K.
Figure 3.2.41: “ZERO” button on HACH DR/2010 Spectrophotometer
11. Within seven minutes after the timer beeps, wipe the prepared sample and place it
into the cell holder. Close the light shield.
Figure 3.2.42: Sample cell is wiped and placed into the cell holder

65
12. Press: READ. The display will show: Reading….then the result in mg/L
potassium will be displayed. (Note: Clean the cells with soap and a brush.)
Figure 3.2.43: “READ” button on HACH DR 2010 Spectrophotometer

66
3.3 Testing on Effectiveness of Seaweed Liquid Fertilizer
Cuphea hyssopifolia, which is a flowering plant, is used in this experiment to test for
the effectiveness of seaweed extract used as liquid fertilizer. As mentioned under
literature research section 2.3.2, C. hyssopifolia is easily grown in well-drained soils
under full sun, with regular watering. It is not advisable to apply fertilizer on young
Cuphea plants due to the excessive amount of nutrients present. Therefore, the
matured Cuphea plants are used in this experiment.
There are several factors that affect the plant growth, which include the exposure to
sunlight, amount of watering provided and volume of fertilizer applied. Since we are
carrying out testing on the effectiveness of seaweed fertilizer, two main possible
manipulating parameters for an effective fertilizer include the concentration or
volume of fertilizer applied to the plants. However, the concentration of fertilizer
applied to the plants is kept constant as highly concentrated fertilizer brings about
undesirable effects on the plants. A high concentration of fertilizer in the soil will
raise the water potential of soil water and hence, water will leave the plant root cells
by osmosis. As a result, in this experiment, the constant parameters include the
amount of exposure to sunlight, amount of watering provided and concentration of
fertilizer applied to the plants. The effectiveness of seaweed fertilizer is tested by
varying only one parameter, which is the volume of fertilizer applied to the plants.
After the elemental testing of seaweed extracted is conducted, the N:P:K rating of
seaweed extract is computed to be 1.091 : 0.003 : 0.128. In order to formulate a
balanced NPK fertilizer, addition of fertilizer enhancer is required. As discussed
under results and discussion section 4.1.3, the amount (in weight) of enhancer
required to be added into the seaweed extract is computed with the help of a fertilizer
model we designed on Microsoft Excel. With reference to Table 4.1.17, 9.8 g of
enhancer is required to be added into 100 mL of raw seaweed extract. Therefore, with
50 mL of seaweed extract, 4.9 g of enhancer is required to be added.

67
Six different volumes of seaweed fertilizer, ranging from 2.19 mL to 13.17 mL, are
applied to the respective rows of Cuphea plants. These volumes are varied based on
the amount of nitrogen (N), phosphorous (P) and potassium (K) contents present, in
order to determine the optimum volume of fertilizer required by the plants. The
sample size is selected to be 10 pots of Cuphea plants for each volume of enhanced
seaweed fertilizer applied. The pots are labeled with “I.D. #A-XX”, in which “A”
represents the row number whereas “XX” represents the pot number of the same row.
Group I.D. Volume of dilute enhanced seaweed fertilizer applied (mL)
#0 0.00
#1 2.19
#2 4.39
#3 6.58
#4 8.78
#5 10.97
#6 13.17
#7 Commercial Fertilizer (3.5 mL)
Table 3.3.1: Volumes of enhanced seaweed fertilizer applied respectively
Refer to Section 4.1.4 Amount of fertilizer to apply to plants on how the above values
were came up with.
The plant growth is monitored based on two main parameters, which include the
number of yellow leaves present on the plants and appearance rating of the plants.
The number of yellow leaves present on the plants indicates nitrogen deficiency, as
nitrogen is a part of chlorophyll, which gives plants their green color. Thus, when
plants do not receive enough nitrogen, their leaves lose their normal green color and
turn yellow. On the other hand, by rating the appearance of the plants on a scale of 1
to 5 allows us to monitor plant development qualitatively. The rubric used for the
appearance rating scale of 1 to 5 is attached in Appendix F.
Furthermore, the performance of seaweed liquid fertilizer is compared with that of
commercial seaweed liquid fertilizer available in the market. In this section, the
materials and apparatus used are listed, as well as the procedures carried out for
testing the effectiveness of seaweed liquid fertilizer.

68
3.3.1 List of Materials and Apparatus for Testing on Effectiveness of Seaweed
Liquid Fertilizer

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