AERATION AND BUBBLING SYSTEM FOR

DEGASSING IN BIOREACTOR

ANTHONY DEGULLACION

FACULTY OF ENGINEERING
UNIVERSITI MALAYSIA SABAH
2020
AERATION AND BUBBLING SYSTEM FOR
DEGASSING IN BIOREACTOR

ANTHONY DEGULLACION

THESIS SUBMITTED IN FULFILMENT FOR

THE DIPLOMA IN PROCESS ENGINEERING
(OIL & GAS OPERATIONS)

FACULTY OF ENGINEERING
UNIVERSITI MALAYSIA SABAH
2020
 DECLARATION

DECLARATION

I hereby declare that this thesis was made by myself and this work is not submitted

for an application for any degree to any other university or institution. I confirm that

the thesis submitted are my own except that the contribution of reference from

researches.

30 December 2020 _____________________

ANTHONY DEGULLACION

EK18160040

ii
 CERTIFICATION

CERTIFICATION

NAME : ANTHONY DEGULLACION

MATRIC NO. : EK18160040

TITLE : AERATION AND BUBBLING SYSTEM FOR DEGASSING IN

BIOREACTOR

DEGREE : DIPLOMA IN PROCESS ENGINEERING

(OIL & GAS OPERATIONS)

VIVA DATE : 5th JANUARY 2020

DECLARED BY:

1. SUPERVISOR
Dr. Emma Suali Signature

______________________

2. CO-SUPERVISOR
Dr. Chiam Chel Ken Signature

______________________

iii
 ACKNOWLEDGEMENT
ACKNOWLEDGEMENT
Firstly, I humble grateful to God for the good health, wellbeing and continuous energy
that were helpful for me to finish this report. Nevertheless, I would like to express
my deepest gratitude for our main supervisor, Dr. Emma for all her sincere guidance,
support and encouragement throughout the final year project. Besides that, I have
also gained new experiences and learnt many things during the project thanks to Dr.
Emma. I also wished to thank all the laboratory staffs for their guidance and
instruction during laboratory work. I wanted to express my appreciation for the staffs
involved at the Borneo Marine Research Institute for the guidance on microalgae-
related issue and allowing me to take a sample for my project. Not to forget, I
sincerely would like to thank my family as they have a big part in helping to finish
my project through financial support, encouragement and constructive critics to fix
any mistakes. Lastly, I am also very grateful to all my friends including my course
mates as they were beside me at times during the project giving all the positive
comments and full support.

ANTHONY DEGULLACION
30 December 2020

iv
 ABSTRACT
ABSTRACT
This aim of this study strongly focused on the aeration and bubbling system in a self-
fabricated photobioreactor. Aeration is one of the important factors for microalgae
growth and cultivation while bubbling focused on the uptake of CO2 and O2. Through
findings and researches, FBRM technology is an appropriate tool for measuring the
size of the bubble and facilitates more understanding of changes in the size of the
bubble based on differences in the parameters of impact. One of the problems
encountered during the study was that the experiment was conducted in a house
instead of in a laboratory which led to insufficient of proper equipment and
technology to obtain accurate result. Besides that, a high-definition (HD) camera was
not available which is important for capturing high quality images of the bubbling
size. There are three main objectives that is greatly focused to achieve. The method
used to provide aeration and bubbling were by using a simple set-up consist of air
pump, control valve and flexible tube as a bubbling column. The size of the bubbles
was determined by controlling air flowrates using the control valve. For the second
objective, the results were that the largest size of the bubble obtained was 0.5 cm
when the valve opening was set to 100 % or fully opened while the smallest size of
the bubble obtained was 0.2 cm when the valve opening was set to 25 %. After that,
the bubbling size was compared by using two different bubbling columns with respect
to air flowrates. The results showed a clear comparison whereby at valve opening of
25, 50, 75 and 100 %, the size of the bubble measured were 0.2, 0.3, 0.4 and 0.5
cm respectively when bubbling column A was used. However, at valve opening of 25,
50, 75 and 100 %, the size of the bubble measured were 0.2, 0.4, 0.5 and 0.6 cm
respectively when using bubbling column B. This study was concluded successfully
with all three main objectives is achieved.

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 ABSTRAK
ABSTRAK
(SISTEM PENGUDARAAN DAN GELEMBUNG UNTUK PENYINGKIRAN GAS
DALAM BIOREAKTOR)

Tujuan kajian ini sangat memfokuskan pada sistem pengudaraan dan

penggelembung pada photobioreactor buatan sendiri. Aerasi adalah salah satu faktor
penting untuk pertumbuhan dan penanaman mikroalga sambil menggelegak yang
difokuskan pada pengambilan CO2 dan O2. Melalui penemuan dan penyelidikan,
teknologi FBRM adalah alat yang tepat untuk mengukur ukuran gelembung dan
memfasilitasi lebih banyak pemahaman mengenai perubahan ukuran gelembung
berdasarkan perbezaan parameter hentaman. Salah satu masalah yang dihadapi
selama kajian ini adalah bahawa eksperimen dilakukan di rumah dan bukan di
makmal yang menyebabkan peralatan dan teknologi tidak mencukupi untuk
mendapatkan hasil yang tepat. Selain itu, kamera definisi tinggi (HD) tidak tersedia
yang penting untuk menangkap gambar berkualiti tinggi dengan ukuran gelembung.
Terdapat tiga objektif utama yang sangat difokuskan untuk dicapai. Metode yang
digunakan untuk menyediakan pengudaraan dan gelembung adalah dengan
menggunakan susunan sederhana yang terdiri dari pam udara, injap kawalan dan
tiub fleksibel sebagai tiang gelembung. Ukuran gelembung ditentukan dengan
mengawal aliran udara menggunakan injap kawalan. Untuk objektif kedua, hasil
eksperimen diperoleh ialah ukuran gelembung terbesar yang diperoleh ialah 0.4 cm
ketika bukaan injap diatur ke 100 % atau dibuka sepenuhnya sementara ukuran
gelembung terkecil yang diperoleh adalah 0.2 cm ketika bukaan injap diatur ke 25%.
Selepas itu, ukuran gelembung dibandingkan dengan menggunakan dua tiang
gelembung yang berbeza berkenaan dengan kadar aliran udara. Hasil kajian
menunjukkan perbandingan yang jelas di mana pada bukaan injap 25, 50, 75 dan
100 %, ukuran gelembung yang diukur masing-masing adalah 0.2, 0.3, 0.4 dan 0.5
cm apabila bubbling column A digunakan. Walau bagaimanapun, pada bukaan injap
25, 50, 75 dan 100%, ukuran gelembung yang diukur masing-masing adalah 0.2,
0.4, 0.5 dan 0.6 cm apabila bubbling column B digunakan. Kajian ini berjaya
diselesaikan dengan ketiga-tiga objektif utama tercapai.

vi
 TABLE OF CONTENTS
CONTENTS
Page

TITLE i

DECLARATION ii

CERTIFICATION iii

ACKNOWLEDGEMENT iv

ABSTRACT v

ABSTRAK vi

CONTENTS vii

LIST OF TABLE x

LIST OF FIGURES x

LIST OF ABBREVIATIONS xi

CHAPTER 1 1

INTRODUCTION 1

1.1 Research Background 1

1.2 Problem Statement 2
1.3 Objectives 3
1.4 Scope of work 3
1.4 Thesis structure 4
CHAPTER 2 5

LITERATURE REVIEW 5

2.1 Background of Nannochloropsis sp. (microalgae) 5

2.1.1 Characteristics of Nannochloropsis sp. 5

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 2.1.2 Uses of Nannochloropsis sp. 6
2.1.2 Cultivation of Nannochloropsis sp. 6
2.2 Bioreactor 7
2.2.1 Photobioreactor 8
2.2.2 Self-fabricated photobioreactor 8
2.1.2 Aeration and bubbling system 8
2.3 Factors of microalgae growth 9
2.3.1 Light 9
2.3.2 Temperature 9
2.3.3 pH and Salinity 10
2.3.4 Nutrients 10
2.3.5 Mixing 11
2.4 Components of microalgae 11
2.4.1 Lipids 11
2.4.2 Carbohydrates 12
2.4.3 Protein 12
CHAPTER 3 13

METHODOLOGY 13

3.1 Methodology Flow Chart 13

3.2 Materials 14
3.2.1 Microalgae 14
3.2.2 Feedstock 14
3.3 Equipment 14
3.4 Preparation of Nannochloropsis sp. 14
3.5 Preparation of feedstocks 14
3.6 Experimental set-up 15
3.7 Method of determining the air flowrates using the control valve 16
CHAPTER 4 19

RESULT AND DISCUSSION 19

4.1 Effect of air flowrate on bubbling size 19

viii
4.2 Comparison based on bubbling size with air flowrate using two different
bubbling columns 26
CHAPTER 5 32

CONCLUSION AND RECOMMENDATIONS 32

5.1 Conclusion 32
5.1 Recommendations 33
REFERENCES 34

APPENDIX A 37

PHOTOS TAKEN DURING THE STUDY 37

Figure A 1: Flexible tube as the bubbling column 37

Figure A 2: Air pump 37

Figure A 3: Nannochloropsis sp. 38

Figure A 4: Control valve to control the air flowrate 38

Figure A 4: Clear plastic bottle containing water 39

Figure A 5: Flexible tube connected to the control valve and air pump 39

Figure A 6: Fluorescent light 39

Figure A 7: Small needle to make holes 40

Figure A 8: Metal compass divider to make holes 40

ix
 LIST OF TABLE

Page

Table 4.1. Changes in size of bubbles (mm) when valve opening 19

(%) is increasing.

Table 4.2 Comparison of bubbling size between bubbling column A 26

and B with respect to air flowrate.

LIST OF FIGURES

Page

Figure 3.1 Experiment flow chart 13

Figure 3.6 Experimental set-up 15

Figure 3.7.1 Valve opening of 0 % at angle of 0° 16

Figure 3.7.2 Valve opening of 25 % at angle of 45° 16

Figure 3.7.3 Valve opening of 50 % at angle of 90° 17

Figure 3.7.4 Valve opening of 75 % at angle of 135° 17

Figure 3.7.5 Valve opening of 100 % at angle of 180° 18

Figure 4.1.1 Image of bubbling size at valve opening of 25 % 20

Figure 4.1.2 Image of bubbling size at valve opening of 50 % 21

Figure 4.1.3 Image of bubbling size at valve opening of 75 % 22

Figure 4.1.4 Image of bubbling size at valve opening of 100 % 23

x
Figure 4.1.5 Digital images of bubbles-fibres of water 25

Figure 4.1.6 Bubble size distribution at various air flowrates 25

Image of bubbling size at valve opening of 25 % when

Figure 4.2.1 using bubbling column B 28

Image of bubbling size at valve opening of 50 % when

Figure 4.2.2 using bubbling column B 29

Image of bubbling size at valve opening of 75 % when

Figure 4.2.3 using bubbling column B 30

Image of bubbling size at valve opening of 100 % when

Figure 4.2.4 using bubbling column B 31

LIST OF ABBREVIATIONS

N2 - Nitrogen

CO2 - Carbon dioxide

-
FBRM Focused Beam Reflectance Measurement
-
O2 Oxygen

HD - High definition

rbcL - Ribulose biphosphate carboxylate large

DNA - Deoxyribonucleic acid

rDNA - Ribosomal deoxyribonucleic acid

EPA - Eicosapentaenoic acid

PBR - Photobioreactor

HCO3 -
Bicarbonate
P - Phosphorus

xi
 CHAPTER 1

INTRODUCTION

1.1 Research Background

Bioreactors are vessels providing biological, biochemical and biomechanical

specifications for the optimum growth of fermenting microorganisms and industrial
biochemical reactions for the synthesis of the required items. One of the most
important functions of bioreactors is providing dissolved oxygen to cells continuously
through a process called aeration. A research study led by Cheng et al. (2019) shows
that the influence of pump power has greater effect on the bubble formation and
mixing time than the gas flow rate. After they examined the bubble formation diameter
and time, it was found out that using the ceramic shell aerator was more suitable than
the rubber strip aerator.

Next, a research study investigated by Oliver et al. (2014) proved that small
bubbles of N2 and carbonic anhydrase (CA) significantly increased rates of degassing
and indirectly the production of nesquehonite. Nesquehonite precipitation occurred
during both rapid degassing of CO 2 induced by sparging small nitrogen bubbles and
during slow degassing caused by the introduction of large nitrogen bubbles. Apart from
that, another study investigated by Druzinec et al. (2015) explained that micro- bubble
aeration efficiently generates sufficiently high k La values that facilitate an effective
supply of oxygen during cultivation. The FBRM technology is a suitable tool for bubble
size measurements and allows further understanding of the changes in bubble size
based on variations of influencing parameters.

Furthermore, aeration is included in the main title of this experiment and it is

also considered as one of the most important factors of microalgae growth. Another
study conducted by Chiu et al. (2009) investigated deeper regarding the effect of CO 2
aeration on lipid accumulation and CO2 utilization of Nannochloropsis oculata. From the
investigation, it is found that the culture aerated with 2 % of CO 2 showed an optimal
growth potential. The amount of biomass produced was high and the specific growth
rate was enhanced when Nannochloropsis oculata culture was aerated with 2% of CO2,
compared with those in the culture aerated with air. The researchers stated that
Nannochloropsis sp., had the best growth rate under condition of aeration which rich
in CO2 compared with air aeration. Significant inhibition of high CO2 aeration, 5 to 15 %,
was also reported that the concentration of CO 2 aeration above 5 % may be harmful
to microalgae cells and inhibit microalgae development (Chang and Yang, 2003).

1.2 Problem Statement

When designing a self-fabricated photobioreactor, it may not operate efficiently and

lack of important components as it is a low budget photobioreactor. During the
experiment, I have encountered a few problems and may have obtained inaccurate
results. As my part is to set up an aeration and bubbling system in the photobioreactor,
the focus will be achieving the objectives. In addition, this experiment was conducted
at my home instead of in a lab. Thus, some of the suitable equipment available in the
lab were not available at my home. Due to that, I had to buy a low-budget equipment
for the set-up of aeration and bubbling system. Being unable to provide aeration would
cause problems specifically affect the CO2 and O2 uptake.

Furthermore, the problems that I faced was to determine the valve opening as
there was no accurate indicators due to low budget control valve. Moreover, there was
no suitable and modern equipment to capture the images of the bubbles and to
determine its characteristics. The images were captured using a phone camera instead
using a high-definition (HD) camera. The quality of the images was not good enough
compared to the HD camera. The results of bubbling size for both second and third
objective was determined manually.

2
1.3 Objectives

This study aims to set up and validate aeration and bubbling system in bioreactor. The
specific objectives of this study are as follows:

i. To set up an aeration and bubbling system that made up from flexible tube
with few holes made on it.
ii. To evaluate effect of air pump flowrate to the bubbling size.
iii. To compare bubbling size with air pump flowrate with using different bubbling
column.

1.4 Scope of work

The scope of this study is based on the objectives that will be achieved. For the first
objective, the equipment to be use for aeration and bubbling system is limited with
low budget equipment. A flexible was used as a bubbling column to provide aeration
and bubbling in the self-fabricated photobioreactor.

As for the second objective, images and videos of the size of the bubbles was
captured and recorded repeatedly using a phone camera to obtain the best result. The
valve opening was determined by adjusting it from low to high to achieve the objective.
Only a single control valve is used in the experiment.

Lastly, for the third objective, the objective is achieved by using two similar
flexible tube but with different bubbling columns. The length was the same for both
flexible tube with each measured 0.5 m. Each bubbling column is differentiated by
using a small needle and metal compass divider respectively to make small diameter
holes. The amount of small diameter holes was the same for each bubbling column
which had 5 small diameter holes on each side which also means 10 small diameter
holes in total.

3
1.4 Thesis structure

Chapter 1 presents a simple introduction containing related issues regarding the project
with a few studies from researchers mentioned. It also contains the problem statement
during the experiment, the main objectives and scope of this project.

Chapter 2 is the literature review or background studies which related to this

project and contains the important knowledge to understand deeper about the
experiment.

Chapter 3 illustrated the materials and method used in this experiment. This
chapter described on how the experiment is conducted step by step and what materials
were used in this experiment. In this chapter, each flow chart for three main objectives
were stated and discussed properly in detail. The preparation of microalgae used,
Nannochloropsis sp. was told with more detailed information. The important part in
this chapter is the experimental set-up so a detailed view of the set-up can be
understood clearly.

Chapter 4 discussed about the results and discussion obtained in this study. All
the findings are discussed in this chapter and table of results is stated for further
observations.

Chapter 5 provides a conclusion of the whole experiment. This chapter talked

about the achievement of the main objectives of this study and the recommendations
for future project.

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 CHAPTER 2

LITERATURE REVIEW

2.1 Background of Nannochloropsis sp. (microalgae)

Nannochloropsis sp. is a genus of microalgae from the phylum Ochrophyta, class

Eustigmatophyceae. In the beginning when the microalgae first found, it was known
as “marine Chlorella”, it was identified based on its ultrastructure and named
Nannochloropsis later by Maruyama et al. (1986). Nannochloropsis sp. can be found in
both freshwater and marine environments. However, we obtained the microalgae
specifically from the culture collection of Borneo Marine Research Institute, Universiti
Malaysia Sabah. Nannochloropsis sp. have been used for several decades to produce
nutraceuticals and feed supplements (Rodolfi et al., 2009).

Nannochloropsis sp. is one of the potential generation of microalgae having

higher eicosapentaenoic acid (EPA), biomass and lipid productivities. Therefore, this
type of microalgae is used in the experiment to investigate further and cultivated in a
self-fabricated photobioreactor.

2.1.1 Characteristics of Nannochloropsis sp.

The characteristics of Nannochloropsis sp. are that it is a unicellular type and planktonic,
with its size ranges from 2 to 4 μm diameter in subspherical or 3 to 4 × 1.5 μm in
cylindrical cells (Hibbert, 1980). They have major pigments such as chlorophyll a, beta-
carotene, violaxanthin, and vaucheriaxanthin, with minor pigments being
canthaxanthin and astaxanthin (Lubián et al., 2000). Furthermore, what makes it vary
from other microalgae is that Nannochloropsis sp. mainly comprise of chlorophyll a but
lack of chlorophyll b and chlorophyll c.

However, since they do not have distinct morphological features that separate
them from those 5 species mentioned, characterizations can only be done using

5
genomic analyses such as rbcL chloroplast DNA and 18S rDNA instead of standard
microscopy.

2.1.2 Uses of Nannochloropsis sp.

Up until this modern day, Nannochloropsis sp. is greatly popular as it it mainly used
for aquaculture feed and food supplements due to it richness in concentrations of
photosynthetic pigments, proteins and high levels of long-chain polyunsaturated fatty
acids (including omega-3 fatty acids), especially EPA whereby it is important for
nutraceuticals and aquaculture industries (Krienitz and Wirth, 2006).

In addition, it is considered a potential model microalgal due to its high

photosynthetic efficiency, high lipid productivity, well-established genetic database and
relatively mature technology for large-scale outdoor cultivation systems. Thus, because
of its ease of growth and high oil content, this made Nannochloropsis sp. suitable for
algal biofuel production and as a renewable biodiesel feedstock.

Moreover, Nannochloropsis sp. is also known to help prevent global warming

or act as a greenhouse because of its fast and high absorption of CO 2, which gain more
interest besides production of valuable and energy products through green processes
(Rodolfi et al., 2009).

2.1.2 Cultivation of Nannochloropsis sp.

In general, microalgae are cultivated in many ways which are photoautotrophically,

heterotrophically and mixotrophically either in open ponds or closed bioreactors. The
process of cultivation of microalgae would be better using direct natural sunlight as it
gives more light intensity. However, the rate of cell growth may slow down due to poor
light penetration in high cell density.

Photoautotrophic cultivation is the most common way to grow microalgae due

to low cost and eco sustainable properties. Energy can be produced from sunlight and
carbon dioxide from power plants, making it easier to offset environmental issues
(Huang et al.,2010). Closed bioreactors seem to be more manageable than open ponds

6
and benefit the growth of microalgae and lipid production. A tubular bioreactor is the
most commonly used commercial closed bioreactor system (Chisti et al.,2007). They
are normally made of transparent materials, which are polypropylene acrylic or
polyvinyl chloride pipes with a thickness of a few millimetres, allowing for adequate
absorption of light (Safi et al.,2014). The method for mixing and agitating microalgae
is to use an air pump to create air bubbles, and illumination can be obtained from
natural sunlight or by using artificial light, e.g. fluorescent light (Briassoulis et al.,2010).

Some microalgae can grow heterotrophically using organic carbon source but
without any source of light. For example, glucose, glycerol and acetate are those
organic carbon source for heterotrophic culture (Huang et al.,2010). Both cell growth
and biosynthesis of products are greatly affected by nutrients and environmental
factors.

Nannochloropsis sp. is capable of growing mixotrophically in the presence of

organic carbon sources under light conditions. Therefore, cell growth may not be
entirely dependent on light or organic carbon.

2.2 Bioreactor

Bioreactors are very known widely used as a vessel in which biological reaction or
change of organisms takes place. Two process can be involved which are aerobic and
anaerobic. Other than that, the biological systems are including enzymes, tissues,
animal or plant cells. The bioreactor works by providing an optimum external
environment as so to meet the requirements of the biological reaction system so that
a high yield of the bioprocess is obtained.

Commonly, the shape of the bioreactor is cylindrical with different sizes

depending on the volume of organisms used and usually made of stainless steel. Cell
culturing can also undergo its process in this vessel to grow microalgae as an example.
Besides that, photobioreactor is a common type of bioreactor that absorbs any form of
light source whether it is natural sunlight or artificial light. However, PBRs also can be
any types of container or plastic bottle which is transparent which can allow light to
penetrate through it.

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2.2.1 Photobioreactor

Photobioreactor is described an enclosed system that used to cultivate microalgae

through photosynthesis whereby it has an illumination designed to utilize light.
Photobioreactor has the potential to overcome a variety of constraints usually faced by
open pond culture architecture. The scale of the bioreactor is more compact than the
open pond scheme, allowing more productive land use. Indoor-operated
photobioreactors necessarily have smaller sizes because artificial light is required.
Designs can either be plastic tubular systems or stainless-steel fermenter-like reactors
with internal lighting to allow optimum light intensity. Two most common
photobioreactor used is tubular photobioreactor and horizontal photobioreactor.

2.2.2 Self-fabricated photobioreactor

As mentioned before, anything that is translucent either a container or plastic bottle

can be made as a PBR. In this project, a plastic bottle (1.5 L) is used to design a self-
fabricated photobioreactor. The plastic bottle used in this experiment is transparent
enough for light go through it and big enough for to put microalgae used in the
experiment.

2.1.2 Aeration and bubbling system

During cultivation, dissolved oxygen is one the important key to substantial growth and
production of microalgae. One of the crucial parts in bioreactors is to provide dissolved
oxygen (DO) to cell culture continuously through a process called aeration. The process
normally occurs when oxygen is diffused through overlay to the cell culture medium
interface or oxygen produced by the sparger dissolves in the cell culture through
convection with the help of agitation. A sparger is an equipment for which it helps to
supply adequate oxygen to the growing cells. Agitation disperses oxygen bubbles and
facilitates mass transmission of gas bubbles through the gas-liquid (cell culture medium)
interface.

The bubble column reactor cannot be used for an extremely viscous medium.
The pattern of gas bubbles in the bubble column reactor depends on the superficial

8
velocity of the gas. The gas velocity should be 1-4 cm per second for uniform bubbles
in the medium to ensure proper mixing. If the gas velocity is higher or lower than the
uniform bubbles, it will not be formed, therefore, when the bubbles coalesce create
variations in the fluid density that disrupt the air flow rate.

2.3 Factors of microalgae growth

Generally, there are important parameters regarding the growth of microalgae which
are commonly light, temperature, pH and salinity, nutrients and turbulence. However,
these factors may depend on the type of microalgae as some of them may need
different requirements. With these conditions applied, the microalgae can adapt with
ease as well as growing rapidly and produce more lipids.

2.3.1 Light

Light is a crucial factor as it influences the microalgae growth under these three
different conditions which are light limitation, light saturation and light inhibition.
Microalgae growth can be increase with the increase of light intensity under condition
light restriction. When light saturation occurs, photosynthetic activity declines as the
accumulation of photons reaches the electron turnover, thereby inhibiting
photosynthesis. Microalgae become photoinhibited when the light intensity is
marginally greater than the light intensity at the specific growth rate. Due to the
excessive light source, photoinhibition phenomena can usually cause reversible harm
to the photosynthetic method (Rubio et al.,2003). Nannochloropsis sp. can achieve a
maximum growth rate in phototrophic and mixotrophic cultures under blue light
condition (Das et al.,2011). The duration and intensity of light, therefore, directly
affects the growth and photosynthesis of microalgae.

2.3.2 Temperature

Temperature have a very important role in microalgae growth. Temperature also

impacts the gross photosynthetic behaviour of microalgae. In view of the prevailing
temperature conditions, microalgae strains should be chosen as this enhances the
growth of the strain under study.

9
 A change in temperature also affects the nutrient absorption and cellular
chemical composition of microalgae. If temperatures below 16°C or over 35°C are
considered lethal for the growth of microalgae (Prasath et al., 2015). Temperature
influences the activity and reaction rates of the intracellular enzyme, which will impact
algal photosynthesis, respiration speed, affect microalgae growth and reduce its
distribution (Tan et al., 2009).

2.3.3 pH and Salinity

Water pH was closely linked to microalgae formation. The pH, which is already
discussed in the photosynthetic portion, will be modified via photosynthetic. The pH
value can also influence the growth rate of microalgae, making it easier for microalgae
to absorb CO2 in the atmosphere when the rising condition is alkaline, which can
produce more biomass (Zang et al., 2011 & Melack, 1981). Microalgae has its own
salinity adjustment system. Microalgae in seawater can usually withstand higher
salinity than microalgae in freshwater (Zhu et al., 2003). Research findings have shown
that microalgae have their own optimum growth salinity, when salinity higher or lower
than this is detrimental to microalgae growth rates.

2.3.4 Nutrients

Nitrogen is considered a building block for proteins and nucleic acids, while phosphorus
is a component of phospholipids. If these macronutrients are reduced, the metabolic
pathways begin to change (Ceballos et al., 2013). A researcher claimed that when the
N/P ratio reaches 16, phosphorus was considered a limiting factor and the nitrogen
content needed to be regulated in order to maximize the increasing condition of
microalgae. The requirement for an optimum amount of phosphorus was considered
to be beneficial to the growth of microalgae. If the total phosphorus content is a little
less than 0.045 mg/L or more than 1.65 mg/L, then growth was prohibited. If the total
phosphorus content is 0.02mg/L, the growth of microalgae has been increased (Ren
et al., 2014).

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2.3.5 Mixing

Generally, microalgae grow well in the lake or stream due to the dynamics of the water
(Wang, 2006). Dynamic is an important criterion when designing PBRs for microalgae.
Gas mixing can be used in the PBRs as so to have equal light source distribution and
nutrients to every microalgae cell. In culturing microalgae, both temperature and
illumination are important and relying on each other. Shading is an issue in the growth
of microalgae, prohibits the efficient absorption of light by microalgae, and affects the
production of biomass. To avoid that, mixing is a common method to ensure even
distribution of light to all the microalgae cells, thereby improving the light regime
(Rocha, Garcia et al., 2003). Therefore, it is important that proper mixing is provided
to the microalgae and this can be done by gas mixing when photo bioreactors are used
for cultivation of microalgae (Ren et al., 2014).

2.4 Components of microalgae

Microalgae-derived bioproducts can be categorized into three major groups, namely

lipids, carbohydrates and protein.

2.4.1 Lipids

Microalgae have been recognized for their lipid content richness, where the lipid
content of dried microalgae biomass is about 20 to 50 %, but the lipid content of
microalgae can be increased to 80 % with unique optimized cultivation technique
(Chisti Y et al.,2007). Generally, there are two major applications for microalgae-
derived lipids, which are biodiesel and food supplement. Due to its carbon neutrality
and the use of feedstock that does not compete with our current food supply for energy,
microalgae biodiesel is considered as the green fuel for the future. On the other hand,
as food additives, lipids derived from microalgae can also be used to provide additional
nutritional factors in human food (Kings et al.,2017).

11
2.4.2 Carbohydrates

Microalgae cells are filled with various complex carbohydrates that can be readily used
as feedstock for the development of bioethanol, such as cellulose, agarose, starch, and
glycogen (Khan et al.,). Bioethanol developed from micro-algae carbohydrates can be
used directly without significant modification by the currently available internal
combustion engine. In addition, bioethanol fuel's high-octane rating and oxygen
content translate well into higher engine efficiency and decreased emission rate
(Quader et al.,2017).

2.4.3 Protein

Microalgae are also rich in protein where the yield of their protein is said to be
equivalent to that of traditional sources of protein such as corn, milk and animal meat.
Spirulina has been cultivated and sold for human consumption as protein supplements
in the form of tablets and capsules (Grahl et al.,2018). The crude extract of T. suecica
has been reported to contain proteins with emulsifying, foaming and gelation
properties that may open up a variety of food industry application options (Garcia et
al.,2018). R-phycoerythrin is another type of protein that can be derived from
microalgae, which is a type of phycobiliprotein that is typically used in diagnostic and
research fields as a fluoresces dye (Sekar et al.,2008).

12
 CHAPTER 3

METHODOLOGY

3.1 Methodology Flow Chart

In this study, all three objectives as stated previously are fulfilled according to the flow
chart below as shown in Figure 3.1.

Figure 3.1: Experiment flow chart

Preparation of feedstock and pre culture of Nannochloropsis sp. for

experiment

Preparation of equipment for experimental set up of aeration and

bubbling system in a self-fabricated photobioreactor

Testing and validation of the set up

Adjustment on the control valve from low to high air flowrates and
observation on the bubbling size

Determining comparison of bubbling size using two different

bubbling columns

Analysis on data acquired

13
3.2 Materials

3.2.1 Microalgae

The microalgae used in this study are Nannochloropsis sp. and it was obtained from
culture collection of Borneo Marine Research Institute, Universiti Malaysia Sabah.

3.2.2 Feedstock

The medium used as a feedstock for microalgae are mostly have nitrogen and
phosphorus content in it. The feedstocks used are Ca(NO 3)2.4H2O, KH2PO4,
MgSO4.7H2O, NaHCO3, NaNO3, Na2HPO4.12H2O and etc.

3.3 Equipment

A transparent plastic bottle (1.5 L) is used as a photobioreactor with air pump used for
aeration, control valve for controlling the air flowrates and flexible tube as a bubbling
column. Other than that, a long fluorescent light is used as an artificial light instead of
using natural sunlight for illumination for microalgae.

3.4 Preparation of Nannochloropsis sp.

The sample is freshly obtained and pre-cultured from the lab which then poured about
500 ml into a plastic bottle. Distilled water is added to the sample about 250 ml which
the volume combined is 750 ml in the plastic bottle and stirred later.

3.5 Preparation of feedstocks

The feedstock is prepared in the lab using the manual given by the lab instructor. The
total volume inside the plastic bottle is 750 ml, so each medium is prepared 75 ml.
After that, the medium is placed inside a small conical flask and sealed properly. Only
0.75 ml is used to feed the microalgae with each medium.

14
3.6 Experimental set-up

The experimental set-up involves the aeration and bubbling inside the transparent
plastic bottle which is the self-fabricated photobioreactor. The microalgae,
Nannochloropsis sp. was poured inside the transparent plastic bottle (1.5 L) and was
cultivated in it. The set-up was placed in an area which have windows, room
temperature and a bit of natural sunlight coming through the windows. The air pump
is placed right beside the plastic bottle and connected with the control valve and a 0.5
m flexible tube which have 10 small diameter holes made on it.

Next, 2 small holes is made on the bubble cap using a driller with slightly bigger
diameter size of the flexible tube to fit through it. The first hole is made for oxygenation
and the other one is to put inside the bubbling column. Besides that, the fluorescent
light is placed behind the plastic bottle. The fluorescent light and the air pump were
turned on constantly until the end of the experiment period which was about 2 weeks.

Figure 3.6 Experimental set-up

15
3.7 Method of determining the air flowrates using the control valve

For the second objective of the experiment, the bubbling size is measured with respect
to air flowrates. So, a simple control valve was used to control the air flowrates from
low to high air flowrates. In Figure 3.7.1, a red line was labelled on the control valve
which it indicated that the valve opening was at 0 % or the valve was fully closed. The
angle of the labelled line was estimated to be at 0° to the right using a protractor.

Figure 3.7.1: Valve opening of 0 % at the angle of 0°

Moreover, the experiment was conducted with 4 sets of different air flowrates
with 25, 50, 75 and 100 % of valve opening. At valve opening of 25 %, Figure 3.7.2
shown that the labelled line was estimated to be at angle of 45° using a protractor.

Figure 3.7.2: Valve opening of 25 % at the angle of 45°

16
 Next, at valve opening of 50%, Figure 3.7.3 shown that the labelled line was
estimated to be at angle of 90° using a protractor. Furthermore, at valve opening of
75 %, Figure 3.7.4 shown that the labelled line was estimated to be at angle of 135°
using a protractor. Finally, at valve opening of 100 % or other means the valve was
fully opened, Figure 3.7.5 shown that the labelled line was estimated to be at angle of
180° using a protractor.

Figure 3.7.3: Valve opening of 50 % at the angle of 90°

Figure 3.7.4: Valve opening of 75 % at the angle of 135°

17
Figure 3.7.5: Valve opening of 100 % at the angle of 180°

18
 CHAPTER 4

RESULT AND DISCUSSION

4.1 Effect of air flowrate on bubbling size

As discussed before, one of main factor that affect the bubbling size is the air flowrate.
Therefore, this experiment for the second objective were determined by focusing the
valve opening (%) as the manipulated variable and the size of bubble in diameter (cm)
as the responding variable. All the data during the observation of the experiment was
recorded in Table 4.1. The image of the size of bubble was taken for each four sets of
valve opening. For ease observation, the experiment was conducted using a
transparent plastic bottle so that the bubbles can be seen clearly.

Table 4.1 Changes in size of bubbles (mm) when valve opening (%) is
increasing

Valve opening (%)

Size of bubble in diameter (cm)

25 0.2

50 0.3

75 0.4

100 0.5

As you can see, Table 4.1 illustrated the changes in size of bubble (cm) when
valve opening (%) is increasing. When the valve opening was set to 25 %, the size of
the bubble obtained is 0.2 cm in diameter. The result obtained was proven in Figure
4.1.1 whereby a bubble was measured using a ruler. Besides that, in Figure 4.1, it is
noticed that the amount of bubbles produced was less as expected when the valve
opening was set to 25 %. Thus, the amount of bubbles was less due to low air flowrate.

19
Figure 4.1.1: Image of bubbling size at valve opening of 25 %

20
Figure 4.1.2: Image of bubbling size at valve opening of 50 %

21
Figure 4.1.3: Image of bubbling size at valve opening of 75 %

22
Figure 4.1.4: Image of bubbling size at valve opening of 100 %

23
 Moreover, when valve opening was set to 50 %, Table 4.1 shown that the size
of bubble increased compared to size of bubble when the valve opening was set to 25
%. The size of the bubble can be observed in Figure 4.2 whereby the size of bubble
measured was approximately 0.3 cm in diameter. It is noticed that an increase in the
valve opening generated an increase in the size of the bubble as well. Besides that,
when observing the amount of bubble produced in Figure 4.2, it seemed more bubbles
produced when there was an increase in the valve opening. Hence, the amount of
bubbles produced increased again when the air flowrate was high.

Next, the valve opening was increased up to 75 % to observe the changes in

the size of the bubble. By referring to Table 4.1, it shows that the size of bubble
obtained shows an increase again in the size compared to size of bubble when valve
opening was set to 50 %. In Figure 4.3, the size of bubble measured was around 0.4
cm in diameter by using a ruler which proved that the size of the bubble became more
larger than the size of bubble obtained in Figure 4.2. Plus, it is noticed that greater
amount of bubbles was produced than before. Therefore, the amount of bubbles
produced were affected again by higher air flowrate.

On the other hand, when valve opening increased again until 100 % which was
the maximum level, the results tabulated in Table 4.1 demonstrated that the size of
bubble obtained again increased than the size of bubble when the valve opening was
set to 75 %. It can be proven in Figure 4.4 whereas the size of bubble measured was
roughly 0.6 cm in diameter by using a ruler. Accordingly, this explained that higher air
flowrate generates larger size of bubbles. As expected, in Figure 4.4 clearly shown a
huge amount of bubbles was produced again than previous result. Consequently, it is
deduced that higher air flowrate generates larger amount of bubbles.

In other researcher study, there are few examples of captured image of

bubbling size at different air flowrates as shown in Figure 4.5. As you can see, many
bubbles are formed when the air flowrate is higher, and the bubble formation is faster
as large bubbles are in sight. In Figure 4.6, the histogram showed a large increase,
specifically at higher air flowrates, which confirmed that higher air flowrates formed
larger bubbles. The maximum bubble size at 2 L/min air flowrate is in the range of

24
1.70 cm to 1.75 cm, whilst at 10 L/min the bubble size can reach up to 2.00 cm to
2.05 cm. Similar trends were observed for both nozzle sizes (Wicaksana et al.,2006).

Figure 4.1.5: Digital images of hollow fibres-bubbles in water

Source: [Wicaksana et al., 2014]

Figure 4.1.6: Bubble size distribution at various air flowrates

Source: [Wicaksana et al., 2014]

25
4.2 Comparison based on bubbling size with air flowrate using two
different bubbling columns

In this experiment, the third objective was to make a comparison between two different
bubbling columns using two similar flexible tube based on their size of bubbles with
respect to air flowrate. Each bubbling columns were categorized as bubbling column A
and bubbling column B and each bubbling columns were made by using different tools
which are a small needle and metal compass divider respectively. Noted that, the
previous bubbling column which was used in the second objective was used as bubbling
column A. For ease observation, the experiment was conducted using a transparent
plastic bottle so that the bubbles can be seen easily.

Table 4.2: Comparison of bubbling size between bubbling column A and B

with respect to air flowrate

Valve opening (%) Size of bubbles in diameter (cm)

Bubbling column A Bubbling column B

25 0.2 0.2

50 0.3 0.4

75 0.4 0.5

100 0.5 0.6

Notes: The results of bubbling size of bubbling column A is same in Table 4.1 due to
same bubbling column was used.

As shown in Table 4.2, when valve opening was set to 25 %, the result indicated
that size of the bubble was the same for bubbling column A and B. It is proven in
Figure 4.7 whereby the size of bubble measured was around 0.2 cm in diameter by
using a ruler. So, no comparison can be made between the two different bubbling
columns. Although, the size of the holes made for each bubbling column was different
as the sharp surface area of the metal compass divider is much bigger than the small
needle.

26
 After that, when valve opening was set to 50 %, a slight increase in the size of
bubble is seen as illustrated in Table 4.2. For bubbling column A, the size of the bubble
measured is 0.3 cm in diameter whereas, for bubbling column B, the size of the bubble
measured is 0.4 cm in diameter using a ruler which was shown in Figure 4.8. It showed
that bubbling column B produced larger bubbling size compared to when using
bubbling column A. Thus, bubbling column B have a greater efficiency to produce
bigger size of bubble. However, a slight increase in the amount of bubbles produced
were seen in Figure 4.8 than in Figure 4.2.

Moving on, when valve opening set to 75 %, Table 4.2 shown that the size of
bubble measured is 0.4 cm in diameter when using bubbling column A whereas the
size of bubble measured when using bubbling column B is 0.5 cm in diameter by using
a ruler as shown in Figure 4.9. The results can be compared whereby the size of bubble
is larger when using bubbling column B while the size of bubble is smaller when using
bubbling column A. In addition, Figure 4.9 shown that greater amount of bubbles were
produced at valve opening of 75 % when using bubbling column B compared to the
amount of bubbles produced at valve opening of 75 % when using bubbling column A
which shown in Figure 4.3.

Finally, when valve opening was set to 100 % which is the maximum level, the
result from Table 4.1 stated that the size of bubble measured when using bubbling
column B increased by a margin of 0.6 cm in diameter whereas the size of bubble
measured when using bubbling column A increased by a margin of 0.5 cm in diameter.
Therefore, from the results obtained, it is clearly stated that bubbling column B
generated larger size of bubble compared to size of bubble when using bubbling
column A. Plus, a tremendous amount of bubbles were produced as shown in Figure
4.10 when using bubbling column B at 100 % of valve opening compared to amount
of bubbles produced when using bubbling column A at 100 % valve opening as shown
in Figure 4.4. The whole experiment also explained that logically larger holes would
generate larger size of bubble.

27
Figure 4.2.1: Image of bubbling size at valve opening of 25 % when using
bubbling column B

28
Figure 4.2.2: Image of bubbling size at valve opening of 50 % when using
bubbling column B

29
Figure 4.2.3: Image of bubbling size at valve opening of 75 % when using
bubbling column B

30
Figure 4.3.3: Image of bubbling size at valve opening of 100 % when using
bubbling column B

31
 CHAPTER 5

CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion

To conclude this whole experiment, the aeration and bubbling system in a self-
fabricated photobioreactor was a success. In this study, all three main objectives was
successfully carried out which are to set up aeration and bubbling system, to evaluate
the effect of air flowrate to bubbling size and to compare bubbling size using two
different bubbling columns with respect to air flowrate. It is a big achievement that the
microalgae, Nannochloropsis sp. was able to be cultivated in a low-budget self-
fabricated photobioreactor with aeration and artificial light provided. The set-up for
aeration and bubbling system may not be fully perfect and had many flaws but it was
enough to generate air bubbles efficiently to provide aeration in the media for CO 2 and
O2 intake for the cultivation of the microalgae.

The effect of air flowrate to the bubbling size was able to be evaluated through
proper observation and analysis on data acquired. Through the results obtained, it is
understood that the air flowrate greatly affected the bubbling size and its
characterisation was shown during the observation. The smallest bubbling size
obtained is 0.2 cm when the valve opening was set to 25 %. While the largest bubbling
size obtained is 0.5 cm when the valve opening was set to 100 %. The results indicated
that the size of the bubbles significantly increased when the control valve is adjusted
slowly from 0 % to 100 %. In addition, higher air flowrates also lead to producing huge
amount of bubbles.

The comparison of bubbling size using two different bubbling columns was
shown in the tabulated result. It is confirmed that each bubbling column produced
different results in the size of the bubble because of its different properties whereby
the bubbling column A was made using a small needle which have smaller sharp
surface area compared to the sharp surface of metal compass divider which was used

32
for designing the bubbling column B. From low to high air flowrates, the results showed
that bubbling column B produced larger bubbling size compared to bubbling size when
bubbling column A was used. These results has been proved whereby the size of the
bubble when bubbling column A was used were 0.2, 0.3, 0.4 and 0.5 cm when the
valve was set to 25, 50, 75 and 100 % respectively meanwhile, the size of the bubble
obtained when bubbling column B was used were 0.2, 0.4, 0.5 and 0.6 cm when the
valve was set to 25, 50, 75 and 100 % respectively. However, all the results obtained
might be not accurate due to lack of modern equipment and some experimental error
may occurred during the experiment.

5.1 Recommendations

Hopefully in the future study, the experiment can be conduct in a more suitable place
with all proper equipment is available to provide accurate results. Other than that, a
high-definition (HD) camera could be used for capturing the images of the bubbling
size for better quality of images and the size of bubble can be determined accurately
using image analysis software. The characteristics of the bubble size can be studied
further to have a better understanding on it. Further studies can be done to prevent
lack of information such as study on the bubbling size formation and distribution with
different methods and calculations relating to this study. Other than that, financial
problem hoped to be resolved so as to buy more and better equipment.

33
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36
 APPENDIX A

PHOTOS TAKEN DURING THE STUDY

Figure A 1: Flexible tube as the bubbling column

Figure A 2: Air pump

37
 Figure A 3: Nannochloropsis sp.

Figure A 4: Control valve to control the air flowrate

38
 Figure A 4: Clear plastic bottle containing water

Figure A 5: Flexible tube connected to the control valve and air pump

Figure A 6: Fluorescent light

39
 Figure A 7: Small needle to make holes

Figure A 8: Metal compass divider to make holes

40