CARBON DIOXIDE CAPTURE BY USING FRESHWATER MICROALGAE

AHMAD NAQIB BIN ABU ASWAD

A report submitted in partial fulfilment of the

requirements for the award of the degree of
Bachelor of Chemical Process Engineering

Malaysia-Japan International Institute Technology Malaysia

UniversitiTeknologi Malaysia

OCTOBER 2018
 TABLE OF CONTENTS

1 INTRODUCTION 1
1.1 Background of the Problem 1
1.2 Problem of Statement 2
1.3 Objectives of the Study 2
1.4 Scope of the Study 2
1.5 Significance of the Study 3
1.6 Thesis Organization 3

2 LITERATURE REVIEW 4
2.1 Microalgae 4
2.2 Algae Culture Technique 4
2.2.1 Batch Culture 5
2.2.2 Continuous Culture 6
2.2.3 Semi-continuous Culture 8
2.3 Culturing Systems for Microalgae 9
2.3.1 Open System 9
2.3.2 Closed System 10
2.4 Microalgae Species 12
2.4.1 Chlorella sp. 12
2.4.2 Chlorella Vulgaris 12
2.5 The Role of CO2 in Microalgae Growth 13
2.5.1 CO2 Concentration Mechanisms (CCMs) 13
2.5.2 Carbon Limitation 14
2.6 Factor Influencing Microalgae Growth 14
2.6.1 Light 15
2.6.2 pH 16
2.6.3 Aeration/Mixing 16
2.6.4 Temperature 16
 2.7 Biomass Measurement 17

3 METHODOLOGY 18
3.1 Research Design 18
3.2 Material Preparation 18
3.2.1 Medium Preparation 18
3.2.2 Freshwater Microalgae Stock Culture 19
3.3 Experimental Setup 21
3.3.1 Freshwater Microalgae Samples 21
3.3.2 pH 22
3.3.3 Light 22
3.3.4 Mixing and Aeration 22
3.3.5 Optical Density (OD) 23
3.4 Process Flow of the Experiment 24

4 EXPECTED RESULTS 25

5 CONCLUSION 26

REFERENCES 27
 CHAPTER 1

INTRODUCTION

1.1 Background of the Problem

Greenhouse gases (especially carbon dioxide CO2) are responsible of keeping

our planet warm and maintaining the life on Earth. If the atmosphere did not contain
carbon dioxide, at that point the normal temperature on Earth would be 30°C cooler
(Chang, 2007). However, if the carbon dioxide level increases it will become a
global concern. It will results in trapping heat, which may threaten the very existence
of the life on Earth. The atmospheric carbon dioxide content it is estimated that has
increased since the twentieth century due to burning of fossil fuels, industrialization
and deforestation (Chang, 2007).

In any case, decreasing fossil fuels with increasing fuel demand is

unavoidable. It is hard to diminish the reliance on fossil fuels and change to other
energy sources, thus it is fundamental to expel carbon dioxide emissions from power
plants, so that energy from burning fossil fuels can be exploited without emitting
carbon dioxide into the atmosphere. The main option for reducing carbon dioxide
emissions is called carbon capture and storage (CCS). Therefore, CO2 fixation via
microalgae is a potential and promisingmethod for CO2 capture and storage (Zhao
and Su, 2014).

1
1.2 Problem of Statement

Currently, the energy industry especially coal industry in Malaysia has the
highest contribution of CO2emission that is 35% compared to other industries.
Recently, the Prime Minister of Malaysia made a pledge for Malaysia to reduce its
emission intensity by 40% by the 2020 from the baseline level in 2005.

To establish the vision of the Prime Minister that had been pledged, carbon
capture process is suggested as an option for reducing anthropogenic CO 2 emissions
and integration between capture, storage, and utilization of CO2 is drastically needed.
However, it is difficult to achieve a sustainable environmental friendly way with the
current carbon capture technology due to its high impact to the environment by their
byproducts.

1.3 Objectives of the Study

(1) To determine the best freshwater microalgae in CO2 capture in terms of

survivability in high concentration of CO2.
(2) To determine the most favourable conditions to secure maximum growth rate
in terms of various type of medium.

1.4 Scope of the Study

This study examines the microalgae growth by selecting the freshwater

microalgae species only, to capture and storage the CO2. Thus, a specific choices of
freshwater microalgae with each of them have their own characteristics will be used
to make a comparison. This study will not cover other factors such as how the
microalgae being used as a biomass and byproducts.

2
1.5 Significance of the Study

This study offers detail. Students will understand better the concepts of
carbon capture using freshwater microalgae. Through specific types of freshwater
microalgae comparison the process can be achieved.

1.6 Thesis Organization

Chapter 1 provides the introduction about the title project. It identifies how
the problem statement can relate with the objective of the research.

Chapter 2 is a review the literature related to the research such as how the
carbon being capture, the steps to growth the freshwater microalgae, measurement of
the growth and the condition of the growth.

Chapter 3 provides the research methods that explain briefly how the
experiment being carried out. It is based on recent study and it will be apply back on
this experiment but with different parameters.

Chapter 4 is the expected results based on the recent research and to

determine is it corresponds together with this experiment.

Chapter 5 is the conclusion that will be made after obtain the final result and
discussions.

Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5

Introduction Literature Methodology Results & Conclusion

Review Discussion

Figure 1.1 Thesis process flow

3
 CHAPTER 2

LITERATURE REVIEW

2.1 Microalgae

Microalgae are made up of eukaryotic cells. Microalgaecells consist of cell

wall, plasmatic membrane, cytoplasm, nucleus and organelles, such as mitochondria,
lysosomes and Golgi (Taher et al., 2011). Microalgae also have plastids, the bodies
with chlorophyll that carry out photosynthesis. Microalgae are microscopic
organisms that usually grow suspended in water and live their way by having
photosyntheticprocess as same as that of higher plants (Hanelt et al., 2007).
Microalgae cultivation is composed of asingle specific strain that is precisely
selected for producingthe desired product and the most beneficial outcome of
thecultivation process. The cultivation conditions, including (1) water media with
optimal pH and temperature, (2) necessarycontained nutrients and (3) CO2 dosed in a
controlledmanner in the presence of sunlight (Klinthong et al., 2015).

2.2 Algae Culture Techniques

Recently, microalgae culture is one of the biotechnologies that are been

developed (Milledge, 2010). Species such as Chlorella, Spirulina, Scenedesmus,
Dunaliella salina, Haematococcus pluviali and Porphyridium cruentum has been
successfully mass cultured on a commercial scale (Chaumont, 1993 and Borowitzka,
1999). Although these microorganisms are abundant in nature they have not yet
made the subject of scientific investigation regarding in extensive research on each
of the algae. It is also known that they have genetic ability to produce a wide range

4
of compounds and chemicals that could be of great commercial value (Grossman,
2005). It is estimated that there are probably well over 30 000 species of microalgae,
only a few hundred of which are cultured in laboratories at present. Only a handful of
them are being researched and assessed their economic potential (Chaumont, 1993).

The biomass that is produced by microalgae is based on specially engineered

facilities, the fundamental design and infrastructure. However, the microalgae need
to achieve a certain growth requirements which are different for each type of
microalgae. The microalgae is also produce different kind of the final product and it
is important to consider the value of the product (Metting, 1996). In general
microalgae can be produced in open systems or closed systems (such as
photobioreactors), and the culturing methods used can be continuous, semi-
continuous or batch (Jorquera et al., 2010).

2.2.1 Batch Culture

A batch culture is a culture growing in a closed system where nothing is

added or taken away (Madigan and Martinko, 2006). The batch culture consists of a
single inoculation of cells into a container of fertilized seawater. The growing period
will last for several days and finally harvesting when the algal population reaches its
maximum or near-maximum density (Lavens and Sorgeloos, 1996). When an
inoculum of cells is added, the batch culture will be kept in an environment, which
favors growth (Lee and Shen, 2007). Generally, the volume of the inoculum which
corresponds with the volume of the preceding stage in the upscaling process amounts
2 – 10% of the final culture volume (Lavens and Sorgeloos, 1996).

The growth of batch culture is divided into three phases; the lag-, the log- and
the stationary phase (Madigan and Martinko, 2006). In practice, algae are transferred
to larger culture volumes prior to reaching the stationary phase and the larger culture
volumes are then brought to a maximum density and harvested (Lavens and
Sorgeloos, 1996). Due to its simplicity and flexibility which allow change in species

5
during culture, batch culture is widely applied not only in research scale but also in
industrial scale.

Figure 2.1 Production Scheme for Batch Culture in Carboy Culture Apparatus
(Lee and Tamaru, 1993)

2.2.2 Continuous Culture

Continuous culturing in which a supply of fertilized seawater is continuously

pumped into a growth chamber and the excess culture is simultaneously washed out.
The benefits Continuous culturing in which a supply of fertilized seawater is
continuously pumped into a growth chamber and the excess culture is simultaneously
washed out. The benefit is that the maintenance of cultures is very close to the
maximum growth rate (Lavens and Sorgeloos, 1996). There are at least two
categories of continuous cultures can be distinguished: (i) turbidostat culture, in
which the algal concentration is kept at a preset level by dilution of the culture with
fresh medium by means of an automatic system. (ii) chemostat culture, in which a
flow of fresh medium is introduced into the culture at a steady, predetermined rate.
The latter adds a limiting vital nutrient such as nitrate at a fixed rate and in this way
6
the growth rate and not the cell density is kept constant. For example, turbidostat
system can produces 30 – 40 L per day at cell densities giving optimal yield for each
flagellate species that is suitable for culture such as Tetraselmis
suecica and Isochrysis galbana (Laing, 1991).

Figure 2.2 Schematic Diagram of a 40 L Continuous Culture Vessel (Laing,

1991)

7
2.2.3 Semi-continuous Culture

The semi-continuous technique prolongs the use of large tank cultures by

partial periodic harvesting followed immediately by topping up to the original
volume and supplementing with nutrients to achieve the original level of enrichment.
The culture is grown up again, partially harvested, etc. Semi-continuous cultures may
be indoors or outdoors, but usually their duration is unpredictable. Due to the
unpredictable duration, competitors, predators and/or contaminants and metabolites
eventually build up, rendering the culture unsuitable for further use. Since the culture
is not harvested completely, the semi-continuous method yields more algae than the
batch method for a given tank size (Lavens and Sorgeloos, 1996).

Figure 2.3 Schematic diagram of a semi-continuous reactor for algal culture: 1.

Perspex tanks; 2. circulating pump; 3. fluorescent lamps; 4. water tank; 5.
nourishment syringe (Rosa et al., 2005).

8
2.3 Culturing Systems for Microalgae

2.3.1 Open System

Open ponds have been used for large-scalemicroalgae cultivation considering

their simple constructionand easy operation.Open ponds can be grouped into natural
systems, such as lakes, lagoons and ponds or artificial ponds and containers, which
can take various forms. These include shallow unstirred, circular stirred and raceway
ponds and tanks (Zittelli et al., 2003). Although open ponds are simple and easy to
operate of a cultivation system, however it is have numerous limitations. These
include poor utilization of light by microalgae cells, high evaporation, low mass
transfer, loss or diffusion of CO2 into the atmosphere, and the large amount of land
required (Ugwu et al., 2008). Moreover, they are susceptible to chemical and
biological contamination from protozoa, bacteria and viruses (Chaumont, 1993). Due
to this contamination, to culture a single species will be depending on the
competitiveness of the microalgae being grown, that is, whether or not they can resist
contamination from other microalgae species. To culture a single species of
microalgae can be successfully using open pond systems, but that their specific needs
must be recognized. In other words, they must be cultivated in a highly selective
environment. For example, Chlorella grows well in nutrient-rich media; Spirulina
requires a high pH and bicarbonate concentration; while D. salina grows best under
highly saline conditions (Soong, 1980; Belay, 1997; Borowitzka and Borowitzka,
1988).

Raceway ponds (or stirred paddle wheel open ponds)are the most famous
open system in current use.A raceway pond is a shallow pond configured as a loop,
around which the algal cultures are circulated, typically using a paddle wheel (Singh
and Sharma, 2012).The 0.2-0.5m pond depth which is shallow allows the microalgae
to receiveequivalent natural sunlight. Nutrients such as nitrate, phosphorus, trace
metals are introduced into the pond after the paddle wheel. As mass transfer of CO2
from the atmosphere is insufficient, purified CO2 or air is usually injected into the
pond through a sparger at the bottom of the pond (Cheng et al., 2015). The

9
paddlewheel is operated to circulate and agitate the microalgae broth and nutrients
(Jorquera et al., 2010). The benefit of a raceway pond is that it has relatively low
capital cost comparedto closed systems and in suitable locations can maintain up to
25 g m-2 d-1biomass productivity (Pulz, 2001).

Figure 2.4 Schematic Diagram of Raceway Pond (Singh and Sharma, 2012)

2.3.2 Closed System

Microalgae can be cultivated in a closed system undercontrolled conditions,

such as light utilization, area requiredand percentage of carbon dioxide. The closed
system can reduce some of the problems that are associated with opensystems.
Photobioreactors are a type of closed pond systemthat are used for microalgae
cultivation, as they can reducecontamination risk from unwanted algae, mold, and
bacteria; control temperature; minimize water evaporation;and remove carbon
dioxide losses (Borowitzka, 2007). In order to improve illumination efficiency
different designs of photobioreactors have been explored, such as flat-plate
photobioreactors, tubular photobioreactors and column photobioreactors. A flat-plate
photobioreactor has a 10- 25 mm light path and large surface area, maximizing
illumination efficiency (Richmond and Cheng-Wu, 2001). Compared with other
photobioreactors, its structure is simple, making it easier to clean and to do
maintenance. For column photobioreactors, gas is injected from the bottom, which
can result in good mixing and agitation of the microalgae (Rasoul-Amini et al.,
10
2011). In tubular photobioreactors are arranged as an array of transparent tubes. To
further enhance the illumination efficiency, a lot of modified designs have been
applied to the photobioreactors such as horizontal tubular and helical tubular
photobioreactors (Molina et al., 2001). However, the disadvantages of this system
are that it is difficult to construct and operateand is costly at large scale.

(A) Flat-plate photobioreactor (B) Column photobioreactor

(C) Helical tubular photobioreactor (Concas et al., 2010)

Figure 2.5 Photobioreactor Designs

11
2.4 Microalgae Species

2.4.1 Chlorella sp.

Chlorella sp. is a genus of single-celled green algae, belonging to the phylum

Chlorophyta. It has a spherical shape, about 2 to 10 μm in diameter, and is without
flagella. Chlorella sp. was initially cultivated for its use as a health food (it contains
β-1, 3-glucan, an immunoactive substance).It is also known for its resistance to
contamination and tolerance to varying growth medium conditions (Borowitzka,
2005). Chlorella sp. has relatively low lipid content but high biomass productivity.
The high biomass productivity results in the high chlorophyll content of this strain
giving it a higher photosynthetic efficiency. It takes around 20 days for the algae to
go from lag phase to exponential phase and then stationary phase (Pirt et al., 1980).

2.4.2 Chlorella Vulgaris

The green alga Chlorella vulgaris is a commonly cultivated species,

according to Spolaore et al. (2005), the annual dry weight production of Chlorella is
about 2000 ton per year. It is a small green, microalgae only 2-10 µm in diameter and
it has a high content of proteins. C. vulgaris is estimated to compose of 51- 58%
protein, 12-17% carbohydrate and 14-22% lipid, but it depends on environmental
conditions under which C. vulgaris has grown and variations can be seen. The lipid
content can be up to 30% under certain conditions (Putt, 2007). Seyfabadi et al.
(2010) reported that the protein concentration of 33- 46% achieved when performing
experiments with different light regimes. C. vulgaris also produces other metabolites
such as pigments and can be grown both autotrophically and heterotrophically
(Seyfabadi et al., 2010). Autotrophic means that the cell collects the energy from the
light through photosynthesis, while heterotrophic growth only requires a carbon
source as energy (Larsdotter, 2006). C. vulgaris has the potential to double in cell
number every 8 hour at a temperature between 20-35oC during autotrophic growth,
provided that nutrients and light is not limiting the growth (Putt, 2007).

12
2.5 The Role of CO2 in Microalgae Growth
2.5.1 CO2 Concentrating Mechanisms (CCMs)

CO2 is converted into reduced carbon by microalgae so that it can be used for
the production of lipids and biomass through photosynthesis, for which the overall
reaction can be summarised as follows (Klinthong et al., 2015):

𝑃ℎ𝑜𝑡𝑜𝑛𝑠 (𝑙𝑖𝑔ℎ𝑡)
𝐶𝑂2 + 𝐻2 𝑂 → [𝐶𝐻2 𝑂]𝑛 + 𝑂2

For microalgae, carbon is a fundamental compositional element which

contribute approximately 50 wt% of the microalgae dry biomass (Klinthong et al.,
2015). In eukaryotic algal cells, inorganic carbon is first fixed by Ribulose
Bisphosphate Carboxylase Oxygenase (RuBisCO, a CO2 fixing enzyme) within the
Calvin cycle, as shown in reaction below (Giordano et al., 2005):

𝑅𝑖𝑏𝑢𝑙𝑜𝑠𝑒 − 1,5 − 𝑏𝑖𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 + 𝐶𝑂2 + 𝐻2 𝑂 → 2 × 𝑔𝑙𝑦𝑐𝑒𝑟𝑎𝑡𝑒 − 3 − 𝑃

However, due to the low affinity of RuBisCO with CO2, microalgae have
evolved CO2 concentrating mechanisms (CCMs) to increases CO2 concentration in
the vicinity of RuBisCO. Figure 2.6 below illustrates the process of CO2 utilization
in eukaryotic algal cells. Firstly, inorganic carbon is delivered into the chloroplast
through CO2 diffusion or active transport of CO2 and HCO3-. Then CO2 is fixed by
RuBisCO in the Calvin cycle (Giordano et al., 2005).

Figure 2.6 Schematic model for inorganic carbon transport and CO2
accumulation processes in eukaryotic algal cells (Giordano et al., 2005)
13
2.5.2 Carbon Limitation

Due to CCMs, the carbon concentration is unlikely to limit photosynthesis in

dilute cell cultures, in which the overall demand for CO2 is not intense. However,
CCMs cannot avoid carbon limitation in dense microalgae cultures in which the
overall demand for CO2 exceeds the supply (Williams and Colman, 1996). A
diffusion of CO2 from air cannot provide sufficient rate, typically to overcome this
higher concentration of CO2 is directly injected into microalgae ponds or
photobioreactors, to ensure a sufficient supply of CO2 is available to fully utilise the
available carboxylase activity of RuBisCO to improve microalgae photosynthetic
efficiency (Duarte et al., 2016). However, excessively high CO2 concentrations can
decrease the medium pH and inhibitory effects. This can eventually limit microalgae
growth (Tang et al., 2011). So it is important to achieve a balance between the CO2
demand of microalgae growth and the CO2 supply in microalgae media. The CO2
demand varies with different microalgae species, the culture density and the
photosynthetic rate. On the other hand, the CO2 supply depends on CO2
concentration in gas, gas flowrate, CO2 mass transfer coefficient and contact surface
of different delivery technologies.

2.6 Factor Influencing Microalgae Growth

The most important parameters regulating algal growth are nutrient quantity
and quality, light, pH, turbulence, salinity and temperature.The most optimal
parameters as well as the tolerated ranges are species specific and a broad
generalization for the most important parameters is given in table below:

14
Table 2.1 A generalized set of conditions for culturing microalgae (Fulks and
Main, 1991)

Parameters Range Optima

Temperature (°C) 16 – 27 18 – 24
Salinity (g/l) 12 – 40 20 – 24
Light intensity (lux) 1,000 – 10,000 (depends 2,500 – 5,000
on volume and density)
Photoperiod (light:dark, 16:8 (minimum)
hours) 24:0 (maximum)
pH 7–9 8.2 – 8.7

2.6.1 Light

Same like plants, microalgae also did the photosynthesis process. Thus, light
is required as the source of energy to drives this reaction and in this regard intensity,
spectral quality and photoperiod need to be considered. Light intensity is important
but it varies with the requirements based on the culture depth and the density of the
algal culture: at higher depths and cell concentrations the light intensity must be
increased to penetrate through the culture (Lavens and Sorgeloos, 1996).

The light can be natural or supplied by fluorescent tubes but when the light
intensity is too high, it may result in photo-inhibition. Also, overheating due to both
natural and artificial illumination should be avoided. It is preferred that the
fluorescent tubes emitting either in the blue or the red light spectrum should be used
as these are the most active portions of the light spectrum for photosynthesis. The
duration of artificial illumination should be minimum 18 h of light per day, although
cultivated phytoplankton develops normally under constant illumination (Lavens and
Sorgeloos, 1996).

15
2.6.2 pH

The range of pH for most cultured algal species is between 7 and 9, with the
optimum range being 8.2-8.7. Usually unsuccessfully culturing of microalgae is due
to failing in maintaining an acceptable pH after been disrupt by many cellular
processes. To avoid this problem, it is recommended to aerate the culture by
supplying air to the culture. In the case of high-density algal culture, the addition of
carbon dioxide allows to correct for increased pH, which may reach limiting values
of up to pH 9 during algal growth (Lavens and Sorgeloos, 1996).

2.6.3 Aeration/Mixing

To prevent sedimentation of the algae, it is suggested that mixing should be

done in order to ensure that all cells of the population are equally exposed to the light
and nutrients, to avoid thermal stratificationand to improve gas exchange between
the culture medium and the air. For aeration, the importance of this is that in air
contains the carbon source for photosynthesis in the form of carbon dioxide.For very
dense cultures, the CO2 originating from the air (containing 0.03% CO2) bubbled
through the culture is limiting the algal growth and pure carbon dioxide may be
supplemented to the air supply. CO2 addition furthermore buffers the water against
pH changes as a result of the CO2/HCO3 - balance. 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 algal species
can tolerate vigorous mixing (Lavens and Sorgeloos, 1996).

2.6.4 Temperature

The optimal temperature for phytoplankton cultures is generally between 20

and 24°C, although this may vary with the composition of the culture medium, the
species and strain cultured. Most commonly cultured species of microalgae tolerate
temperatures between 16 and 27°C. Temperatures lower than 16°C will slow down
growth, whereas those higher than 35°C are lethal for a number of species.If

16
necessary, algal cultures can be cooled by a flow of cold water over the surface of the
culture vessel or by controlling the air temperature with refrigerated air- conditioning
units (Lavens and Sorgeloos, 1996).

Since lamps and sunlight emits heat, there is a relationship between

temperature and light intensity (Richmond, 2007). In addition, the flue gas that
bubbled through the cell suspension also has its own temperature which will also
affect the culture temperature (Welty et al., 2001).

2.7 Biomass Measurement

An indirect measurement of biomass within the culture was chosen to allow

for increased sampling throughout the growth of the algae. Optical density
wasselected as the mostappropriate form ofmeasurement due to the low volume of
sample required (approximately 1 mL persampling event) as well as the low
operational technicality of a spectrophotometer (Boggess, 2014).

Algae can only absorb contain wavelengths of light. Absorption of light can
be maximized by matching the absorbance spectrum for Chlorella vulgaris (Figure
2.7) withthe emission spectrum of the light source. This graphic helps demonstrate
why it wasnecessary, when measuring biomass, to choose a wavelength that the
microalga speciesdoes not absorb. For example, 750 nm was selected due to
Chlorella not absorbing lightat this wavelength (Boggess, 2014).

Figure 2.7 The absorbance spectrum of Chlorella vulgaris grown with water
(Boggess, 2014)
17
 CHAPTER 3

METHODOLOGY

3.1 Research Design

The experiment will be conduct at indoor area, where it will be done in a

room temperature of around 30 to 32°C to simulate the real temperature of Malaysia.
The freshwater microalgae used in this experiment will be cultivated from the
previous stock culture. A new stock culture will be produced in order to mitigate the
possibility of contamination during the actual culture which is based on inoculum
percentage. By using 10% inoculum, a 300 ml of freshwater microalgae (30 ml is
microalgae from stock culture while 270 ml is fresh medium) are growth in 500 ml
flask and it will be carried out in triplicate in order to obtain average results. The
microalgae will take around 10 – 14 days to growth. After that duration, only then
the carbon dioxide gas is directly supply into the flask.

Every 3 days of cultivation, a sample will be taken from the flask to check the
optical density. After 12 days of CO2 supply, the microalgae will be harvested and
the optical density will be recorded for the whole culturing process to determine the
growth of the microalgae.

3.2 Material Preparation

3.2.1 Medium Preparation

The microalgae will be grown on various synthetic media in order to check

which of the medium was able to support the best growth of algae. In order to find

18
out the best culture medium, the microalgae species will be cultured in three different
culture media compositions having different pH, such as BG-11, Bold basal medium
(BBM), and Half strength Chu-10 medium (HC10). However, for first objective only
BBM as the standard medium will be used to determine the best freshwater
microalgae. After obtaining the best algae, only then the best algae will be used with
the other medium to enhance it growth rate. The individual compositions of the three
medium are listed in Table 3.1. Before experimental setup all the media prepared and
sterilized in autoclave at 121°C for 2 hours.

3.2.2 Freshwater Microalgae Stock Culture

For this experiment, two freshwater microalgae species are chosen which are
Chlorella sp. and Chlorella Vulgaris to be tested and to determine their capability in
CO2 capture while surviving the high concentration of CO2. The previous stock
culture of these is used to create a new stock culture for this study. The new stock
culture will be cultured in 500 ml of conical flask with the composition of 300 ml of
fresh medium and 5 ml of the microalgae. In stock culture, the photoperiod is set to
be 24 hours of light for maximize growth and the temperature is around 25°C. The
flask also will be aerated by pumping air into it.

19
Table 3.1 Composition of various culture media in gL-1 (Bajwa et al., 2017)

Bold Basal Medium Half Strength Chu-10 BG-11 Medium, gL-1

(BBM), gL-1 Medium (HC10), gL-1
NaNO3 – 0.25 K2HPO4 – 0.0025 NaNO3 – 1.50
K2HPO4.3H2O – 0.075 Mg SO4.7H20 – 0.0125 K2HPO4 – 0.040
MgSO4.7H2O – 0.075 NaCO3 – 0.010 MgSO4.7H2O – 0.075
CaCl2.2H2O – 0.025 Na2SiO3 – 0.0125 CaCl2.2H2O – 0.036
KH2PO4 – 0.175 CA(NO3)2 – 0.02 Citric acid – 0.06
NaCl – 0.025 FeCl3 – 0.0004 Ferric ammonium
Citrate – 0.001
Acidified Iron solution Trace metal mixture 1ml EDTA (disodium salt) –
having composition (H3BO3 – 0.001
(FeSO4.7H2O – 0.00498; 0.00248;ZnSO4.7H2O –
H2SO4 – 1-2 drops) 0.00023;Na2MoO4.2HO –
0.00007; CuSO4.5H2O –
0.0001; Co(NO3)2.6H2O –
0.00014)
Alkaline EDTA solution Na2CO3 – 0.02
having composition
(EDTA – 0.05; KOH –
0.031)
1ml Trace metal solution
(H3BO3 – 2.86;
MnCl2.4H2O – 1.81;
ZnSO4.7H2O – 0.222;
NaMoO4.2H2O – 0.39;
CuSO4.5H2O – 0.079;
Co(NO3)2.6H2O – 0.0494)

20
3.3 Experimental Setup

Below is the figure of the setup of the experiment that will be conducted in a
room temperature of 30 to 32°C.

Figure 3.1 The Schematic Diagram of the Experimental Setup

The experiment will be conduct at least around 22 to 26 days to obtain

optimum results. The results will be taken in terms of optical density (OD) for each 3
days of cultivation. The experiment will be carried out in triplicate in order to
achieve average value of the results. The light intensity and photoperiod will be in
the ratio of 12:12 hours of light and dark. The air and CO2 supply will be controlled
by flow meter to limit the flowrate by 500 ml/min and 25 ml/min respectively.

3.3.1 Freshwater Microalgae Samples

Previously from the stock culture of Chlorella sp. and Chlorella Vulgaris, it
will be used in the real cultivation which specified parameters. 10% of inoculum has
been chosen where 300 ml microalgae mixture with the composition of 30 ml of
microalgae from the stock culture and 270 ml of fresh medium will be used to
cultivate.
21
3.3.2 pH

The pH of the medium with the microalgae will be in range of 6.5 – 9 due to
the pH of the specified medium with various values. However, the optimum range
should be around the range of 8.2 – 8.7. To achieve those values, an adjustment will
be done by adding HCl or NaOH to change the pH of the medium. This is because in
the case of high-density algal culture, the addition of CO2 will drop the pH value and
also can correct for increased in pH. However, too much CO2 can affect the growth
of the microalgae and it is important to balance both the CO2 concentration and the
pH.

3.3.3 Light

Microalgae share the same characteristic as plant where it also can carry out
photosynthesis. While conduct this experiment, the light source which is fluorescent
light will be provided to the microalgae. Fluorescent tubes emitting either in the blue
or the red light spectrum should be preferred as these are the most active portions of
the light spectrum for photosynthesis. The duration of the artificial illumination will
be controlled by timer and it will be set for 12:12 hours of light and dark for a day.

3.3.4 Mixing and Aeration

The microalgae will be mix daily in order to prevent sedimentation at the

bottom of the flask and to ensure that all cells of the population are equally received
the same light and nutrients. The air will be supply through pump and will be
controlled by flow meter at the rate of 500 ml/min to the flask. The same goes to the
CO2 cylinder tank as it flow rate will be limit by the flow meter and the regulator to
obtain the rate of 25 ml/min.

22
3.3.5 Optical Density (OD)

On each three days of cultivation, a sample will be taken from the flask to
determine the OD. The sample is then place into UV-Vis spectrometer at the
wavelength of 720 nm to check the quantity of the chlorophyll formed. The removal
efficiency of CO2 also will be calculated based on the calculation below. At the end
of 22 – 26 days of cultivation, the microalgae will be harvest and the OD will be
recorded.

𝑅𝑒𝑚𝑜𝑣𝑎𝑙 𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 (%) = (1 − 𝑂𝐷𝑡 ⁄𝑂𝐷0 ) × 100%

Where OD0 is the initial optical density of the culture and ODt is the optical
density of the culture at time t.

23
3.4 Process Flow of the Experiment

Media preparation (BBM, HC10 and BG-11)

Autoclave at 121°C for 2 hours the media and

the apparatus that will be used to cultivate the
microalgae

Prepare the stock culture for the chosen microalgae that

will be used in the experiment by using the previous
stock culture.

For stock culture, temperature of 25°C and 24 hours of

light will be provided to the microalgae in order to
maximize the growth. After 6 - 10 days, the microalgae
will be harvest and store in stock bottle.

For the actual culturing, 10% of inoculum (30 ml of microalgae

with 270 ml of fresh medium) will be cultivate inside 500 ml
flask and will be carried out in triplicate. The temperature will
be set around 30-32°C and air will be pump into the flask. The
photoperiod of 12:12 hours light and dark will be set.

After 10-14 days of culturing, the CO2 gas will be

directly supply into the flask for 12 days. The optical
density will be measured every three days of the
cultivation.

After 12 days, the algae will be harvest and

the OD will be recorded to analyse the growth
of the algae throughout the experiment.

Figure 3.2 Summary of the Process Flow of the Experiment

24
 CHAPTER 4

EXPECTED RESULT

The evaluation of the growth of (OD value), total chlorophyll in Chlorella sp.
and Chlorella vulgaris will be recorded. Each of the microalgae will show variation
in their growth pattern and pigment when in different medium and when introduce to
CO2 gas. In general, HC10 medium and BBM will greatly influence the growth of
the microalgae followed by BG-11 medium. Based on Ilavarasi et al. (2011), he
reported that Chlorella sp. will exhibit maximum growth in HC10 medium. From the
5th day the OD value of Chlorella sp. will gradually increase and reached maximum
value at 25th day. In the meantime, the growth rate in BG-11 will be fairly low
compared to the other medium. Due to that the chlorophyll content correlate with the
growth of the microalgae, it will show that the Chlorella sp.will increased along with
the growth for each day of cultivation. These results should be the same as Chlorella
Vulgaris but it will be different in terms of quantity of yield and survivability of the
strain. When it is introduced with CO2 gas it is reported that Chlorella Vulgaris can
withstand 12% CO2 concentration with 60 ppm SO2 and 100 ppm NO (Radmann et
al., 2011). Chlorella vulgaris also possesses a maximum CO2removal efficiency of
55.3% at 0.15% CO2 but the efficiencieswere reduced to 7–17% and 4–9% under
12% CO2 aeration (de Morais and Costa, 2007). It is reported that Chlorella sp. can
grow at temperatures of up to 42°C, and their tolerance toboth high temperatures and
a high CO2 content makes thempotentially appropriate microbial cells to involve in
CO2 capture for flue gases (Wang et al., 2008).

25
 CHAPTER 5

CONCLUSION

The effect of three different medium namely HC10 medium, BBM and BG-
11 medium on growth and total chlorophyll content will be varied for Chlorella sp.
and Chlorella Vulgaris. From the expected result, HC10 medium being the most
favors the growth of Chlorella sp. However, the addition of CO2 gas into the process
might change the results due to the characteristics of CO2 which will lower the pH
value of the medium and will either lower or higher the growth rate of the
microalgae. It is also depends on the trait of the strain to withstand with the extreme
condition of low pH and high temperature and from this study. It is estimated that
Chlorella sp. can withstand these and survive under these extreme conditions.

26
 REFERENCES

Bajwa, K., Bishnoi, N.R., Kirrolia, A., Sharma, J., Gupta, S. (2017). Comparison of
various growth media composition for physio-biochemical parameters of
biodiesel producing microalgal species (Chlorococcum aquaticum,
Scenedesmus obliquus, Nannochloropsis oculata and Chlorella pyrenoidosa).
European Journal of Biotechnology and Bioscience. Volume 5; Issue 6;
November 2017; Page No. 27-31

Belay, A., (1997). Mass Culture of Spirulina outdoors-The Earthrise Farms

Experience. In Spirulina platensis (Arthrospira), Physiology, Cell Biology
and Biotechnology, Vonshak, A., (Ed), Taylor and Francis, London. pp. 131-
158.

Boggess, C.D., (2014). Optimization Of Growth Parameters For Algal Regrowth

Potential Experiments. Faculty of California Polytechnic State University,
San Luis Obispo

Borowitzka M. A., (2005). Culturing microalgae in outdoor ponds. 205-218. In Algal

culturing techniques. Andersen R. A. (ed) Elsevier Academic Press,
Amsterdam, The Netherlands.

Borowitzka, L.J., and Borowitzka, M.A., (1988). “Dunaliela”, In: Microalgae

Biotechnology, Borowitzka, L.J., and Borowitzka, M.A., (Eds). Cambridge
University, Cambridge, UK, pp. 27-58.

Borowitzka, M.A. (2007). Algal Biotechnology Products and Processes Matching

Science and Economics. J. Appl. Phycol. 4: 267–279.

Borowitzka, M.A., (1999). Commercial Production of Microalgae: Ponds, Tanks,

Tubes and Fermenters. Journal of Biotechnology, 70, pp. 313-321.

Chang, R. (2007). Chemistry Ninth Edition. New York: McGraw-Hill. pp. 763-767.

Chaumont, D., (1993). Biotechnology of Algal Biomass Production: a Review of

Systems for Outdoor Mass Culture. Journal of Applied Phycology, 5, pp. 593-
604.

Cheng, J., Yang, Z., Huang, Y., Huang, L., Hu, L., Xu, D., Zhou, J., & Cen, K.
(2015). Improving growth rate of microalgae in a 1191m(2) raceway pond to
fix CO2 from flue gas in a coal-fired power plant. Bioresource technology,
190, 235-41.
27
Concas, A., M. Pisu, and G. Cao, (2010). Novel simulation model of the solar
collector of BIOCOIL photobioreactors for CO 2 sequestration with
microalgae. Chemical Engineering Journal, 157(2): p. 297-303.

de Morais, M.G. and J.A.V. Costa, (2007). Isolation and selection of microalgae
from coal fired thermoelectric power plant for biofixation of carbon dioxide.
Energy Conversion and Management, 48(7): p. 2169-2173.

Duarte, J.H., L.S. Fanka, and J.A.V. Costa, (2016). Utilization of simulated flue gas
containing CO 2, SO 2, NO and ash for Chlorella fusca cultivation.
Bioresource technology,. 214: p. 159-165.

Fulks, W., and K. L. Main (Eds). (1991). Rotifer and microalgae culture systems:
Proceedings of U.S. – Asia workshop. Honolulu, Hawaii, January 28-31,
Oceanic Institute, Honolulu, HI. PP364.

Giordano, M., J. Beardall, and J.A. Raven, (2005). CO 2 concentrating mechanisms

in algae: mechanisms, environmental modulation, and evolution. Annu. Rev.
Plant Biol., 56: p. 99-131.

Grossman, R.V., (2005). Paths toward Algal Genomics. Plant Physiology 137, pp.
410-427.

Hanelt, D., Bischof, K. and Dunton, K. (2007). Life Strategy, Ecophysiology and
Ecology of Seaweeds in Polar Waters. Rev. Environ. Sci. Biotechnol. 6: 95-
126.

Ilavarasi, A., Mubarakali, D., Praveenkumar, R., Baldev, E., and Thajuddin N.
(2011). Optimization of various Growth Media to a freshwater Microalgae for
Biomass Production. Biotechnology 10(6): 540-545.

Jorquera, O., Kiperstok, A., Sales, E.A., Embirucu, M., and Ghirardi, M.L., (2010).
Comparative Energy Life-Cycle Analyses of Microalgal Biomass Production
in Open Ponds and Photobioreactors. Bioresource Technology, 101, pp. 1406-
1413.

Jorquera, O., Kiperstok, A., Sales, E.A., Embiruçu, M., Maria L. Ghirardi. (2010).
Comparative energy life-cycle analyses of microalgal biomass production in
open ponds and photobioreactors, Bioresource Technology, Volume 101,
Issue 4, pp. 1406-1413, ISSN 0960-8524

Klinthong, W., Yang, Y.H., Huang, C.H., Tan, C.S., (2015). Microalgae and Their
Application in CO2 Capture and Renewable Energy. A Review. Aerosol and
Air Quality Research 15: 712-742.

28
Laing, I., (1991). Cultivation of Marine Unicellular Algae. MAFF Laboratory Leaflet
Number 67. Directorate of Fisheries Research Lowestoft, UK. 31 pp.

Larsdotter, K. (2006). Wastewater treatment with microalgae – a literature review.

Vatten 62:31-38.

Lavens, P., and Sorgeloos, P., (1996). Fisheries and Aquaculture Department.
Fisheries Technical Paper. No. 361. ROME. FAO.. 295p

Lee, C.S., and Tamaru, C.S., (1993). Live Larval Food Production at the Oceanic
Institute, Hawaii. In: CRC Handbook of mariculture. Volume 1. Crustacean
Aquaculture, 2nd Edition. McVey J.P. (Ed.) CRC Press, Inc., Boca Raton,
Florida, USA, pp. 15-28.

Lee, Y., and Shen, H., (2007). “3 Basic Culturing Techniques”, in Handbook of
Microalgal Culture – Biotechnology and Applied Phycology, Oxford,
Blackwell Science Ltd, pp. 40-56.

Madigan, M., and Martinko, J., (2006). Brock Biology of Microorganisms (Eleventh
Edition), Upper Saddle River, NJ 07458: Pearson Prentice Hall.

Metting, F.B., (1996). Biodiversity and Application of Microalgae. Journal of

Industrial Microbiology and Biotechnology, 17, 5-6, pp. 477-489.

Milledge, J.J., (2010). Commercial Application of Microalgae other than as Biofuels

a Brief Review. Reviews in Environmental Science and Biotechnology. pp. 1-
11.

Molina, E., Fernández, J., Acién, F.G., & Chisti, Y. (2001). Tubular photobioreactor
design for algal cultures. Journal of biotechnology, 92 2, 113-31.

Pulz, O., (2001). Photobioreactors: production systems for phototrophic

microorganisms. Applied microbiology and biotechnology, 57(3): p. 287-293.

Putt, R., (2007). Algae as a biodiesel feedstock: a feasibility assessment. Center for
Microfibrous Materials Manufacturing, Department of Chemical
Engineering, Auburn University, Alabama

Radmann, M., Elisangela & Vieira Camerini, Felipe & Duarte Santos, Thaisa &
Costa, Jorge Alberto. (2011). Isolation and application of SOX and NOX
resitant microalgae in biofixation of CO2 from thermoelectricity plants.
Energy Conversion and Management. 52. 3132–3136.
10.1016/j.enconman.2011.04.021.

29
Rasoul-Amini, S., Montazeri-Najafabady, N., Mohammad Ali Mobasher, Samira
Hoseini-Alhashemi, Younes Ghasemi, (2011). Chlorella sp.: A new strain
with highly saturated fatty acids for biodiesel production in bubble-column
photobioreactor. Applied Energy, 88(10): p. 3354-3356.

Richmond, A. and Z. Cheng-Wu, (2001). Optimization of a flat plate glass reactor

for mass production of Nannochloropsis sp. outdoors. Journal of
biotechnology, 85(3): p. 259-269.

Richmond, A., (2007). “8 Biological Principles of Mass Cultivation,” in Handbook

of Microalgal Culture – Biotechnology and Applied Phycology, Oxford,
Blackwell Science Ltd, pp. 125-177.

Rosa, A., Deidda, D., Serra, A., Deiana, M., Dessi, M.A., and Pompei, R., (2005).
Omega-3 Fatty Acid Composition and Biological Activity of Three
Microalgae Species. Journal of Food, Agriculture & Environment, Vol. 3(2),
pp. 120-124.

S. John Pirt, Y.-K., Amos Richmond and Margaret Watts Pirt (1980). "The
Photosynthetic Efficiency of Chlorella Biomass Growth with Reference to
Solar Energy Utilisation " J. Chem. Tech Biotechnolo. 30: 25-34.

Seyfabadi, J., Ramezanpour, Z. & Khoeyi, Z.A. (2010). Protein, fatty acid and
pigment content of Chlorella vulgaris under different light regimes. Journal
of applied phycology, 23, 721-726.

Singh R., and S. Sharma., (2012). Development of suitable photobioreactor for algae
production–A review. Renewable and Sustainable Energy Reviews, 16(4): p.
2347-2353.

Soong, P., (1980). Production and Development of Chlorella and Spirulina in

Taiwani. In Shelef and Soeder (Eds), Algae Biomass, Elsevier/North-Holland
Biomedical Press, Amsterdam, 97-113.

Spolaore, P., Joannis-Cassan, C., Duran, E. & Isambert, A. (2005). Commercial

applications of microalgae. Journal of bioscience and bioengineering 101:2,
87-96.

Taher, H., Al-Zuhair, S., Al-Marzouqui, A.H., Haik, Y. and Farid, M.M. (2011). A
Review of Enzymatic Transesterification of Microalgal Oil-based Biodiesel
Using Supercritical Technology. Enzyme Res. 3011: 468495, doi:
10.4061/6011/468798.

Tang, Dahai & Han, Wei & Li, Penglin & Miao, Xiaoling & Zhong, Jian-Jiang.
(2010). CO2 Biofixation and Fatty Acid Composition of Scenedesmus
30
 obliquus and Chlorella pyrenoidosa in Response to Different CO2 Levels.
Bioresource technology. 102. 3071-6. 10.1016/j.biortech.2010.10.047.

Ugwu, C.U., Aoyagi, H., and Uchiyama, H., (2008). Photo Bioreactors for Mass
Cultivation of Algae. Bioresource Technology, 99, pp. 4021-4028.

Wang, B., Li, Y., Wu, N. and Lan, Q. (2008). CO2 Biomitigation Using Microalgae.
Appl. Microbiol. Biotechnol. 79: 707–718.

Welty, J., Wicks, R.W. and Rorrer, G., (2001). Fundamentals of Momentum, Heat
and Mass Transfer 4th Edition, Hoboken, NJ: John Wiley & Sons, Inc.

Williams, T. and B. Colman, (1996). The effects of pH and dissolved inorganic

carbon on external carbonic anhydrase activity in Chlorella saccharophila.
Plant, Cell & Environment, 19(4): p. 485-489.

Zhao, B., and Su, Y. (2014). Process Effect of Microalgal Carbon Dioxide Fixation
and Biomass Production : A Review. Renewable Sustainable Energy Rev. 31:
121-132.

Zittelli, G.C., Roddfi, C., and Treclici, M.R., (2003). Mass Cultivation of
Nannochloripsis sp in Annular Reactors. Journal of Applied Phycology, 15,
pp. 107-114.

31