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AUTHOR’S DECLARATION

“I hereby declare that this report is the result of my own work except for quotations
and summaries which have been duly acknowledge”

Nurul Syazmimi Bt Hamzah 12 April 2013


2010286288

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SUPERVISOR’S CERTIFICATION

“I hereby declare that I have read this thesis and in my opinion


this project report is sufficient in terms of scope and quality
for the award of Diploma of Chemical Engineering”

Signature :……………………………………….……….
Name :………………………………………….…….
Date :…………………………………………….….

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Accepted:

Signed :………………………………………
Date :……………………………………….

Coordinator
Encik Muhammad Zahiruddin Bin Ramli
Faculty of Chemical Engineering
Universiti Teknologi MARA
Pulau Pinang

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ACKNOWLEDGEMENT

I take this opportunity to express my profound gratitude and deep regards to


my supervisor Madam Nurul Huda Bt Amri for his exemplary guidance, monitoring
and constant encouragement throughout the course of this thesis. The blessing,
help and guidance given by her time to time shall carry me a long way in the journey
of life on which I am about to embark.

I also take this opportunity to express a deep sense of gratitude to University


of science Malaysia for supplying microalgae strain for me to done my study. In
addition, special thank to Sir Mohamed Syazwan Bin Osman which provided the
useful information to carry out this study.

Lastly, I thank almighty, my parents, sisters and friends for their for their
cordial support, valuable information and guidance, which helped me in completing
this task. Without all the help, this project would not be possible to finish on time.

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LIST OF CONTENTS

Page

AUTHOR’S DECLARATION .................................................................................... ii

SUPERVISOR’S CERTIFICATION.......................................................................... iii

ACKNOWLEDGEMENT ........................................................................................... v

LIST OF TABLES.................................................................................................. viii

LIST OF FIGURES .................................................................................................. ix

LIST OF PLATES ..................................................................................................... x

LIST OF SYMBOLS ................................................................................................ xi

LIST OF ABBREVIATION ...................................................................................... xii

ABSTRACT ........................................................................................................... xiii

CHAPTER 1 .............................................................................................................1

1.1 REASEARCH BACKGOUND ........................................................................1


1.2 PROBLEM STATEMENT ..............................................................................3
1.3 OBJECTIVE ..................................................................................................5

CHAPTER 2 .............................................................................................................6

2.1 ALGAE ..........................................................................................................6


2.2 MACROALGAE .............................................................................................6
2.3 LIGHT ...........................................................................................................7
2.4 AERATION OR MIXING OF MICROALGAE ..................................................8
2.5 NUTRIENT SUPPLY .....................................................................................9
2.6 CHEMICAL COMPOSITION OF MICROALGAE ............................................9
2.7 ALGAE CULTIVATION METHOD ................................................................11
2.8 OPEN POND ...............................................................................................11
2.9 PHOTOBIOREACTORS..............................................................................12
2.10 ALGAE HARVESTING ................................................................................13
2.11 DIFFERENTIAL BETWEEN MICROALGAE HARVESTING METHOD ........13

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2.12 MICROALGAE FLOCCULATION METHOD ................................................14
2.13 CHEMICAL FLOCCULANTS .......................................................................14
2.14 CHITOSAN..................................................................................................15
2.15 AUTOFLOCCULATION ...............................................................................15
2.16 RESPONSE SURFACE METHODOLOGY ..................................................16
2.17 BOX BEHNKEN DESIGN ............................................................................16
2.18 EFFECT OF PH ON FLOCCULATION EFFICIENCY ..................................17
2.19 EFFECT OF CHEMICAL DOSAGE..............................................................17
2.20 EFFECT OF AGITATION SPEED ................................................................19

CHAPTER 3 ...........................................................................................................20

3.1 OVERVIEW .................................................................................................20


3.2 PREPARATION STOCK SOLUTION ..........................................................21
3.3 STERILIZATION PROCESS........................................................................22
3.4 SUB CULTURING MICROALGAE STRAIN. ................................................23
3.5 MICROALGAE GROWTH CULTURE ..........................................................23
3.6 EXPERIMENTAL DESIGN AND OPTIMIZATION (RSM ANLYSIS) .............24
3.7 MICROALGAE HARVESTING PROCESS...................................................24

CHAPTER 4 ...........................................................................................................27

4.1 OVERVIEW .................................................................................................27


4.2 DEVELOPMENT OF REGRESSION MODEL EQUATION ..........................27
4.3 STATICAL ANALYSIS .................................................................................29
4.4 FLOCCULATION EFFICIENCY OF MICROALGAE .....................................31
4.3 PROCESS OPTIMIZATION .........................................................................34
4.3 HIGHEST FLOCCULATION EFFICIENCY CONDITION ..............................34
5.1 CONCLUSION ............................................................................................36

CHAPTER 6 ...........................................................................................................37

6.1 RECOMMENDATION..................................................................................37

LIST OF REFRANCE .............................................................................................38

APPENDICES A .....................................................................................................41

APPENDICES B .....................................................................................................42

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LIST OF TABLES

Page

Table 2.1: Feature and electricity consumption for different artificial light sources
( Chen et el.,2011). ...................................................................................8

Table 2.2: Chemicals contain inside microalgae (Spolaore, 2005). ...........................9

Table 2.3: Microalgae and other biofuels feedstock (Mata et al., 2010)...................10

Table 2.4: Advantages and disadvantages of open pond system. ...........................11

Table 2.5: Advantages and disadvantages of photobioreactor. ...............................12

Table 3.1: Recipe of Bold’s Basal Medium (Anderson, 2005)..................................21

Table 3.2: Independent variable and their coded level. ...........................................25

Table 3.3: Respond surface methodology analysis data. ........................................26

Table 4.1: Experimental design matrixes and result. ...............................................28

Table 4.2: Analysis of variance (ANOVA) for response surface quadratic model for
flocculation efficiency. ............................................................................30

Table 4.3: Model validation. ....................................................................................34

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LIST OF FIGURES

Page

Figure 2.1: The effect of pH on flocculation efficiency (Wu et al., 2012) ..................17

Figure 2.2: The effect of interphases height with time of different flocculants
concentration (Granados et al., 2012)...................................................18

Figure 2.3: The effect of flocculants dose to biomass recovery


(Granados et al., 2012). .........................................................................18

Figure 2.4: Effect of mixing rate on the removal of microalgae cells


(Ahmad et al., 2011). ...........................................................................19

Figure 3.1: Flow chart of overall experimental work ................................................20

Figure 4.1: Predicted versus actual experimental flocculation efficiency. ................31

Figure 4.2: Three-dimensional response surface plot of pH and CaCl2 dosage on


Chlorella sp flocculation efficiency (agitation speed = 200 rpm). ............32

Figure 4.3: Three-dimensional response surface plot of pH and agitation speed on


Chlorella sp flocculation efficiency (CaCl2 dosage = 0.11). ....................33

Figure 4.3: Three-dimensional response surface plot of CaCl2 dosage and agitation
speed on Chlorella sp flocculation efficiency (pH = 8.50). ......................33

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LIST OF PLATES

Page

Plate 2.1: Three basic classification of macroalgae (Andrade et al., 2005)................7

Plate 3.1: (a) The microalgae strain was exposed to 24 hour light supply.
(b) The aeration was done to increases the algae growth rate .................24

Plate 4.1: (a) Chlorella sp before pH addition of CaCl2. (b) Chlorella sp after
addition of CaCl2......................................................................................27

Plate A.1: Autoclave machine ……………………………………………………………41


Plate A.2: Analytical balance…………………………………………………………….41
Plate A.3: Micropipette……………………………………………………………………41
Plate A.4: Calcium Chloride……………………………………………………………...41
Plate A.5: Spectrophotometer…………………………………………………………...41
Plate A.6: Magnetic stirrer……………………………………………………………......41
Plate B.1: BBM medium…………………………………………………………….........42
Plate B.2: Sterilization process………………………………………………………….42
Plate B.3: Bacterial removal…………………………………………………………......42
Plate B.4: Subculturing process………………………………………………………....42
Plate B.5: Aeration process………………………………………………………….......42
Plate B.6: Measuring OD………………………………………………………………....42
Plate B.7: Addition of CaCl2………………………………………………………………43
Plate B.8: 10 minute mixing process…………………………………………………....43
Plate B.9: After flocculation process…………………………………………………….43

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LIST OF SYMBOLS

m Mass g

V Volume L/mL

W Power W

minute Time Min

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LIST OF ABBREVIATION

RSM Respond Surface Methodology

rpm Rotation speed

OD Optical density

BBM Bold Bassal Medium

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ABSTRACT

Microalgae feedstock are gaining interest in the present day energy scenario
due to their fast growth potential coupled with relatively high lipid, carbohydrate and
nutrients contents which render them as excellent source for biofuels such as
biodiesel, bioethanol and biomethane as well as a number of other valuable
pharmaceutical products. However, the critical part of producing microalgae product
is cost of harvesting process. Thus, flocculation process was choosen as it is the
cheapest microalgae harvesting process compared to other. In this study, the CaCl2
act as flocculants agent because the less toxicity compared to other chemical
flocculants. This experiment was done to determine the effect of flocculation
efficiency by adjusting the pH using NaOH and HCl, CaCl2 dosage and agitation
speed to achieve the optimum condition of harvesting the Chlorella sp. That variable
was tested based on the response surface methodology method (RSM) using box
banhken design. From the result, the optimum condition for Chlorella sp freshwater
was 10.50 in pH, 0.14 CaCl2 dosage and 200 rpm for agitation speed. RSM model
stated that the most effecting variable which effected flocculation efficiency is pH
and dosage. So CaCl2 can act as a good flocculants due to highest flocculation
efficiency which is 97.59%.

Keywords: microalgae, biofuels, Calcium Chloride, RSM.

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CHAPTER 1

INTRODUCTION

1.1 REASEARCH BACKGOUND

Nowadays, fossil fuels are necessary in human daily lives which producing energy
for transportation and generate electricity. About 80% of global energy demand is
produced from fossil fuels. However, lack of fossil fuels source influence
governments and research institutions making a great effort to develop new fuels. In
this case, biofuels are rapidly being developed from plantation oil crops, waste
vegetable oil and animal fat. Higher demand on food processing industry from those
sources unable to meet supplication for biofuels production. Therefore, some
species of microalgae, with high growth rate and high lipid content, appear to be
attractive alternatives as a feedstock for biodiesel production. Microalgae grow at a
remarkable rate 100 times faster than terrestrial plants and can double their biomass
in less than one day. Other than that, some microalgae strains are able to
accumulate large quantity of lipid inside their cells which can be converted to
biodiesel (Chisti, 2007). A microalgae biomass production lies between 15 to
25 tonne/ha/year with an assumption of 30% lipid content in microalgae cells. This is
equivalent to of 4.5 – 7.5 tonne/ha/year lipid production which is higher compared to
the production of oil from soybean, oil palm and jatropha (Kee & Lee, 2012).

Currently, extensive research has been done to classify the suitable


microalgae strains that can grow under high concentration of CO 2 while producing
lipid for subsequent biodiesel production. This renewable energy currently non-toxic,
biodegradable and lower emission of green house gas which contribute to global
warming (Demirbas, 2009a). Selected microalgae strains should higher in growth
rate and biomass productivity, high tolerance to detect amount of acidic components
from flue gases such as NOx and SOx and able to maintain their growth even under

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extreme culture conditions for example high temperature of water, contaminant and
pH (Brennan & Owende, 2010). A few recent studies have reported that Chlorella
sp., Scenedesmus sp., and Botryococcus braunii are among the microalgae strains
that have higher capability to consume CO2 emission with typical in the range of
200–1300 mg/L/day (Zhao et al., 2011).

Basically, algae can be defined as simple plant but lack of root, stem and
leave. It divided into two type which microalgae and macroalgae. Microalgae are
prokaryotic microorganisms which have simple structure like Cynobacteria. While
macroalgae are multicellular organisms consisting of a leaf like thallus instead of
roots, stems, and leaves (Jung et al., 2012). Both micro and macroalgae is
photosynthetic organism that requires light, carbon dioxide, water and inorganic
salts. To minimize the culturing cost, biodiesel production must depend on the sun
that can be obtained daily and for free with constant supply of light. Furthermore, the
inorganic elements that are needed for the growth of algae medium must consist of
nitrogen, phosphate, and carbon dioxide. In industrial scale microalgae commonly
being culture in open systems which allow relatively inexpensive but subject to
contamination that can limited the cultivation system. Beside that microalgae also
can be cultivate in closed system which can successfully control each of the
parameter for example temperature, light and nutrient. However, these cultivation
methods are not suitable for large industrial scale because higher in installation cost
(Harun et al., 2010).

Chlorella sp freshwater is one of the green microalgae species that can


growth in extreme condition. The production of oil lipid of the algae was dependent
on the cultivation system and harvesting of the algae. This type of microalgae was
higher in growth rate and biomass productivity, high tolerance to detect amount of
acidic components compared to other species for example Chlamydomonas and
Scenedesmus. It is also known due to its ability to produce highly valuable
biochemical or health food. So that, Chlorella sp are usually used in the production
of biofuels due to high higher oil lipid contains (Chisti, 2007).

After microalgae reached the stationary phase it is need to be separate from


water to recover their biomass for other biofuels processing. According to Greenwell
et al (2010) microalgae species with a size range of 1–50 µm in diameter always
form stable suspensions in growth medium due to their negative surface charge.
Hence, the separation and recovery of microalgae biomass from growth medium is a

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critical step in the microalgae biomass production process. There are several
methods that have been developed for harvesting microalgae which is
centrifugation, filtration, gravity sedimentation, electrophoresis and flocculation. The
common methods used for harvesting microalgae are flocculation due to lower in
cost for big scale biofuels production.

Flocculation is the addition chemical known as flocculants which is used to


amass microalgae cell from water. It is considered to be an effective and convenient
process, which allows rapid treatment of large quantities of microalgae cultures (Oh
et al., 2001). Basically, flocculation can be done either using a chemical additive
which is metal salt, polymer or chitosan. In short, there are many different type of
chemical flocculants such as Aluminium sulphate, Al2(SO4)3; Ferric chloride (FeCl2)
and Ferric sulphate (Fe2 (SO4)3 but higher in toxicity contain. Therefore, in this work
flocculation process of microalgae has been done using calcium chloride (CaCl2)
which is low in cost and less hazardous to environment compared to other chemical
flocculants.

1.2 PROBLEM STATEMENT

Microalgae are now commercially being cultured to replace other biofuels production
source. In order to perform the desired solid–liquid separation, suitable harvesting
method may involve physical, chemical or biological ways. This separation was
done to remove large quantities of water and to process large algae biomass
volumes. Various efforts have been made to increases lipid productivity of algae
cultivation (Wijffels & Barbosa, 2010). Thus, harvesting of algae biomass,
dewatering to concentrate and recover algae from dilute growth medium are
important to commercial scale operations which is the most energy intensive
aspects of the algae industry (Brentner et al., 2011). Further, to ensure the
economic and environmental sustainability of algae based fuels and materials it is
necessary to choose efficient harvesting methods in terms of material and energy
demands.

There are several effective ways to separate microalgae biomass from water
such as centrifugation and microfiltration. However, these methods is higher in cost
due to exceptional amount of energy consume hence not applicable in big scale
microalgae industry. Among all harvesting method, flocculation are common

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dewatering techniques which generally considered to be reliable and high-yielding
(Uduman et al., 2010). Flocculation is used to aggregate the microalgae cells to
increase the effective particle size. The most economic flocculation process is by
combining gravity sedimentation or flotation because it can concentrate the dilute
algae suspension to a thick slurry containing 2–7% solid material (Brennan &
Owende, 2010).

Generally, the mechanism of flocculation is the aggregation of suspended


cells into large particles due to interaction of flocculants with the surface change of
the cells. The large size of cells which coagulate will settle down cause by gravity
concept. In industrial scale, flocculants used should be inexpensive, less toxicity and
effective. Most studies on flocculation of microalgae carried out so far have focused
on a single species cultured under one particular condition. However, flocculations
depends on the properties of microalgae cell surfaces and these properties differ
between species depending on culture conditions. The cell surface to biomass ratio
increases with decreasing cell size, therefore smaller species will require a higher
flocculants dose to harvest the same amount of biomass than larger species.

The surface change on the algae can be countered by the addition of


chemical know as flocculants. Organic polymers is one of the which is
biodegradable and less toxic offers an alternative and environmental friendly but it
is not effective for marine microalgae due to inhabition by high ionic strength of
seawater (Bilanovic et al., 1988). Other than that, chitosan also used in flocculation
method which is cationic polyelectrolyte obtained by deacetylation of chitin (Renault
et al., 2009). However it is to expansive to be used for big scale production and
variable amount of chitosan will be required for different algae species thus resulting
in varies flocculation efficiencies.

In addition, chemical flocculation provides an economic solution to harvest


microalgae biomass. Nevertheless, high concentrations of chemical flocculants are
necessary to achieve satisfactory result and thus, being not environmentally benign.
Furthermore, chemical flocculants which mainly derived from multivalent salts for
example polyaluminium chloride can potentially contaminate the microalgae
biomass and bringing adverse effects towards the final product quality. Positive
charge of chemical for example aluminum sulfate and ferric chloride are often used
for charge neutralization but can potentially contaminate the microalgae biomass
and bringing adverse effects towards the final product quality which increases in

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toxicity contain. Alkaline iron(III) hydroxide may also be used as a flocculants but
has the same toxicity problems. Toxic flocculants are also unacceptable because
they do not allow whole algae or residues after oil extraction to be used as feed, or
to be used as feedstock for further fermentation.

Thus an alternative, chemical flocculants which is calcium chloride has been


choosen in this work to investigate the flocculation efficiency of Chlorella sp
freshwater. This is because calcium chloride has a good chemical property which is
less toxicity compare to other chemical flocculants and it is widely used in
wastewater treatment plant to remove fluorine. The efficiency of calcium chloride as
chemical flocculants that has been tested on chlorella freshwater was determined by
adjusting the pH value, dosage of the calcium chloride and agitation speed for mixed
the flocculants and Chlorella sp.

1.3 OBJECTIVE

The objectives of this study are:

1. To study the effect of pH, dosage of calcium chloride and agitation speed
on the flocculation process of Chlorella sp freshwater with CaCl2 as
flocculants.

2. The optimize the harvesting condition such as pH, dosage of CaCl2 and
agitation speed on the flocculation process of Chlorella sp freshwater
using respond surface methodology (RSM) analysis with respond to the
Chlorella sp. flocculation efficiency.

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CHAPTER 2

LITERATURE REVIEW

2.1 ALGAE

Algae is a group of aquatic, photosynthetic, eukaryotic organisms from


unicellular to multicellular forms and generally possess chlorophyll but lack true
roots, stems and leaves. It usually found in damp places or bodies of water and thus
are common in terrestrial as well as aquatic environment. It can be in a range from
the microscopic (microalgae) that live in water as single cells or in colonies, to large
seaweeds (macroalgae), such as giant kelp which is more than one hundred feet in
length (Rebolloso et al., 2001). Algae growth using photosynthesis process that
required sunlight, carbon dioxide, nitrate, phosphate and water which is some
bacteria convert solar energy into chemical energy. This energy is store in the form
of oils, carbohydrates, and proteins. The more efficient a particular plant converting
solar to chemical energy, the better it is from a biodiesel perspective and algae are
among the most photosynthetically efficient plants on earth (Lam et al., 2012)

2.2 MACROALGAE

Macroalgae are multicellular photosynthetic organisms consist of a leaf like


thallus instead of roots, stems, and leaves. Macroalgae are classified as green, red,
and brown algae, according to the thallus color derived from natural pigments and
chlorophylls as shown in Figure 2.1. Green algae, known as phylum Chlorophyta
has the same ratio of chlorophyll as land plants. While, red algae (Rhodophyta) is
the largest and most diverse group of tropical and temperate marine algae. Most of
them exist in tropical marine environments. Their dominant pigment is phycoerythrin,
which rich shades of red, orange and blue. Identification of red macroalgae can be

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difficult in some species, such as Gracilaria and some have slight variations
depending on the location and depth. On the other hand, brown algae are classified
as Phaeophyceae under phylum Chrysophyta. Their principal photosynthetic
pigments are chlorophyll a and c, b-carotene, and other xanthophylls. Those
macroalgae a now used to produce bioenergy for example biogas, bioethanol and
biobutanol (Jung et al., 2012).

GREEN RED BROWN


MACROALGAE MACROALGAE MACROALGAE

Plate 2.1: Three basic classification of macroalgae (Andrade et al., 2005).

2.3 LIGHT

In both indoor and outdoor microalgae cultivation systems, the light source
and light intensity are critical factors affecting the performance of the phototrophic
growth of microalgae. For outdoor cultivation, sunlight is the major light source while
some innovative artificial light sources for example LED and optical fiber are of
interest for indoor cultivation systems. In addition, it is also possible to transmit solar
energy from outside to illuminate indoor photobioreactors, such as solar-energy-
excited optical fiber systems (OF-solar). In industrial prospect, many light sources
have been applied to microalgae cultivation culture based on cost and efficiency.
Table 2.1 summarize the features and electricity consumption of using different light
sources for microalgae growth ( Chen et el.,2011).

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Table 2.1: Feature and electricity consumption for different artificial light sources
( Chen et el.,2011).

Light source Feature Operation Electricity


stability consumption
of the light
source (kW.h)
Conventional Higher biomass productivity, high High 40.32
artificial light stability, large illumination, low
sources constructing cost
LED Lower energy consumption, lower High 20.16
heat generation, longer life-
expectancy, tolerate higher
frequency of on–off switching,
higher stability, low constructing
cost
LED/OF-solar No electricity consumption, good High 0
combined with light path, uniform light
wind power or distribution, lower space
solar panel requirement,low contamination
risk

2.4 AERATION OR MIXING OF MICROALGAE

Mixing is necessary to prevent sedimentation of the algae, to ensure that all


cells of the population are equally exposed to the light and nutrients, to avoid
thermal stratification for example in outdoor cultures and to improve gas exchange
between the culture medium and the air. The importance part of this process is air
supply contains the carbon source for photosynthesis in the form of carbon dioxide
which is one of the parameter that can affect the microalgae growth. For very dense
cultures, the CO2 originating from the air bubbled through the culture is limiting the
algal growth and pure carbon dioxide may be supplemented to the air supply.
Depending on the scale of the culture system, mixing is achieved by stirring daily by
hand, aerating or using paddle wheels and jet pumps. However, it should be noted
that not all algae species can tolerate vigorous mixing (Lavens, 2010).

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2.5 NUTRIENT SUPPLY

Growing algae requires consideration of three primary nutrients which


carbon, nitrogen which in the form of nitrate, and phosphorus. These nutrients are
normally from chemical or inorganic fertilizers that are used to achieve promising
growth rate of microalgae and obtain bulk quantity of biomass. The use of chemical
fertilizer has the advantage of reducing contamination in culturing medium and thus
promotes water reutilization to re-culture microalgae. In addition, microalgae also
need trace metal include silica, calcium, magnesium, potassium, iron, manganese,
sulfur, zinc, copper and cobalt as micronutrients (Anderson, 2005).

2.6 CHEMICAL COMPOSITION OF MICROALGAE

The actual composition of microalgae is depending on the environmental


parameter which is light, temperature and algae media. Basically, the original
chemical composition of microalgae is 46 % for carbon dioxide, 10 % nitrogen, 1 %
phosphate and 43 % for other chemical. However, for producing microalgae product
different in chemical contain inside each of microalgae was examined based on
percentage of lipids, carbohydrate and protein contain as listed in Table 2.2.

Table 2.2: Chemicals contain inside microalgae (Spolaore, 2005).

COMMODITY PROTEIN CARBOHYDRATE LIPID

Chlamydomonas rheinhardii 48 17 21
Chlorella vulgaris 51-58 12-17 14 - 22
Dunaliella salina 57 32 6-8
Porphyridium cruentum 28-39 40-57 9 - 14
Spirulina maxima 60-71 13-16 6-7
Synechococcus sp 63 15 11

Furthermore, microalgae biomass also used to produce bioethanol. Protein


and carbohydrate inside microalgae are similar to most biomass such as corn and
sugar cane which is used to generate bioethanol. On the other hand, corn and sugar
cane biomass specifically used in production of food and required large land so new

9
way was investigate by creating ethanol using microalgae (Harun et al., 2010).
Other applications of microalgae are production of food supply due to higher protein
level. Protein level that contain in microalgae species consider them as
unconventional source of protein which having batter amino acid pattern compares
with other food proteins (Soletto, 2005). As the cells are capable of synthesizing all
amino acids they can provide necessary proteins to humans and animals
(Guil-Guerrroro, 2004).

Furthermore, for lipid contain in microalgae it used to produce sustainable


energy which is biofuels. Many microalgae species can be induced to accumulate
substantial quantities of lipids thus contributing to a high oil yield. The comparison of
biofuels production from microalgae and other plant source can be looked at Table
2.3 (Mata et al., 2010). Algae based technologies can reduce greenhouse gas
emissions from coal-fired power plants and other carbon intensive industrial
processes due to higher carbon dioxide releases in the air. All existing processes for
biodiesel production from microalgae include a production unit where cells are
grown, followed by the separation of the cells from the growing media and
subsequent lipids extraction. Then, biodiesel or other biofuels are produced same as
existing process and technology used for other biofuels feedstock.

Table 2.3: Microalgae and other biofuels feedstock (Mata et al., 2010).

Plant source Seed oil Oil yield Land use Biodiesel


content (l oil/ha (m2 year/kg productivity
(% oil by wt year) biodiesel) (kgbiodiesel/ha
in biomass) year)

Corn/Maize 44 172 66 152


Soybean 18 636 16 562
Jatropha 28 741 15 656
Sunflower 40 1070 11 946
Palm oil 36 5366 2 4747
Microalgae 30 58 700 0.2 51 927
(low oil content)
Microalgae 70 136 900 0.1 121 104
(high oil content)

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2.7 ALGAE CULTIVATION METHOD

Apart from nutrients supplement, microalgae can be cultivate either using


closed system or open system which important role to determine the successfulness
in growing the algae. The criteria that should be consider are illumination area, gas–
liquid transfer, land area requirement, ease of operation, contamination level, capital
and production cost. Raceway open pond and photobioreactor used are two
common methods for algae cultivation. Nevertheless, usually for commercial scale
only raceway pond apply due to production of high-value nutritious products for
animal feed and low in term of capital and production cost (Man & Keat, 2012).

2.8 OPEN POND

The open ponds are quite common method for culturing microalgae. The
open system came in many different shapes and forms, each having certain
strength and weakness as showed in Table 2.4. Basically algae can growth in
ponds, lakes, lagoons, and other water entrapment habitats. The design of raceway
open pond systems are shallow ponds which can easily absorb CO2 to dissolved in
water (Energy, 2002). The purpose of constructing the shallow depth pond is to
ensure that all algae are exposed to the sunlight. Paddle wheels also exist in this
pond system to gently mix all the algae, water, and nutrients around a racetrack
(Benemam et al., 2006). For the process flow, nutrients and water are constantly fed
into the pond and algae that contain water are withdrawn simultaneously.

Table 2.4: Advantages and disadvantages of open pond system.

ADVANTAGE DISADVANTAGES
o Suitable for algae that require o Open pond system exhibits higher
extreme conditions to survive algae cell density
Example : Spirulina sp. o Large land space and water used
o easy inoculation of new pond with a o Easy to contact with contamination
desired concentration and strain of or attack by invasive algae
algae through an outflow pipe species, bacterial, and others

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2.9 PHOTOBIOREACTORS

Photobioreactors is closed systems that have the ability of producing algae


while performing other tasks such as scrubbing power plant flue gases or removing
nutrients from wastewater treatment (Carlsson, 2007). Table 2.5 summarize several
goodness the closed culture environment. Photobioreactor come in many different
designs but the most common type is tubular photobioreactor which the most
satisfactory for producing algal biomass on the scale needed for biofuels production.
According to Becker (2004), photobioreactors that driven by artificial light are
already economical for large scale products such as pharmaceuticals which can be
combined with the production of biodiesel to reduce the cost.

Table 2.5: Advantages and disadvantages of photobioreactor.

ADVANTAGE DISADVANTAGES
o better control of cultivation o expensive to maintain
conditions o the distribution of light in the
o yield higher productivity and algae culture leading to inhibition
reproducibility in the total algae growth
o easy inoculation of new pond o requires some specialties for
with a desired concentration operations
and strain of algae through an o operated on small-scale units
outflow pipe due to difficulty in maintaining
o reduce contamination risk the mass transfer

Photobioreactors has shorter harvest time which is 2 to 4 weeks, and surface


volume ratio higher in the range of 25 to 125/m than open ponds (Lee, 2001; Wang,
2008). For the frequency of light and dark zone cycling, it is influence by intensity of
turbulence, cell concentration, optical properties of the culture, tube diameter, and
the external irradiance level. Tubular design has a number of clear transparent
tubes in diameter of less than 10 cm to maximize sunlight penetration that usually
aligned with the sun’s rays. A section of the algae is usually harvested after it
passes through the solar collection tubes, making continuous algal culture possible
(Chisti, 2007).

12
2.10 ALGAE HARVESTING

The important parts of producing the microalgae product are harvesting


process. A major challenge in downstream processing of microalgae lies in
separating the microalgae from their growth medium with the size range 1 - 50 µm
and density similar as the growth medium. A high biomass concentration leads to
mutual shading of the microalgae cells and thus a reduction in productivity.
Therefore, biomass concentrations in microalgae cultures are usually low from 0.5
g/L in open pond reactors to about 5 g/L in photobioreactors. It always form stable
suspension in growth medium due to it negative surface charge especially during
the growth phase (Milledge & Heaven, 2012). The harvesting method could be
divided into bulk harvesting and thickening. Bulk harvesting done by separating
microalgae from suspension using natural gravity sedimentation process,
flocculation, and flotation. According to Lam and Lee (2012) thickening involved
centrifugation and filtration process to concentrate microalgae slurry after bulk
harvesting process.

2.11 DIFFERENTIAL BETWEEN MICROALGAE HARVESTING METHOD

There are several major technique used for the harvesting microalgae which
is filtration, centrifugation, gravity sedimentation and flocculation. In detail, filtration
techniques are the process of running microalgae through filter that trapping the
microalgae until thick paste form. Nevertheless, rapid clogging, continues
backwashing, cost arising from pumping and membrane replacement are major
problem for this method. Furthermore, most commercial organizations use
centrifugation, the traditional method for harvesting microalgae but it is an energy
intensive process as it consumes a great deal of electric power (Chen et al., 2011).
Centrifugation required large capital investment with complicated processing and
need a higher energy input. For harvesting process that involved gravity
sedimentation, it is limited for large microalgae cell. Among all those process the
flocculation process are most preferred in industry because it allows rapid treatment
of large quantities of microalgae culture. By focusing on flocculation method many
thing is being consider for example type of flocculants, efficiency of the selected
flocculants and pH of the microalgae.

13
2.12 MICROALGAE FLOCCULATION METHOD

Flocculation is normally used in conjunction with other harvesting methods.


Increasing the size of particles by the aggregation of algal cells through flocculation
can increase the rate of settling or flotation. Flocculation has been suggested as a
superior method to separate algae as it can handle large quantities of suspension
and a wide range of microalgae. Evaluation of several harvesting methods showed
that flocculation combined with flotation or sedimentation and subsequent further
dewatering by centrifugation or filtration is the most promising cost and energy
efficient alternative (Schenk et al., 2008). Flocculation can be induced in different
ways which is chemical flocculation, chitosan and autoflocculation with Ca2+ ion
which is involve self flocculation of Chlorella sp.

2.13 CHEMICAL FLOCCULANTS

Microalgae cells are negatively charged, as a result of adsorption of ions


originating from organic matter and dissociation or ionization of surface functional
groups. Disrupting the stability of the system can obtain the successful microalgae
harvesting can be obtained. Addition of a coagulant, like iron-based or aluminum-
based coagulants will neutralize or reduce the surface charge of Scenedesmus and
Chlorella sp via charge neutralization (Grima et al., 2003). Microalgae can also be
flocculated by inorganic flocculants at sufficiently low pH (Uduman et al., 2010).
However, despite its advantages of using inorganic flocculants it may suffers from a
large concentration of inorganic flocculants is needed to cause solid–liquid
separation of the microalgae, thereby producing a large quantity of sludge.
However, the use of these inorganic coagulants produces a large volume of sludge
that can kill and prevent the growth of the microalgae (Chen et al., 2011). Although
alum has been shown to be an effective coagulant for some microalgae species,
flocculation by metal salts may be unacceptable if the biomass is to be used in
aquaculture, animal feed or fertilizer. The major component of alum and acrylamide
may also have human health implications, such as involvement in Alzheimer’s
disease and carcinogenesis. Because inorganic salts and organic polymers are not
suitable for harvesting live microalgae, a bionatural and environmentally friendly
chitosan appears to be a good coagulant for this purpose (Ahmad et al., 2011).

14
2.14 CHITOSAN

Chitosan has been recommended for use as a flocculant because it is non-


toxic, non-corrosive and safe to handle. It is biodegradable and biocompatible and
has attractive adsorption properties, flocculating ability and polyelectrolicity.
Additionally, it can be regenerated in a number of applications. The high cationic
charge density of chitosan allows it to strongly adsorb negative regions on other
particles and effectively destabilize them. This mechanism may act either through
polymer bridging or charge neutralization (electrostatic patch effects) that have been
found elsewhere. Other than that, chitosan also used in flocculation method which is
cationic polyelectrolyte obtained by deacetylation of chitin (Renault et al., 2009).
However it is to expansive to be used for big scale production and variable amount
of chitosan will be required for different algae species thus resulting in varies
flocculation efficiencies.

2.15 AUTOFLOCCULATION

Flocculation often occurs spontaneously in microalgae cultures when pH


increases above 9. This type of flocculation is often referred as autoflocculation
because it occurs spontaneously in microalgae cultures as a result of a pH increase
due to photosynthetic CO2 depletion. Autoflocculation is associated with the
formation of calcium or magnesium precipitates. Depending on the conditions, these
precipitates carry positive surface charges and can induce flocculation through
charge neutralization and/or sweeping flocculation. Calcium phosphate precipitates
are positively charged when calcium ions are in excess of phosphate ions and
interact with the negative surface charge of microalgae cells which cause the
autoflocculation to happen. To increases the flocculation efficiency, the addition of
Ca2+ ion or Mg2+ into the medium is one of the effective way to increases flocculation
efficiency because calcium ion inside microalgae medium are not enough in
enhance the flocculation even through the pH was adjusted (Salim et al., 2011).

15
2.16 RESPONSE SURFACE METHODOLOGY

Response Surface Methodology (RSM) is an experimental technique


invented to find the optimal response within the specified ranges of the factors
selected. These designs are capable of fitting a second order prediction equation for
the response. RSM is a statistics based procedure and is a powerful experimental
design tool to recognize the performance of composite systems. Response surface
methodology (RSM) is an empirical modeling technique used to evaluate the
relationship between a set of controllable experimental factors and observed results.
This optimization process involves three major steps which is performing statistically
designed experiments, estimating the coefficients in a mathematical model and
predicting the response and checking the adequacy of the model (Aslan & Cebeci,
2007). The flocculation efficiency of Chlorella sp freshwater was analyze using the
box behnken design to search the optimum condition of the flocculation.

2.17 BOX BEHNKEN DESIGN

Box–Behnken designs are a class of rotatable or nearly rotatable second-


order designs based on three-level, incomplete factorial designs. The number of
experiments (N) required for a full Box–Behnken design are given by the
formula N = 2 k (k − 1) + C0, where k is the number of factors and C0 is the number
of central points. In the optimization analysis, the target criteria were set as
maximum values for the optical density which present the flocculation efficiency
(Prakash Maran et al., 2013). The experimental with the higher desirability was
selected to be verified. The experimental data were analyzed using statistical
software which is Design Expert version 6.0.6 (STAT_EASE Inc., Minneapolis, USA)
for regression analysis to fit the second degree polynomial equation and also for the
evaluation of the statically significance of the equation develop
(Ahmad & Alrozi, 2011).

16
2.18 EFFECT OF pH ON FLOCCULATION EFFICIENCY

Flocculation using pH changes can be performed artificially by modifying the


pH of the culture with the addition of alkali (NaOH) or acid (HCl) or naturally
increasing the pH produced by cells under no pH control or adverse growth
conditions. Whatever the method, pH increase within the range from 8.5 to 12.5
allows the recovery of microalgae. According to Wu et al. (2012), the freshwater
microalgae cells began to agglomerate when the pH was adjusted up to about 8.6.
Based on Figure 2.1, it indicates that as pH increased to 10.6, the efficiencies were
greatly raised to more than 90% and reached plateaus. However, for flocculation
process of Chlorella sp freshwater species showed that the highly efficient pH of
microalgae are in the range of 11.5 to 12 but less efficient if the pH value exceed 12.

Figure 2.1: The effect of pH on flocculation efficiency (Wu et al., 2012)

2.19 EFFECT OF CHEMICAL DOSAGE

According to Granados et al.(2012), doses of flocculants were expressed as


mg of flocculants per liter of microalgae culture. Figure 2.2 represent the effect of
interphase high with time on different flocculants concentration. This figure showed
that if no flocculants was added, no interphase formed, and so the height of the
concentrated zone remained at the initial value. When flocculants was added, flocs
appeared and an interphase was clearly observed. The interphase height
diminished with time. So that the higher the flocculants doses, the higher the height
variation slope of the interphase. Figure 2.3 showed the biomass recovery for

17
different concentration of flocculants. The experimental tested show that, the higher
the flocculants doses, the higher the biomass recovery.

Figure 2.2: The effect of interphases height with time of different flocculants
concentration (Granados et al., 2012).

Figure 2.3: The effect of flocculants dose to biomass recovery


(Granados et al., 2012).

18
2.20 EFFECT OF AGITATION SPEED

Figure 2.4 demonstrates that agitation speed is crucial in the coagulation of


microalgae cells by chitosan coagulation. Referring to research made by Ahmad et
al. (2011), this figure shows that at a mixing speed of 150 rpm, the 10 ppm chitosan
dose resulted in the coagulation of 99.3 ± 0.4% of the microalgae cells. This rate is
sufficient for all of the microalgae cells to be absorbed by the chitosan. However,
when the mixing rate was increased further, the removal percentage decreased
slightly, reaching 97.8 ± 1.1% at a rate of 250 rpm. This study was applying the
mixing speed of 0 – 350 rpm. Nevertheless, the most efficient mixing speed is 150
rpm which mean the medium speed range are more effective speed. This
phenomenon is caused by the restabilization of the cells at the fast mixing speed.
High-speed mixing tends to break the flocs, causing the coagulated cells introduced
again into the medium.

Figure 2.4: Effect of mixing rate on the removal of microalgae cells


(Ahmad et al., 2011).

19
CHAPTER 3

METHODOLOGY

3.1 OVERVIEW

Chlorella sp freshwater strain was obtained from the Laboratory of Microalgae at


University of science Malaysia. Liquid Bold’s Basal Medium (BBM) was used to
cultivate this type of microalgae because each microalgae strain grow in different
media depends on their need on chemical composition which suite with their
function and structure. After cultivation process, flocculation method was proceed
with calcium chloride (CaCl2) as flocculants to study the effect pH, chemical dosage
and speed of rotation on flocculation efficiency and to determine the optimum
harvesting condition of Chlorella sp using Box Benhken design which is respond
surface methodology (RSM) method. Figure 3.1 shows a flow chart of overall
experimental work.

Preparation of microalgae
stock culture

BBM sterilization process

Microalgae sub culturing


process

Increasing microalgae growth


rate

Flocculation of microalgae for


harvesting process.

Figure 3.1: Flow chart of overall experimental work

20
After weighting all desired chemical, each macronutrient was mixed in 1 L
distilled water according to the weight listed in Table 3.1. For FeSO4 • 7H2O and
H2SO4, both was mixed in 1 L distilled water as acidified iron solution. Boron solution
which is H3BO4 also added in 1 L distilled water. Furthermore, another five chemical
left was mixed as trace metal solution which can be added at sufficiently high levels
to support vigorous algae. Each 1 L BBM mixture only needs 1 mL trace metal
solution on their growth. All those four part in BBM was mixed in 1 L distilled water in
conical flask.

3.2 PREPARATION STOCK SOLUTION

The selection and preparation of culture media is very important for culturing
microalgae to optimize the yield of phytoplankton in the culture. This medium consist
of five main parts which are macronutrient and alkaline EDTA solution, acidified iron
solution, boron solution, trace metals solution as micronutrient. Each part consist of
different chemical composition which is important to culture Chlorella sp. Table 3.1
shows the complete chemical composition and concentration for each part of the
medium including the microalgae nutrients which is nitrate and phosphate.

Table 3.1: Recipe of Bold’s Basal Medium (Anderson, 2005).

Component Stock Quantity Concen- Chemical


solution used tration in brand
(g.L-1 Final
dH2O) Medium (M)
Macronutrients
NaNO3 25.00 10mL 2.94 × 10-3
CaCl2 · 2H2O 2.50 10 mL 1.70 × 10-4
MgSO4 · 7H2O 7.50 10 mL 3.04 × 10-4 R&M
K2HPO4 7.50 10 mL 4.31 × 10-4 chemical
-4
KH2PO4 17.50 10 mL 1.29 × 10
NaCl 2.50 10 mL 4.28 × 10-4
Alkaline EDTA solution 1mL
EDTA 50.00 1.71 × 10-4 R&M
-4
KOH 31.00 5.53 × 10 chemical

21
Table 3.1: Continued (Anderson, 2005)

Acidified Iron Solution 1mL 1.79 × 10-5


FeSO4 · 7H2O 4.98 1 mL R&M
H2SO4 chemical
Boron Solution 1mL
H3BO3 11.42 1.84 × 10-4
Trace Metals Solution 1 mL
ZnSO4 · 7H2O 8.82 3.07 × 10-5
MnCl2 · 4H2O 1.44 7.28 × 10-6 R&M
MoO3 0.71 4.93 × 10-6 chemical
CuSO4 · 5H2O 1.57 6.29 × 10-6
Co(NO3)2 · 6H2O 0.49 1.68 × 10-6

3.3 STERILIZATION PROCESS

After BBM medium ready, next step is sterilization process. This process
was done to remove or killing all microorganisms. Sterilization is very important to
maintain living organisms as isolated strains in culture. Sterile technique, in
combination with sterile equipment and supplies done to minimize contamination so
that this experiment free of potential variables caused by unwanted organisms.
Autoclaving is the most effective and popular way to sterilize heat resistant materials
and is generally used to sterilize liquids. An autoclave machine (Hirayama) as
shown in Appendix A was run at temperature 121ºC in 2 and half hour for
sterilization process of BBM before microalgae was culture inside the medium.
Enough water levels inside the autoclave machine were needed before placing the
basket containing the media to be autoclave. The mode button then pushed to
select liquid or solid form and the process was started. After the sterilization was
done the media was taken out and be cool. The container was remove from the
autoclave at temperature is about 50°C ˗ 60°C. Faster cooling process mixture can
also be done by placed it inside water bath because higher temperature may
condense the medium. Machine was turn off after sterilization process completed.

22
3.4 SUB CULTURING MICROALGAE STRAIN.

The working area of transferring microalgae to the medium must be done in


non-bacterial environment. Thus, 70% ethanol was used to wipe the working area to
ensure no bacterial can interfere the medium and the original strain. The Bunsen
burner was place between conical flasks that contain the BBM medium and
Chlorella sp strain. After organizing all materials, the Bunsen burner was lighted.
Ethanol was spray to hands which using sterile disposable gloves to make sure no
bacteria can affect the sub culturing process. The mouth of the conical flask that
filled with culture is bringing close to the Bunsen burner to be flame. A few mL of
culture in ratio 1:10 (volume microalgae strain: volume of the BBM) was suck using
the micropipette. After suction process the conical flask was flame before closed
with aluminum foil to ensure no bacteria left within the process. The tip of the pipette
must be dissociated from source of flame to ensure no microalgae die due to high
temperature. The micropipette which contains Chlorella sp strain immediately
carried careful to make sure micropipette tip never touches the container surface or
anything else. The micropipette tip was slowly inserted into the conical flask that
contains BBM without touching the pipette against the conical flask mouth. Before
transferring the microalgae, the mouth of the BBM conical flask also be flame to kill
bacteria that may disturbed the microalgae culture. The figure of sub culturing
microalgae step can be refer in Appendix B.

3.5 MICROALGAE GROWTH CULTURE

Based on Plate 3.1 (a) and (b) microalgae culture need 24 hour light source
to growth, so 8 Watt light was supplied to ensure the microalgae receive enough
light to successfully growth. Air conditioning was also 24 hour supply to the
microalgae in the temperature range of 20ºC – 23ºC to enhance the growth.
Furthermore, to increase the microalgae growth rate the aeration process was apply
because providing sufficient O2 is often the single most limiting factor for achieving a
rapid growth rate and a high cell concentration. This process was done using a
pump (LifeTech) in one week to achieve maximum growth rate.

23
(a) (b)

Plate 3.1: (a) The microalgae strain was exposed to 24 hour light supply. (b)
The aeration was done to increases the algae growth rate

3.6 EXPERIMENTAL DESIGN AND OPTIMIZATION (RSM ANLYSIS)

Response surface methodology (RSM) is a collection of statistical and


mathematical techniques that uses quantitative data from appropriate experiments
to determine regression model equation and operating conditions which are useful
for developing, improving and processes. In this work, a standard RSM design know
as Box Bahnken was applied to study the variables of flocculation efficiency for
Chlorella sp freshwater. The experimental data were analyzed using statistical
software which is Design Expert version 6.0.6 (STAT_EASE Inc., Minneapolis, USA)
for regression analysis to fit the second degree polynomial equation and also for the
evaluation of the statically significance of the equation develop.

3.7 MICROALGAE HARVESTING PROCESS.

The harvesting process was continued to study the efficiency of the


flocculants used. After Chlorella sp reached it matured phase, the flocculation
process using calcium chloride (CaCl2) which act as flocculants was tested on 34
Chlorella sp sample using suggested box bhanken design data from response
surface methodology (RSM) method. The matured microalgae strain was adjusted in
three range of pH which is 5, 8.5 and 12 using 0.1M NaOH and 0.1M HCl. After the
required pH value was reached, each 100 mL sample was run according to the
condition stated in Table 3.3 with 10 minute agitation time for each sample. The
sample then left for 1 to 2 hours to let it settle down on the bottom of the 100mL

24
schott bottle. An aliquot of medium was taken for measuring the flocculation
efficiency at the height of two-thirds from the bottom. Spectrometer at wavelength of
550 µm was used to determine the optical density of Chlorella sp which represent
the flocculation efficiency of microalgae harvesting process. First and second 17
sample was run based on the selected value of pH, chemical dosage and agitation
speeds that listed in Table 3.3 which suggested by box behnken from RSM design.
The absorption value was measured three times to take the average measurement.
The stirrer (IKA©) was used to adjust the stirring speed (50rpm, 200rpm, 350rpm)
according to sample tested. In addition, the CaCl2 dosage was weighted using
analytical balance base on three selected weight (0.02g, 0.11g, 0.20g). These three
variables together with their respective ranges were chosen based on preliminary
studies as given in Table 3.2.

Table 3.2: Independent variable and their coded level.

Variable Code Unit Coded variable coded


-1 0 1
Ph A - 5 8.5 12
CaCl2 dosage B g 0.02 0.11 0.2
Agitation speed C rpm 50 200 350

The Equation (3.1), (3.2) and (3.3) was used to calculate the average optical
density which represent the flocculation efficiency for each sample and final
flocculation efficiency of Chlorella sp freshwater.

Average absorption = 1st reading + 2nd reading + 3rd reading (3.1)


3

% flocculation efficiency for each sample = initial - final × 100 (3.2)


Initial

Final flocculation = 1st sample + 2nd sample (3.3)


2

25
Table 3.3: Respond surface methodology analysis data.

Std Run Block Factor 1 Factor 2 Factor 3


A : pH B : CaCl2 C : Agitation
dosage speed
g rpm
6 1 Block 1 12 0.11 50.00
4 2 Block 1 12 0.20 200.00
3 3 Block 1 5 0.20 200.00
13 4 Block 1 8.50 0.11 200.00
12 5 Block 1 8.50 0.20 350.00
8 6 Block 1 12.00 0.11 350.00
16 7 Block 1 8.50 0.11 200.00
17 8 Block 1 8.50 0.11 200.00
2 9 Block 1 12.00 0.02 200.00
1 10 Block 1 5.00 0.02 200.00
7 11 Block 1 5.00 0.11 350.00
14 12 Block 1 8.50 0.11 200.00
11 13 Block 1 8.50 0.02 350.00
5 14 Block 1 5.00 0.11 50.00
15 15 Block 1 8.50 0.11 200.00
10 16 Block 1 8.50 0.02 50.00
9 17 Block 1 8.50 0.20 50.00

26
CHAPTER 4

RESULT AND DISCUSSION

4.1 OVERVIEW

The detail results of the studies are presented and discuss as in this chapter.
This chapter was divided into a few sections to explain the result in details. First
sections are focusing on the analysis of respond surface methodology by using
design expert software. The Box banhken data was used to determine the effect of
harvesting variable which is pH, CaCl2 dosage and agitation speed and optimum
condition of getting the highest flocculation efficiency for Chlorella sp. freshwater.
Plate 4.1 show the stage of harvesting process.

(a) (b)

Plate 4.1: (a) Chlorella sp before pH addition of CaCl2. (b) Chlorella sp after
addition of CaCl2.

4.2 DEVELOPMENT OF REGRESSION MODEL EQUATION

In this work, the box behnken design was used to create a polynomial
regression equation in order to analyze the correlation between the microalgae
harvesting parameter and optical density which represent the flocculation efficiency.

27
These designs lead to studying the effects of three factors in a single block of 17
sets of test conditions and five central points. The central point which is 8.5 in pH,
0.11 in dosage of CaCl2 and 200 rpm for the mixing speed was run to determine the
experimental error and the reproducibility of the data. The order of the experiments
was fully randomized. Three levels were attributed to each factor which is coded as -
1, 0, and 1. According to the sequential model sum of square, the model was
selected based on the highest order polynomial where the additional terms were
significant and the model not alised. Table 4.1 shows the complete design matrixes
together with response value obtain from the experimental work. The flocculation
efficiency was found in the range of 9.31% to 97.21%.

Table 4.1: Experimental design matrixes and result.

STD RUN BLOCK HARVESTING MICROALGAE RESPOND


PARAMETER %
Factor 1 Factor 2 Factor 3 Flocculation
A : pH B : CaCl2 C: Efficiency
dosage Agitation
g.mL-1 speed
rpm
6 1 Block 1 12 0.11 50 95.26
4 2 Block 1 12 0.2 200 97.21
3 3 Block 1 5 0.2 200 13.44
13 4 Block 1 8.5 0.11 200 66.12
12 5 Block 1 8.5 0.2 350 89.66
8 6 Block 1 12 0.11 350 96.69
16 7 Block 1 8.5 0.11 200 76.77
17 8 Block 1 8.5 0.11 200 89.12
2 9 Block 1 12 0.02 200 29.39
1 10 Block 1 5 0.02 200 13.01
7 11 Block 1 5 0.11 350 10.35
14 12 Block 1 8.5 0.11 200 86.84
1 13 Block 1 8.5 0.02 350 12.93

28
Table 4.1: Continued.

5 14 Block 1 5 0.11 50 10.63


15 15 Block 1 8.5 0.11 200 87.28
10 16 Block 1 8.5 0.2 50 89.79
9 17 Block 1 8.5 0.02 50 9.31

The quadratic model was suggested by the software for percent of the
flocculation efficiency. The final empirical formula models for percent of flocculation
efficiency of the Chlorella sp. in term of coded factor that design to fit the response
are represented in Equation (4.1).

% Flocculation efficiency = 81.23 + 33.89A + 28.18B + 0.58C – 20.08A2 – 22.89B2


– 7.9C2 + 16.85AB + 0.43AC – 0.94BC (4.1)

The coefficient with one factor represent the effect of the particular factor,
while the coefficients with two factor and those with second order term represent the
interaction between two factor and quadratic effect respectively. The quality of the
models developed was evaluated based on the correlation coefficients, R2 value. In
fact, the models developed seem to be the best at high R2 statistics which is closer
to unity as it will give predicted value closer to the actual value for the responses
(Ahmad & Alrozi, 2011). In this experiment, the R2 value for the Equation (4.1) is
0.9142 which indicated that 91.4% of the total variations in the flocculation efficiency
were attributed to the experimental variables studied. The R2 value relatively high,
indicating that the predicted values for flocculation were accurate and closer to its
actual value.

4.3 STATICAL ANALYSIS

The results of the surface quadratic model in the form of analysis of variance
(ANOVA) are given in Table 4.2 for flocculation efficiency of microalgae. ANOVA is
required to justify the significance and adequacy of the model. The mean squares
were obtained by dividing the sum of the square of each of the variation source, the
model and the error variance by respective degrees of freedom. If the value of
Prob.>F less than 0.05, the model terms consider as significant (Ahmad & Alrozi,
2011). From Table 4.2 the model F-value is equal to 8.28 and Prob.>F of 0.0054

29
implied that this model was significant. In this study, the significant source of
variation are A, B, A2 and B2 while C, C2, AB, AC and BC are insignificant to the
respond. From the tabulated data it shown that the models from the Equation (4.1)
were adequate to predict the flocculation efficiency within the range of variables
studies.

Table 4.2: Analysis of variance (ANOVA) for response surface quadratic model for
flocculation efficiency.

Source of Sum of Degree of Mean F-value Prob.> Comment


variations square freedom square F

Model 21251.19 9 2361.24 8.28 0.0054 significant


A 9188.363 1 9188.363 32.23 0.0008
B 6353.23 1 6353.23 22.29 0.0022
C 2.700402 1 2.700402 9.473E-003 0.9252
A2 1696.965 1 1696.965 5.95 0.0448
B2 2205.742 1 2205.742 7.74 0.0272
C2 263.9869 1 263.9869 0.93 0.3679
AB 1135.119 1 1135.119 3.98 0.0862
AC 0.735241 1 0.735241 2.579E-003 0.9609
BC 3.5126 1 3.5126 0.012 0.9147

Furthermore, Figure 4.1 shows the predicted value versus the experimental
value for the response which is flocculation efficiency. It can be seen that the
models develop were successful in capturing the correlation between the harvesting
parameter to the response when the predicted values obtain were quite close to the
experimental value.

30
R2 = 0.9142

Figure 4.1: Predicted versus actual experimental flocculation efficiency.

4.4 FLOCCULATION EFFICIENCY OF MICROALGAE

Based on the Table 4.2, the highest F-value for the model is 32.23 refer to
pH which found to have the greatest effect on the flocculation efficiency with the
Prob.>F value of 0.0008. For the pH and CaCl2 dosage, it consider as moderate
effect to flocculation efficiency which is significant and showed almost similar effects
on the response. However, the effect of agitation speed on the response was
relatively insignificant. From the Figure 4.2 and 4.3 it showed that the increasing of
pH also one of the factors to achieved higher flocculation efficiency. The freshwater
microalgae cells began to agglomerate when the pH was adjusted up to about 8.6.
As pH increased to 10.6, the efficiencies were greatly raised to more than 90%
(Wu et al., 2012). Figure 4.2 demonstrates the effect of pH and CaCl2 dosage on
flocculation efficiency with fixed agitation speed at 200 rpm give the result of higher
flocculation efficiency. According to Castrillo et al. (2013), the higher the pH value
which is more than 10.8, the more effective the process rose with increase of Ca2+
dosage which is consider as better flocculants agent .

31
Figure 4.2: Three-dimensional response surface plot of pH and CaCl2 dosage on
Chlorella sp flocculation efficiency (agitation speed = 200 rpm).

In Figure 4.3, it showed the effect of agitation speed with the pH value by
maintaining the CaCl2 dosage which is also linearly increases the flocculation
efficiency. However, agitation speed has a little effect on flocculation efficiency this
is because of the pH adjustment process. The increasing of medium pH value can
cause autoflocculations where autoflocculation is process of adjusting the medium
pH to let the microalgae settle down by itselfs. This process was effectively modified
by the addition of Ca2+ ion to increases the flocculation efficiency because the Ca2+
already exist in the medium which also can act as flocculants but in higher pH value.
So that, the agitation was needed to efficiently mixed the microalgae with Ca2+ to
accumulate the microalgae 10 minute of CaCl2 reaction with microalgae.

Furthermore, Figure 4.4 illustrated that the effect of agitation speed and
dosage by fixed pH value of 8.5. From the figure, the relation between agitation
speeds with CaCl2 dosage can be seen when moderate CaCl2 dosage used which is
represent the higher performance of agitation speed. As the agitation speed
increases the flocculation efficiency decreases which due to restabilization of the
cells at the fast mixing speed. High speed mixing tends to break the flocs, causing
the coagulated cells introduced again into the medium (Ahmad et al., 2011).

32
Figure 4.3: Three-dimensional response surface plot of pH and agitation speed on
Chlorella sp flocculation efficiency (CaCl2 dosage = 0.11).

Figure 4.3: Three-dimensional response surface plot of CaCl2 dosage and agitation
speed on Chlorella sp flocculation efficiency (pH = 8.50).

33
4.3 PROCESS OPTIMIZATION

Box behnken analysis has been used to optimize the parameters affecting
the flocculation efficiency responses. Therefore, the function of desirability was
applied using Design-Expert software (STAT-EASE Inc., Minneapolis, USA) in order
to compromise between these responses (Ahmad & Alrozi, 2011). In this
optimization analysis, the target criteria was set as maximum values for both
responses while the values of the variables were set in the ranges being studied.
The predicted and experimental results of optical density obtained at optimum
conditions are shown in Table 4.3. The optimum flocculation efficiency was obtained
by using pH of 10.50, CaCl2 dosage of 0.14 and mixing rate of 198.98 rpm which
showed the flocculants efficiency of 97.59%. It was observed that the experimental
values obtained were in good agreement with the values predicted from the models,
with relatively small errors, which were only 2.20%.

Table 4.3: Model validation.

Model A B C Flocculation efficiency (%)


-1
desirability (g.mL ) (rpm) Predicted Experimental Error
(%)
1.000 10.50 0.14 198.98 99.79 97.59 2.20

4.3 HIGHEST FLOCCULATION EFFICIENCY CONDITION

According to the study conducted by Wu et al. (2012), a Chlorella sp.


microalgae which is freshwater microalgae cells will began to agglomerate when the
pH was adjusted up to about 8.6. As pH increased to 10.6, the efficiencies were
greatly raised to more than 90%. The result obtain was quite similar compared to
this study which found that at pH of 10.5 the flocculation yielded 97.59% efficiency.
Furthermore, the calcium ion dosage of 0.14 determined as optimum concentration
for Chlorella sp to achieve the 99.79% flocculation efficiency. This is because the
calcium ion already exists in the microalgae BBM medium which is used to culture

34
these microalgae. The flocculation actually can be done without adding the
flocculants because it just depending on the existing metal ions (Ca and Mg ions) in
the medium. Flocculation often occurs spontaneously in microalgae cultures when
pH increases above 9. CaCl2 was added only to increases the flocculation efficiency.
The phenomena call as autoflocculations. Since, agitation speed not gave a
significant effect in this study, medium agitation speed which is 198.98 rpm for the
experimental study is which is near to the mixing speed study by Ahmad et al.,
(2011) that take 150 rpm as the optimum agitation speed which is between the
speed range of 0 – 350 rpm.

35
CHAPTER 5

CONCLUSION

5.1 CONCLUSION

A microalgae is one of the useful nature sources to fulfill the biofuels energy
demand around the world. Higher cost of microalgae biomass recovery be the most
problematic factor for microalgae production. Several harvesting method already
exist in industrial scale but flocculation process was choose to be study in this
experiment. The calcium chloride was selected to act as a flocculants for harvesting
Chlorella sp freshwater. From autoflocculation theory, the higher microalgae pH can
enhance the flocculation process due to Ca and Mg ion inside the medium
concentration. So to increases the flocculation efficiency addition of CaCl2 was
needed.

Response surface methodology was successfully used to determine the


relationship between the pH, CaCl2 dosage and agitation speed with optical density
which represent the flocculation efficiency. The quality of the models developed was
evaluated based on the correlation coefficients, R2 value. Higher R2 statistic near to
unity present the best model development which 0.9142 for this experiment. This
value indicate that the predicted values for flocculation were accurate and closer to it
actual value.

Based on the suggested optimum condition which is 10.5 in pH, 0.14 of


CaCl2 dosage and 198.98 rpm in agitation speed were in good agreement.The
values predicted with the efficiency of 97.57% differ 2.20% from the predicted value.
Besides that, from the ANOVA analysis shows that the model was significant for pH
and CaCl2 dosage. This is mean those variable is the most effecting factor that
enhances the flocculation efficiency. In conclusion, the microalgae harvesting
process can be done using CaCl2 as flocculants agent which successfully achive
higher flocculation efficiency which is 97.59%

36
CHAPTER 6

RECOMENDATION

6.1 RECOMMENDATION

There are several suggestion to future research for studying microalgae harvesting
process of Chlorella sp freshwater which are:

a) Used different flocculants type for example chitosan or polymer to


determined the optimum flocculants condition for harvesting Chlorella sp
freshwater.

b) Used other positive charge chemical which Mg2+ ion or Zn2+ to react with
negatively charge microalgae to determine the optimum condition for efficient
flocculation process.

c) Study the different parameter which affects the harvesting process such as
temperature and concentration of microalgae strain.

d) Study the biomass recovery for each sample tested to determine exact value
of the microalgae that being flocculate.

37
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APPENDICES A

Plate A1: Autoclave machine Plate A2: Analytical balance

Plate A3: Microppette Plate A4: Calcium Chloride

Plate A5: Spectrophotometer. Plate A6 : Magnetic Stirrer

41
APPENDICES B

Plate B.1: BBM medium Plate B.2: Sterilization process

Plate B.3: Sub culturing process Plate B.4: Bacterial removal

Plate B.5: Aeration process Plate B.6: Measuring the OD

42
Plate B.7: addition of CaCl2 Plate B.8: agitation in 10 minute

Plate B.9: After flocculation

43