Batch and Continuous Studies of Chlorella Vulgaris in Photo-Biore

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February 2013
Batch and Continuous Studies of Chlorella Vulgaris

in Photo-Bioreactors
Claudia Sacasa Castellanos
The University of Western Ontario
Supervisor
Amarjeet Bassi
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BATCH AND CONTINUOUS STUDIES OF Chlorella vulgaris IN PHOTOBIOREACTORS

(Spine title: Batch & Continuous Study of C. vulgaris in Photo-bioreactors)
(Thesis format: Monograph)
by
Graduate Program in Chemical and Biochemical Engineering
A thesis submitted in partial fulfillment

of the requirements for the degree of
M.E.Sc. in Chemical and Biochemical Engineering
The School of Graduate and Postdoctoral Studies

London, Ontario, Canada
January 2013
Claudia Sacasa Castellanos 2013
THE UNIVERSITY OF WESTERN ONTARIO

School of Graduate and Postdoctoral Studies
CERTIFICATE OF EXAMINATION
Supervisor
Examiners
______________________________
Dr. Amarjeet Bassi
______________________________
Dr. Ernest Yanful
Supervisory Committee
______________________________
Dr. Shahzad Barghi
______________________________
Dr. Mita Ray
______________________________
Dr. Dimitre Karamanev
The thesis by

entitled:
Batch and Continuous Studies of Chlorella vulgaris in

Photo-bioreactors
is accepted in partial fulfillment of the
requirements for the degree of
M.E.Sc. in Chemical and Biochemical Engineering
______________________
Date
_______________________________
Chair of the Thesis Examination Board
ii
Abstract
Microalgae are unicellular, photosynthetic microorganisms, they have minimal nutrient
requirements and are being used as source materials for a variety of products such as
protein rich nutritional supplements, pharmaceutical chemicals and pigments (used in
food and cosmetics). They also grow extremely fast, in comparison to plants and many
microalgae strains are exceedingly rich in oil which makes them great candidates for the
production of biodiesel. The strain Chlorella vulgaris, has shown a great promise as a
source of oil and as being able to help in the treatment of wastewater. Despite all the
advantages , the high cost of production in bioreactors still remains a limitation and much
research is required. The production of microalgae (Chlorella vulgaris) in different photobioreactor configurations (4L flask, tubular and stirred tank reactor) was investigated,
using different media formulations and in both batch and continuous operation (for PBR
and CSTR). The effect of dilution rate and mixing speed were both studied in the case of
CSTR. The highest overall specific growth rate () was found when growing C. vulgaris
in continuous operation in a tubular photo-bioreactor, = 0.7 d-1, which also reached the
maximum biomass concentration of 0.79 g/L.
Keywords
Microalgae, Chlorella vulgaris, tubular photo-bioreactor, stirred tank reactor, continuous
operation
iii
Acknowledgments
My sincere gratefulness goes to my supervisor Dr. Amarjeet Bassi. His help, support and
understanding, financially and emotionally, that made it possible for me to be successful
in the pursue and finalization of this project. I am thankful for his encouragement to keep
going and to constantly learn new things.
I would like to thank Dr. Ajay Ray, who was my co-supervisor for part of my research,
and Joanna Blom for his guidance and great support.
I am grateful for the patience of Mr. Brian Dennis as I asked him question after question
and for giving me access to material and equipment essential to my research.
I would also like to thank the members of the labs I visited and worked in for helping me
with my experiments and for providing intelligent insights that made my work easier,
thank you for being so friendly.
Lastly but not least I am very grateful for the support of my family, of my mom Maribel
and my brother Carlos, and of all the friends that are like family to me, to Jsus Moreira
and Carolyn Moore for always being there for me and most importantly to my little sister
Magaly who has been and will continue to be the main motivation for me to do better.
iv
Table of Contents
CERTIFICATE OF EXAMINATION ........................................................................... ii
Abstract .............................................................................................................................. iii
Acknowledgments .............................................................................................................. iv
Table of Contents ................................................................................................................ v
List of Tables ................................................................................................................... viii
List of Figures .................................................................................................................... ix
List of Appendices ............................................................................................................. xi
Nomenclature .................................................................................................................... xii
Chapter 1 ............................................................................................................................. 1
1 Introduction .................................................................................................................... 1
1.1 Research Objectives ................................................................................................ 2
1.2 Thesis Overview ..................................................................................................... 2
Chapter 2 ............................................................................................................................. 3
2 Literature Review ........................................................................................................... 3
2.1 Introduction on Microalgae..................................................................................... 3
2.1.1
What are Microalgae ................................................................................... 3
2.1.2
Current Uses of Microalgae ........................................................................ 3
2.2 Factors Affecting Microalgal Growth ..................................................................... 7

2.2.1
Nutrient Composition of Media .................................................................. 7
2.2.2
Light ............................................................................................................ 8
2.2.3
pH ................................................................................................................ 8
2.3 Microalgae Cultivation ........................................................................................... 9

2.3.1
Microalgae Growth Kinetics ....................................................................... 9
2.3.2
Photo-bioreactor Design ........................................................................... 12
2.3.3
Tubular Photo-bioreactors ........................................................................ 14
2.3.4
Continuously Stirred Tank Reactors ......................................................... 15
2.3.5
Batch and Continuous Cultivation ............................................................ 17
2.4 Importance of This Study...................................................................................... 18

Chapter 3 ........................................................................................................................... 19
3 Materials and Methods ................................................................................................. 19
3.1 Microalgae Strain .................................................................................................. 19
3.2 Growth Media ....................................................................................................... 19
3.2.1
Bolds Basal Media ................................................................................... 19
3.2.2
Other Media Formulation ......................................................................... 21
3.3 Inoculum and Strain Maintenance ........................................................................ 22

3.4 Photo-bioreactor Set-up ........................................................................................ 22
3.4.1
4 L Conical Flasks .................................................................................... 22
3.4.2
Tubular Photo-bioreactor .......................................................................... 23
3.4.3
Continuously Stirred Tank (CSTR) Photo-bioreactor .............................. 25
3.4.4
Air Supply ................................................................................................. 26
3.4.5
Lighting ..................................................................................................... 26
3.5 Sampling of Photo-bioreactor ............................................................................... 28

3.6 Analysis of Samples .............................................................................................. 28
3.6.1
Cell Count ................................................................................................. 28
3.6.2
pH .............................................................................................................. 28
3.7 Determination of Dry Weight and Biomass Concentration .................................. 28

3.8 Determination of CO2 Concentration .................................................................... 29
3.9 Nutrient Analysis .................................................................................................. 30
vi
3.9.1
Nitrates ...................................................................................................... 30
3.9.2
Phosphates................................................................................................. 30
Chapter 4 ........................................................................................................................... 31
4 Results and Discussions ............................................................................................... 31
4.1 Batch Cultivation in 4L Flasks ............................................................................. 31
4.1.1
Modified Bolds Media with NaHCO3 ..................................................... 31
4.2 Batch Cultivation in Tubular PBR. ....................................................................... 33

4.2.1
Batch Cultivation in C30 and PCS-2 Media ............................................. 33
4.2.2
Batch Cultivation in Bolds Media ........................................................... 35
4.3 Batch Cultivation in Stirred Tank Photo-bioreactor. ............................................ 39

4.4 Batch Cultivation in Tubular and Continuously Stirred Tank Photo-bioreactors . 42
4.5 Semi-Continuous Cultivation in a Tubular Photo-bioreactor ............................... 44
4.6 CSTR..................................................................................................................... 46
4.6.1
Effect of Agitation Speed on Cell Growth ................................................ 46
4.6.2
Effect of Dilution Rate on Cell Growth .................................................... 48
Chapter 5 ........................................................................................................................... 51
5 Conclusions and Recommendations ............................................................................ 51
5.1 Future Work .......................................................................................................... 52
References ......................................................................................................................... 54
Appendices ........................................................................................................................ 63
Curriculum Vitae .............................................................................................................. 68
vii
List of Tables
Table 2.1: Comparison of crop-dependent and algae biodiesel (Schenk et al., 2008). ........ 6
Table 2.2: Summary of continuous and semi-continuous cultivation of microalgae......... 13
Table 3.1: Modified Bolds Basal Media (Andersen, 2005). ............................................. 20
Table 3.2: Other Media Formulations (Perez-Garcia et al., 2011). ................................... 21
Table 4.1: Comparison of Chlorella vulgaris growth in Tubular PBR with Modified
Bolds Media ...................................................................................................................... 36
Table 4.2: Parameters and results of CSTR experiments................................................... 40
Table 4.3: Comparison of Batch Studies with Chlorella vulgaris on different photobioreactor configurations. .................................................................................................. 43
Table 4.4: Steady state biomass concentration at different dilution rates in a tubular
photo-bioreactor ................................................................................................................. 46
Table 4.5: Biomass concentration at different mixing speeds in a CSTR ......................... 48
Table 4.6: Biomass concentration at different dilution rates in a CSTR ........................... 50
viii
List of Figures
Figure 2.1: Microscopic image of Chlorella vulgaris UTEX 2714 ..................................... 3
Figure 2.2: Various commercial applications of microalgae ............................................... 4
Figure 2.3: Carbon dioxide equilibrium in water (Becker, 1994) ........................................ 9
Figure 2.4: Microalgae batch growth profile ..................................................................... 10
Figure 2.5: Tubular photo-bioreactor diagram ................................................................... 15
Figure 2.6: Examples of CSTR configurations .................................................................. 16
Figure 3.1: 4L Flask Set-Up............................................................................................... 23
Figure 3.2: Tubular Photo-bioreactor Diagram ................................................................. 24
Figure 3.3: Diagram of Ring Sparger ................................................................................ 24
Figure 3.4: CSTR Diagram. ............................................................................................... 25
Figure 3.5. Light Set-Up on Tubular Photo-bioreactor...................................................... 27
Figure 3.6. Light Set-up On Stirred Tank Photo-bioreactor ............................................. 27
Figure 4.1: Biomass concentration of C. vulgaris in 4L flasks.......................................... 32
Figure 4.2: Change of pH with the addition of NaHCO3 ................................................... 33
Figure 4.3: Chlorella vulgaris growth in C30 (A) and PCS-2 (B) media. ......................... 34
Figure 4.4: Comparison of (A) specific growth rate and (B) doubling time. .................... 37
Figure 4.5: Comparison of change in biomass concentration of experiments with (A) CO2
enriched and (B) no added CO2 in air. ............................................................................... 38
Figure 4.6: Effect of mixing speed on Chlorella vulgaris cultivated in a CSTR .............. 41
ix
Figure 4.7: Turbulence inside the stirred tank reactor at different mixing (Kazim, 2012) 42
Figure 4.8: Semi-continuous cultivation and nutrient consumption of C. vulgaris in a
tubular photo-bioreactor. .................................................................................................... 44
Figure 4.9: Steady state behaviour of C. vulgaris. ............................................................. 45
Figure 4.10: Effect of Mixing Speed on Cell Growth and Biomass Concentration .......... 47
Figure 4.11: Effect of dilution rate on biomass concentration of Chlorella vulgaris in a
CSTR.................................................................................................................................. 49
Figure 4.12: Biomass concentration vs dilution rate in a CSTR........................................ 50
List of Appendices
Appendix A: Equipment and Materials ............................................................................. 63
Appendix B: Calibration Curve for CO2 ............................................................................ 65
Appendix C: Determination of Dry Weight and Biomass Concentration ......................... 66
Appendix D: Sample Calculations ..................................................................................... 67
xi
Nomenclature
D
Dilution rate
days-1
Volumetric flow rate
L/min
mV
Millivolt
,
Cell number, cell or biomass concentration
cells, g/L
,
Initial cell number, cell or biomass
cells, g/L
concentration

Rate of cell formation
cells/day
Time
days
Initial time
days
td
Doubling time
days
Volume
liters
Specific growth rate
d-1
Greek Letters
xii
Chapter 1
Introduction
The depletion of fossils fuel supplies and the contribution of these to the accumulation of
CO2 in the atmosphere make it a priority to find renewable, carbon neutral, transport fuels
for environmental and economic sustainability. Biodiesel is considered as a possible
substitute or extender of conventional diesel fuel, it is quite similar and compatible with
conventional diesel fuel in its main characteristics. Biodiesel derived from oil crops is a
potential renewable and carbon neutral alternative to petroleum fuels. Unfortunately,
biodiesel from oil crops, waste cooking oil and animal fat cannot realistically satisfy even
a small fraction of the existing demand for transport fuels (Chisti, 2007; GonzlezFernndez et al., 2012).
Microalgae are unicellular, photosynthetic microorganisms, they have minimal nutrient
requirements and are being used as source materials for a variety of products such as
protein rich nutritional supplements, pharmaceutical chemicals and pigments (used in
food and cosmetics) (Becker, 1994). They also grow extremely fast, in comparison to
plants and many microalgae strains are exceedingly rich in oil which makes them great
candidates for the production of biodiesel. Using microalgae to produce biodiesel will not
compromise production of human food, animal feed and other products as when oil crops
are used. There are many strains that have shown promise, specifically the strain
Chlorella vulgaris has shown a great promise as a source of oil. However, the high cost of
production in bioreactors still remains a limitation and much research is required. In this
study, the production of microalgae (Chlorella vulgaris) in 4L flasks, a tubular photobioreactor and a CSTR, in batch, semi-continuous and continuous operation was
investigated.
1.1
Research Objectives
Investigate the effect of nutrient media formulations on the growth of Chlorella
vulgaris
Investigate batch and continuous cultivation of Chlorella vulgaris in different

photo-bioreactor configurations.
Investigate the effect of different dilution rates on the biomass concentration of

Chlorella vulgaris when grown in a tubular photo-bioreactor.
Investigate the effect of different mixing speeds on the biomass concentration of

Chlorella vulgaris when grown in a Continuously Stirred Tank Reactor (CSTR).
Investigate the effect of different dilution rates on the biomass concentration of

Chlorella vulgaris when grown in a Continuously Stirred Tank Reactor (CSTR).
1.2
Thesis Overview
This thesis consists of five chapters. Chapter 1 contains a brief introduction to the
research topic. Chapter 2 contains a more detailed literature review relevant to the
research such as basic information of microalgae, their place and importance in the
modern world, photo-bioreactor technology that is currently used for their proper
cultivation, the pros and cons, and how the research of this thesis fits into current
research. In chapter 3, materials and methods listed including descriptions of the different
photo-bioreactor configurations and parameters monitored. In chapter 4, results obtained
are shown, analyzed and discussed. The final chapter (chapter 5) includes the conclusions
that can be drawn from the results obtained as well as recommendations for future work.
Chapter 2
Literature Revi
Review
2.1
Introduction on M
Microalgae
2.1.1
What are Microalgae
Microalgae are microscopic photosynthetic microorganisms. They are a type of

eukaryotic cell and they contain similar organelles such as chloroplasts, nucleus, etc.
Microalgae are generally more efficient than land based plants in utilizing sun light, CO2,
water and other nutrients which translate to higher biomass yields and higher growth
rates. They can also grow in a variety of aquatic environments and can be grown without
the
he use of fertilizers and pesticides which results in less waste and pollution (Gouveia and
SpringerLink, 2011; Richmond, 2004)
2004).
Figure 2.1 shows an image of Chlorella vulgaris UTEX 2714, which was used on the
study mentioned in this thesis. It has been found that this strain of microalga can vary in
diameter from 4-5m (Chandhok, 2010)
2010).
Figure 2.1:: Microscopic image of Chlorella vulgaris UTEX 2714
2.1.2
Current Uses of M
Microalgae
The are multiple areas in where microalgae are used,, the specifics will depend on the
needs. The whole algal biomass can be used as a source of protein or valuable chemicals
(pigments, enzymes) can be extracted. Several studies have shown the potential of
microalgae as a therapeutica
therapeutical agents, preparations containing Spirulina
ulina have shown to
help wound cicatrization, Scenedesmus- containing preparations have been tested and
shown effects on the treatment of certain skin diseases such as eczema and some algal
compounds have shown inhibitory effects on the HIV virus (Becker, 1994; Clement et al.,
1967; Gustafson et al., 1989). Microalgae are already used as a source of nutritional
supplements, as an additive for cosmetics, in the treatment of wastewater and as a
potential source of oil for biofuels (Aslan and Kapdan, 2006; Becker, 1994; Feng et al.,
2011; Gellenbeck, 2012) Figure 2.2 shows a summary of various commercial applications
of microalgae.
Figure 2.2: Various commercial applications of microalgae (Varfolomeev and

Wasserman, 2011).
2.1.2.1
Source of Oil for Biodiesel
Reduction of CO2 emissions is expedient but even a 30% reduction (as agreed by some
international legislations), is still not enough to stabilize the CO2 levels into a safe zone.
This requires the development of alternative biofuels, of which biodiesel and bioethanol
are the ones with the greatest market potential at the moment. Biodiesel is a popular
alternative to petroleum based diesel; it can be used in regular diesel engines. It is ecofriendly, non toxic, biodegradable and when burned, due to its low sulphur content, it
produces fewer emissions than its petroleum counterpart. It can be prepared from
renewable sources like edible and non edible vegetable oils, animal fats and even waste
cooking oils. The most common concern with first generation biofuels is that as
production capacities increase, so does their competition with agriculture for arable lands
used for food production. As an example, current biodiesel supplies from oil-production
crops, supplemented with small amounts of animal fat and waste cooking oil, only
account for an estimated 0.3% (approx. 12 million tons in 2007) current global oil
consumption and cannot even come close to satisfying the existing and future demand for
transport fuels. Currently approximately 8% of plant-based oil production is used as
biodiesel and this has already contributed to an increase in price of oil crops over the last
few years. Second generation biofuels (such as lignocellulosic and microalgal biofuel
systems) have the potential to overcome many of the limitations.(Bajpai and Tyagi, 2006;
Schenk et al., 2008; Srivastava and Prasad, 2000). Biodiesel made from different
vegetable oils has comparable properties as conventional diesels and can be used instead
or as blended fuels in diesels engines. (Lang et al., 2001)
In Table 2.1 it can be seen that some strains of microalgae are reported to produce 15-300
times more oil per area, for biodiesel production, than traditional oil crops. Microalgae
also have a shorter average harvesting cycle of around 10 days, traditional oil crops are
only harvested once or twice a year. The cost of biodiesel could be reduced if one
considers non-edible oils such as microalgae oil (Chisti, 2007; Schenk et al., 2008;
Srivastava and Prasad, 2000).
Table 2.1: Comparison of crop-dependent and algae biodiesel (Schenk et al., 2008).
Biodiesel
(L/ha/year)
Land needed for

cultivation
(ha x 106)
Global land
needed for
cultivation (%)
Global arable
land needed for
cultivation (%)
Soybean
446
10,932
73.4
551.6
Sunflower
952
5,121
34.4
254.4
Rapeseed/Canola
1,190
4,097
27.5
206.7
Jathropa
1,892
2,577
17.3
130 (0a)
5,950
819
5.5
41.3
Algae (10g m day at

30% TAG)
12,000
406
2.7
20.5 (0a)
Algae (50 g m-2 day-1 at

50% TAG)
98,500
49
0.3
2.5
Crop
Palm
-2
-1
If algal ponds and bioreactors are situated on non-arable land; jathropa is mainly grown on marginal land.
TAG: Triacylglycerides
Oil content in microalgae can vary widely from strain to strain. Chlorella protothecoides
has been found to produce 29.4% of lipids (dry basis), when growing in nature, and up to
55.2% in a study where it was grown autotrophically (Demirbas, 2009; Miao and Wu,
2006). Properties of microalgal oil indicate that its biodiesel has comparable
characteristics and might perform better, in cold temperatures, than biodiesel from
vegetable oils and animal fats (Demirbas, 2009; Miao and Wu, 2006). Different studies
have shown Chlorella to have promise as a source for oil in the production of biodiesel
(Ahn et al., 2012; Feng et al., 2012; Sirisansaneeyakul et al., 2011; Subramaniam et al.,
2010)
2.1.2.2
Wastewater Treatment
Microalgae have been successfully used as part of the treatment of different types of
wastewater, they can then help in the removal of pollutants including some carcinogenic
chemicals. In some cases pre-treatment maybe necessary since dark color and high fat
content of the wastewater has been found to inhibit the growth of microalgae but this
makes it possible to effectively use microalgae in the treatment of different animal farm
effluents (Heidari et al., 2011; Hodaifa et al., 2012; Muoz and Guieysse, 2006; Muoz et
al., 2005; Schenk et al., 2008) .
2.2
Factors Affecting Microalgal Growth
2.2.1
Nutrient Composition of Media
Media composition has an immense effect on both the growth rate and the final
concentration of microalgae. Microalgae are known to grow more abundantly in nutrientrich (eutrophic) waters leading frequently to algal blooms (Schenk et al., 2008). In a
comparison study of the growth of Chlorella sorokiniana, in TAP (Tris-AcetatePhosphate) media, with either 15g/L of NH4Cl or 1.5 g/L of NaNO3, it was found that the
maximum biomass concentration (4.4 g/L) was similar on both cases when using 5% CO2
enriched air but the pH decline was more drastic in the case of the original TAP
formulation (with NH4Cl) (Kumar and Das, 2012). (Feng et al., 2011) observed a
depletion of ammonium (as NH4Cl) in artificial wastewater within 3 days of culture. The
growth of Chlorella vulgaris was not limited, even after the depletion of ammonium,
which leads to confirm that microalgae utilized ammonium first and then the nitrate
present in the inoculum.
A change of the concentration of nitrates (as KNO3) in the culture medium, when
growing different strains of Chlorella, had little effect on the biomass concentration and
lipid production possibly due to the fact that the lowest concentration tested (1.24 g/L)
was already high enough to promote growth (Sirisansaneeyakul et al., 2011)
A study published in 1982 showed that relatively low concentrations of NaHCO3 (0.5M
or 4.2 g/L) and bubbling with CO2 enriched air (1.5%), favored the growth of Chlorella
vulgaris in a mixed culture with Spirulina platensis, this being independent of the pH,
which ranged between 8.5 and 9 (Richmond et al., 1982). Another study by (Zheng et al.,
2011), showed similar results where the growth of Chlorella vulgaris, with NaHCO3 at a
concentration of 40mmol/L (3.36 g/L), yielded 0.67 g/L of dried biomass. Addition of
NaHCO3 to Basal media, showed an increase of biomass production by Chlorella
vulgaris in a concentration range of 0.1-1.6 g/L, as the concentration of NaHCO3
increased so did the biomass production, reaching its maximum (0.6 g/L of biomass) at a
concentration of 1.2 g/L of NaHCO3 (Yeh et al., 2010). The fixation of dissolved CO2, in
the form of bicarbonate using an alkali NaOH, has shown to increase the biomass
production of Chlorella sp. in comparison to the addition of CO2 alone (Aishvarya et al.,
2012).
It has been noted that the lipid content of some microalgae increases with the
supplementation of NaHCO3 to the media at different concentrations and bubbling with
CO2 enriched air. Maximum growth rate and lipid production in the case of Chlorella was
seen at a NaHCO3 concentration of 75ppm (75 mg/L) and with a concentration of CO2 of
4758ppm (Devgoswami et al., 2011).
2.2.2
Light
Duration of light periods is an important criteria to be considered in the design of a photobioreactor set-up. Photoperiods have a significant effect on the photosynthetic activity
and growth rates in a typical microalgae cultivation system but it should be noted that
excessive light exposure can lead to waste of electricity and cell growth inhibition. With a
photoperiod of 12h nearly doubling the microalgae biomass concentration in comparison
with 3h, 6h, and 9h light periods (Lam and Lee, 2012). It has been found that growth rates
and maximum cell density of microalgae increase proportionally to light period duration,
even at different light intensities (Amini Khoeyi et al., 2012; Jacob-Lopes et al., 2009).
An exception to this was discovered with a 12:12h (light:dark) photoperiod, in which
higher productivities and maximum cell density values where obtained, in comparison
with the 14:10h (light:dark) (Jacob-Lopes et al., 2009)
2.2.3
pH
Initial culture pH can have a mayor on biomass concentration and lipid production of
some strains of microalgae, it can also cause the precipitation of some calcium salts if it
exceeds a value of 9.0 (Becker, 1994; Sirisansaneeyakul et al., 2011). (Mayo, 1997)
found that Chlorella vulgaris can tolerate a pH as low as 3.0. At the same time, high final
pH (pH=11) caused negative effects on the growth of another strain of Chlorella vulgaris
(Yeh et al., 2010).
Figure 2.3 shows the different forms of CO2 that can be available in water, the amount of
each form that can be found in a solution at any given time will depend greatly on the pH
of the solution. In a poorly buffered system, the assimilation of CO2 by the growing
microalgae causes a shift on the equilibrium shown on Figure 2.3 which results in an
increase of pH values due to the excretion of OH- by the algae into the media. It is
important to maintain pH within an adequate range to avoid the loss of carbon dioxide
present in the media (Becker, 1994).

Figure 2.3: Carbon dioxide equilibrium in water (Becker, 1994)
2.3
Microalgae Cultivation
Different approaches can be taken when looking to grow microalgae in large volumes.
Mainly outdoor ponds, with light supplied by the sun, and photo-bioreactors, which can
be outdoor or indoor and light can be supplied by electric lights. Species control is better
achieved under closed conditions that are very common in laboratory setting. Closed
systems provide a better opportunity meet specific demands and in the control and
optimization cell growth parameters. In the case of photo-bioreactors, many design
considerations need to be made depending on what the end goal is. There are basic design
features that should be considered regardless of the configuration; like the materials used
for the set-up, source of light, how will the algae circulate through the reactor, how to
provide CO2, and how to control other parameters such as pH, temperature and nutrient
concentration in the media (Andersen, 2005; Chisti, 2007). A study on Chlorella vulgaris
by (Lam and Lee, 2012) found that the most adequate growth was under indoor
conditions and utilizing inorganic nutrient media. There are various configurations that
have been built and tested for the growth of microalgae.
2.3.1
Microalgae Growth Kinetics
To estimate growth rates, one must have a series of measurements, at different times, that
will permit the calculation of the rate of change in biomass concentration. Cell number
should be counted, either through a direct method, as through light microscopy with a
hemacytometer, or indirectly through biomass concentration (as dry weight) or optical
10
density, as long as this measurements correlate linearly with the number of cells
(Andersen, 2005; Becker, 1994)
Under a typical homogenous batch regime (in a closed system), microalgae will pass
through the following growth phases, illustrated in Figure 2.4:
A. Adaptation (lag phase)
B. Exponential growth phase (log phase)
C. Stationary phase
D. Logarithmic death phase
C
BIOMASS
CONCENTRATION
D
B
TIME
Figure 2.4: Microalgae batch growth profile

The individual phases, shown in Figure 2.4, are not always clearly defined; their length or
slope might change according to the culture conditions. During lag phase (phase A) the
microalgae cells are adapting to the new environment conditions, at the end of the lag
phase, the cells are well adjusted and then start to multiply rapidly, this is the exponential
phase (phase B), in this phase, the number of living cells, doubles regularly with time
(Bailey and Ollis, 1977; Becker, 1994). During this period, equations (2.1) and (2.2)
describe cell growth.

or

(2.1)
11
or
(2.2)
Where and are the number of cells (or cell concentration) at different times and ,
where is the initial time and is the specific growth rate.
From the integrated form of equation (2.1) the following is obtained:

or

(2.3)

From equation (2.3) it can deduced that the time required for cell population to double
( ) is given by:

During the exponential phase
ln 2

(2.4)
or can be used to characterize the state of the cell
population.
In an open system, media with fresh nutrients is added in at the same rate that medium is
withdrawn from the culture; this keeps growth parameters and cell concentration at a
constant level. The following mass balance can be applied to any component of the
system:
!" #$ !
%%# # &'&"( !" #$ "(#*! $#( &'&"(
(2.5)
!" #$ +# ,-%# .%/% &'&"( 0
The above can be reformulated as:

$ $
1 0
(2.6)
Where $ is the volumetric flow rate, is the cell or biomass concentration in the feed stream,
is the cell concentration inside the system, 1 is the culture volume and is the rate of
cell formation. If it is assumed that the growth rate is much higher than cell death rate and
cell death is neglected, if denotes the specific growth rate ( ), then it can be
substituted in equation (2.6) to obtain the following (Bailey and Ollis, 1977):
12
3 3
(2.7)
The ratio of the volumetric flow rate, at which fresh medium is added to the culture, is
referred to as the dilution rate 3, so that:
3 $/1
(2.8)
When the liquid feed stream to a continuous culture consists only of nutrients, so that
0, meaning the algal cell concentration is constant in the system, equation (2.9) and
(2.10) are fulfilled (Becker, 1994).
0
3
2.3.2
(2.9)
(2.10)
Photo-bioreactor Design
There are many possible photbioreactor (PBR) configurations. Table 2.2 shows a
summary of research performed in various PBR configurations using different
microorganisms and obtaining a variety of results.
Tubular PBRs are the most popular configurations and can be arranged in several
different patterns (straight, bent, spirals) and in different orientations (horizontal, vertical,
inclined at different angles) (Wang et al., 2012).
13
Table 2.2: Summary of continuous and semi-continuous cultivation of microalgae

Author
(Feng et al., 2011)
(Su et al., 2012)
(Tang et al., 2012a)
Species
Chlorella vulgaris
Thermosynechococ
cus sp
Chlorella
minutissima,
(Hulatt and
Thomas, 2011)
Scenedesmus
(Henrard et al.,
2011)
(Wang et al.,
2010a)
Cyanobium sp.
Chlorella vulgaris
obliquus
D. tertiolecta
Type
Artificial
Wastewater
Media
Media Supplement
Glucose , NH4Cl
cyanobacterium
freshwater
freshwater
Fitzgerald media
Modified Bold 3N
Jaworski
15% CO2
3 x Reg. NaNO3
1
-1
1.7g L d
Biomass
Productivity
Biomass
Concentration (g/L)
1.72 (batch)
Tubular
Light Source
30
Temp. (C)
36
500
1.8
0.726
2.5-3.5
1.2
1.38
CSTR
Fence + Tank
Tubular
4L flasks
Circular LED
Sunlight
-1
25
4
% CO2
Growth Rate (d )
37%
Fluorescent lamp
30
to control pH
0.03-0.23
-1
Lipid productivity/
Diluted Manure
0.28-.89 (cont.)
tubular
PBR Design
BG11
0.4 -1.6 g/L
NaHCO3
absorbed by NaOH
solution
Working Vol. (L)
freshwater
252
2 (for pH control)
0.127
13.5 % dry weight
content
Dilution Rate
pH
0.5-1.5
1.4 d-1
0.076-0.64 d-1
Not specified
8-10
10-11.5
7.8 (initial)
7.0 0.2
Not constant
0.05-0.3 d-1
14
2.3.3
Tubular Photo-bioreactors
Tubular photo-bioreactors with shallow light paths are commonly used to grow
photosynthetic microorganisms; it is possible to reduce their diameter to allow for a
higher surface-volume ratio. Air agitated bubble columns and airlift bioreactors have been
studied for the culture of large amounts of microalgae but there are restrictions in height
since it could negatively affect gas transfer and increase residence time of O2 generated
during photosynthesis, which can lead to cell growth inhibition. Mechanical agitation in
photo-bioreactors is known to enhance radial mixing but it can cause damage to the algal
cells, therefore, air bubbling from the bottom of the column (which is common in tubular
PBR) is preferred since it enables efficient CO2 utilization, optimal O2 removal and it is
gentle enough to avoid shear stress damage to the cells even a high superficial gas
velocities (Sobczuk et al., 2006; Snchez Mirn et al., 2002; Vega-Estrada et al., 2005;
Xu et al., 2009). Besides excellent mixing, tubular photo-bioreactors are simple and very
easy to clean (Wang et al., 2012)
A typical vertical column or tubular PBR (Figure 2.5 A) consists of a transparent column
with air inlet at the bottom, usually there is a sparger to allow for smaller an multiple
bubble formation for better mixing of the culture, in some cases there is an added internal
column which creates a airlift photo-bioreactor (Figure 2.5 B). Both configurations have
been extensively studied for the production of microalgae (Garca Camacho et al., 1999;
Krichnavaruk et al., 2007; Kumar and Das, 2012; Wang et al., 2012).
15
Figure 2.5: Tubular photo-bioreactor diagram
2.3.4
Continuously Stirred Tank Reactors
Stirred-tank reactors, being operationally and structurally simple, are ideal devices for
cultivating different types of cells, including microalgae (Yang and Wang, 1992). CSTR
can be beneficial in preliminary studies for ideal growth conditions and determining
different parameters, they are easy to model and systems are available that could easily
monitor and control the main experimental parameters. Whether it is a batch or
continuous run, a continuously stirred tank reactor can come in different capacities and
different configurations (Muoz et al., 2005). On Figure 2.6, two examples can be seen,
example (A) shows a simple flask on top of a stirring plate where the stirring is done by a
magnetic bar, example (B) shows a more complex reactor, where the stirring is done by
impellers and bubbling air. There are many parameters that could be considered when
doing an experiment on a CSTR, one of possible importance in microalgae cultivation is
the mixing speed.
16
Figure 2.6: Examples of CSTR configurations
2.3.4.1
Effect of mixing speed
It is important to know the effect of the agitation speed on microalgae, if too high it could
not only be impairing cell growth but it could be damaging in other ways as by causing
the leakage of important chemicals from within the cell (Funahashi et al., 1999; Ohira et
al., 2012; Sobczuk et al., 2006)
CSTR are used to grow microalgae at different mixing speeds within the wide range of
100 1,200 rpm. The maximum mixing speed that microalgae will tolerate without cell
damage varies and is very dependent on the species (Crdoba-Castro et al., 2012;
Funahashi et al., 1999; Hodaifa et al., 2012; Mazzuca Sobczuk and Chisti, 2010; Mitra et
al., 2012; Sirisansaneeyakul et al., 2011; Sobczuk et al., 2006). In a study of Chlorella sp.
it was found that chlorophyll was more efficiently produced at a mixing speed of 250 rpm
(Funahashi et al., 1999) . But again, the ideal mixing speed will vary according to species
and the chemical that is being investigated.
Both CSTR and tubular photo-bioreactors, have shown similar results when used to grow
microalgae for different purposes (Muoz et al., 2005).
17
2.3.5
Batch and Continuous Cultivation
A batch reactor is a closed system with respect to exchange of matter with its
environment, while a continuous reactor is an open system with mass exchange across its
boundaries (Bailey and Ollis, 1977)
As mentioned in a previous section, the steady-state concentration of cells in a continuous
culture can be determined by the dilution rate, this is a correct assumption when the are
cultures that can grow to high densities (Andersen, 2005)
(McGinn et al., 2012) found that
the biomass productivity, of the microalgae
Scenedesmus sp., was two times greater when growing in a continuous chemostat than in
batch cultivation, with the productivity in the latter being 130 mg L-1 d-1. This is the
general case for various microalgae species grown in continuous or semi-continuous
systems, they tend to show greater productivity when grown continuously or semicontinuously versus when grown in batch (Cern-Garca et al., 2012; Filali et al., 2011;
Sun et al., 2009; Tang et al., 2012b; Wang et al., 2010b)
2.3.5.1
Effect of Dilution Rate
Generally, increasing the dilution rate of a system will lead to an increase of biomass
concentration and cell productivity. (Tang et al., 2012b) found that the productivity of
Chlorella minutissima varied from 39 to 137 mg/L/day (dry mass) as the dilution rate
increased from 0.08 to 0.64 d-1, with the maximum productivity reached at the dilution
rate of 0.33 d-1. In contrast, a study where the microalga Choricystis minor was grown
continuously, found that it could reach a maximum biomass concentration of 2.02 g/L at
the dilution rate of 0.005 h-1 (at 25 C), although this is almost ten times lower a growth
rate than the maximum specific growth rate of 0.046 h-1 reached during the batch study,
the maximum biomass concentration reached in the latter case was only 0.135 g/L
(Mazzuca Sobczuk and Chisti, 2010).
18
2.4
Importance of This Study
Microalgae are already widely used in many commercial areas; they show great potential
as a source for different types of biofuels without competing with food sources and are
easy to grow in photo-bioreactors. Various microalgae strains within the genus Chlorella,
including Chlorella vulgaris, have shown promise in treating waste water, as a source for
nutritional supplements, used in cosmetics and as a source for oil for the production of
biodiesel. Photo-bioreactors provide an easy way to grow microalgae in large volume and
they come in different shapes, sizes and configurations, each of which have their own
advantages and disadvantages. Tubular and continuously stirred tank reactors are choice
photo-bioreactors designs for studying and optimizing the different parameters that affect
microalgae growth. With few additions, any photo-bioreactor can be converted to run
continuously. Continuous cultures in photo-bioreactors have shown to improve overall
biomass production of microalgae hence the importance of studying, not only their
growth in different photo-bioreactor configurations but also the effect that dilution rates
can have in a microalgae system.
19
Chapter 3
Materials and Methods
3.1
Microalgae Strain
The microalga Chlorella vulgaris UTEX 2714 was used for this study. The strain was
obtained through the Culture Collection of Algae from the University of Texas at Austin.
3.2
Growth Media
3.2.1
Bolds Basal Media
A modified version of Bolds Basal Medium, as reported by (Andersen, 2005), the

formulation shown in Table 3.1, was used in most of the experiments. It contains macro
and micro nutrients necessary for the growth of microalgae.
The stocks solutions were prepared as needed and stored in the fridge until final media
preparation.
20
Table 3.1: Modified Bolds Basal Media (Andersen, 2005).

Component
Macronutrients
NaNO3
CaCl22H2O
MgSO47H2O
K2HPO4
KH2PO4
NaCl
Alkaline EDTA Solution
EDTA
KOH
Acidified Iron Solution
FeSO47H2O
H2SO4
Boron Solution
H3BO3
Trace Metal Solution
ZnSO47H2O
MnCl24H2O
CuSO45H2O
3.2.1.1
Stock Solution
(g/L)
Quantity Used
(ml)
25.00
2.50
7.50
7.50
17.50
2.50
10
10
10
10
10
10
1
50.00
31.00
1
4.98
1
1
11.42
1
8.82
1.44
1.57
Addition of NaHCO3
NaHCO3 was added to the regular Bolds media formulation. The solubility of NaHCO3
in water is very low, 9.6g/100ml H2O (Seidell, 1907). NaHCO3 would also form
precipitates if autoclaved with the other media ingredients, therefore it was added after
the media was autoclaved, it was sterilized with a PALL, 0.45 Acrodisc syringe filter.
21
3.2.2
Other Media Formulation
The different media formulations used in experiments are listed in Table 3.2.
Table 3.2: Other Media Formulations (Perez-Garcia et al., 2011).
Components*
Macronutrients
Proteose Peptone
NH4Cl
KNO3
NaNO3
CaCl22H2O
MgSO47H2O
K2HPO4
KH2PO4
NaCl
FeSO47H2O
FeEDTA
Alkaline EDTA Solution
Na2EDTA
KOH
Acidified Iron Solution
FeSO47H2O
H2SO4
Boron Solution
H3BO3
Trace Metal Solution
H3BO3
Na2MoO4
ZnSO47H2O
MnCl24H2O
CuSO45H2O
C30
Proteose
PCS-2
------5g
------2.5g
100mg
1.25g
---2.8mg
----
1g
------250mg
25mg
75mg
75mg
175mg
25mg
-------
---200mg
---250mg
25mg
75mg
75mg
175mg
25mg
-------
-------
-------
63.9mg
31mg
-------
-------
4.98mg
1ml
----
----
11.42mg
2.68mg
0.09mg
0.11mg
1.81mg
0.021mg
2.68mg
0.09mg
0.11mg
1.81mg
0.021mg
------8.82mg
1.44mg
1.57mg
22
3.3
Inoculum and Strain Maintenance
Previous to every photo-bioreactor run, a separate inoculum was prepared. The inoculum
set-up consisted of 4L flasks with a 3L working volume. The growth media was either
Bolds or a combination of Bolds and Proteose media, depending on the experiment. Air
was supplied by either foam stoppers, which allowed gas exchange in small volume flasks
(50-250ml), or through a fish tank air pump (Top Fin 4000, PetSmart, Canada) and stone
sparger for larger volume flasks (1-4L). Light was provided by placing the flasks by a
sunny window.
Strain maintenance was done only in small culture volumes (25-100ml), Bolds or a
combination of Bolds and Proteose media was used, air exchange was allowed through
foam stoppers and flasks were placed away from direct sunlight, to slow down cell
growth.
3.4
Photo-bioreactor Set-up
Three different photo-bioreactors configurations were used for the experiments with
Chlorella vulgaris. These configurations were 4 liter conical flasks, a tubular and a
continuously stirred tank reactor (CSTR)
3.4.1
4 L Conical Flasks
Figure 3.1 shows the set-up for 4L flasks with a working volume of no more than 3.5 L.
The flasks were positioned by a window to provide sunlight. Culture samples were taken
through the sampling port with a sterile syringe. Air was supplied through a fish tank air
pump (Top Fin 4000, PetSmart, Canada). This configuration was easy and quick to setup, and more than one experiment could be run at the same time.
23
(1) Stone sparger, (2) Air filter, (3) Sampling port, (4) Air exhaust
Figure 3.1: 4L Flask Set-Up.
3.4.2
Tubular Photo-bioreactor
The general diagram for the tubular photo-bioreactor is shown on Figure 3.2. The reactor
consisted of tubular sections of different lengths made of plexiglass. The height of the
reactor varied from, depending on the working volume, all set-ups had an inside diameter
of 0.1 m and a wall thickness of 1.0x10-2 m. Pictures of the different volume set ups can
be found on Appendix A.
24
Figure 3.2: Tubular Photo-bioreactor Diagram
3.4.2.1
Sparger
A ring sparger (Figure 3.3) was used on the tubular photo-bioreactor experiments. The
O.D and I.D. were 0.09 m and 0.06 m respectively, with aeration holes measuring
0.15x10-2 m in diameter.
Figure 3.3: Diagram of Ring Sparger
3.4.2.2
Batch and Semi-Continuous Experiments
For samples taken during the batch stage of the experiment (before steady state was
reached) a sampling device consisting of a pipette attached to a syringe through a piece of
25
tubing was used, it allowed for easy sampling through the top of the bioreactor , a picture
can be found in Appendix A.
For all different dilution rates, a set volume of culture was taken out of the PBR, every
two days, directly from a valve at the bottom of the column.
Temperature was measured directly from the culture in the photo-bioreactor with a digithermo digital thermometer (VWR International, Canada), all other analysis were done
from samples removed from the photo-bioreactor.
3.4.3
Continuously Stirred Tank (CSTR) Photo-bioreactor
A Bioflo 3000 fermentor (Eppendorf New Brunswick, Mississauga, ON) was used to
study the effects of mixing speed in microalgae growth. Figure 3.4 shows a diagram of
the CSTR. Maximum capacity was of 14L with a maximum working volume of 10L.
During continuous runs ports (1) and (3), as shown in Figure 3.4, were used for inlet of
media and outlet of culture respectively.
Source: http://www.igz.ch/puebersicht.asp?action=download&fileid=5700
Figure 3.4: CSTR Diagram.

(1) Media inlet, (2) 6-blade Rushton impeller, (3) Culture harvest tube, (4) Ring
sparger
26
3.4.3.1
Monitoring of Parameters
Track & Trend Biocommand (Eppendorf New Brunswick Scientific, Mississauga, ON)
was used to track dissolved oxygen, pH, mixing speed and temperature. The settings of
the software were arranged so that readings were taken every minute for the first hour, at
the start of every experiment, and then every hour after that. The data collected was
extracted every few days and transferred to an excel file.
3.4.3.2
Batch and Continuous Experiments
Samples were taken every 1-2 days during the batch period For the continuous runs on
the CSTR, two low flow micro pumps (VWR International, Canada) were utilized to
maintain a steady state. They were calibrated and adjusted as needed to obtain different
dilution rates.
3.4.4
Air Supply
Pressurized air and CO2 were supplied, mixed and regulated with a custom made multi
tube rotameter (OMEGA Engineering, Quebec, Canada) for all tubular and stirred tank
photo-bioreactor experiments.
3.4.5
Lighting
There was different lighting for the different photo-bioreactor settings. In all cases, light
intensity was measured with a light meter (VWR International, Canada).
3.4.5.1
Tubular PBR
The tubular photo-bioreactor was situated by a large window therefore light was provided
by the sun but supplemented with two tubular fluorescent lamps (Illume, Quebec,
Canada), regular fluorescent light bulbs were replaced with 21watt daylight light bulbs,
which simulated the glow of natural sun light. Light intensity readings were taken on
points 1, 2 and 3 (as shown in Figure 3.5), on the outside surface of the photobiareactor.
The lamps operated to provide a 12hr light supplementation to simulate the regular
night/day light cycle.
27
Figure 3.5. Light Set-Up on Tubular Photo-bioreactor
3.4.5.2
CSTR
No direct sunlight was provided in the case of the CSTR. Four 12.5in, 8watts fluorescent
lights (Bazz, Quebec, Canada) were placed, in pairs, on both sides of the photo-bioreactor
as shown in Figure 3.6. The lamps operated to provide a 12hr light supplementation to
simulate the regular night/day light cycle. Light intensity readings were taken by the
outside surface of the photo-bioreactor, directly in front of where each pair of lamps were
placed.
Figure 3.6. Light Set-up On Stirred Tank Photo-bioreactor
28
3.5
Sampling of Photo-bioreactor
For sampling in the case of all batch and semi-continuous runs, first, previously sterilized
water was added to make up for any evaporation loss, the PBR was left undisturbed for
about 5 to 10 minutes, then the sample was taken.
3.6
Analysis of Samples
3.6.1
Cell Count
Cell count and cell condition were determined through light microscopy (Leica,
Germany) with an improved Neubauer phase hemacytometer (Hausser Scientific, U.S.), a
diagram of the hemacytometer counting grid is presented in Appendix A.
3.6.2
pH
Measured in every sample with a pH meter (Beckman Coulter Inc., U.S.). Except for the
CSTR runs where it was measured in-line. If it pH was out of range, it was adjusted with
either a 1N HCl, 5N HCl or a 5N NaOH solution.
3.7
Determination of Dry Weight and Biomass

Concentration
To determine the dry weight of every sample would not have been efficient, therefore, the
calibration curve, show in Appendix C, was created using the following procedure:
1. 2 samples of 50ml of Chlorella vulgaris culture were measured
2. The first sample was filtered through a 42.5mm round GF/C glass microfiber
(Whatman, U.K.), dried in an oven at 105 C for 24-48hrs until the weight of the
filter was unchanged, and weighted to obtain the dry weight of the sample.
3. The second sample was used for two-fold serial dilution, absorbance at 686nm
was taken.
29
4. The theoretical weight from the serial dilutions of the second sample were
correlated to their absorbance reading. The resulting equation was used for
calculating the theoretical biomass concentration of experimental samples.
3.8
Determination of CO2 Concentration
An ion selective carbon dioxide electrode (Model no: ISE-8750, Omega, Laval, Quebec)
was used to measure the concentration of dissolved carbon dioxide during experiments.
The CO2 electrode was not part of the Bioflo 3000 bioreactor original set-up, it was
incorporated through a specially designed holder that fit into one of the bioreactors ports.
The CO2 electrode was connected to a pH meter (Accumet XL-50, Fisher Scientific,
Ottawa, ON), which at the same time was connected to a computer that recorded and
stored all reading through the Accumet XLComm (Fisher Scientific, Ottawa, ON)
software.
When dissolved carbon dioxide passed through the membrane, it entered the probe in the
form of carbonic acid (H2CO3), which brought a change in the H+ ion concentration in the
refill solution of the electrode. This change in the H+ ion concentration was interpreted as
potential difference and the reading was displayed on the pH meter in millivolts. Higher
mV values meant higher concentrations of dissolved carbon dioxide in the sample.
3.8.1.1
Checking Slope of CO2 Probe
The slope of the carbon dioxide probe assured the proper operation of the probe. In order
to check the slope of the electrode, 5 ml of a sodium citrate buffer solution were added to
45 ml of distilled water in a 100 ml beaker. The solution was stirred and 0.5 ml of
standard carbon dioxide solution (1000 ppm as CO2) was added. As soon as the probe
was immersed inside the solution the negative mV values begin to decrease in magnitude.
Once the mV value stabilized, it was zeroed. Afterwards, 5 ml of the standard carbon
dioxide solution was added in the increments of 1 ml, as soon as the mV value was
stabilized it was determined as the slope of the probe. The purpose of using 0.5ml and
5ml of the same solution was to obtain the change in mV when the CO2 concentration in
the sample increased ten-fold. A slope 30-62mV assured proper operation of the probe.
30
3.8.1.2
Calibration Curve for CO2 Probe
A calibration curve for the carbon dioxide probe was created before the start of every
CSTR experiment. Three standardized carbon dioxide solutions (10, 100 and 1000 ppm
CO2) were used. The reading of the 100ppm solution was used as a zero and a logarithmic
graph, correlating the potential difference (mV) vs. Concentration (ppm as CO2), was
created and the equation generated was used to convert the mV readings into ppm of CO2.
The equation and graphs are shown on Appendix B.
3.9
Nutrient Analysis
For nutrient analysis, an aliquot of no less than 7ml was taken and filtered through a
0.45 syringe filter (Acrodisc, PALL Corporation, Cornwall, UK) and frozen at -20C for
later analysis. At the day of nutrient analysis, samples were thawed out and allowed to
reach room temperature. A DR 2800 spectrophotometer (HACH Company, Colorado,
U.S.) and appropriate test kits were used for the analysis of nutrients (nitrates and
phosphates), the methodology listed for each kit was followed.
3.9.1
Nitrates
TNTplus 836 vials (HACH Company, Colorado, U.S.) high range (5-35 mg/L NO3-N or
22-155 mg/L NO3) and TestN Tube vials (HACH Company, Colorado, U.S.) high range
(0.2 -30 mg/L NO3-N or 0.9-133 mg/L NO3), were used. Maximum theoretical and
expected nitrate concentration was calculated and the filtered sample was diluted as
needed. Method 10206 and method 10020 of the DR2800 spectrophotometer procedures
manual was followed.
3.9.2
Phosphates
TestN Tube vials (HACH Company, Colorado, U.S.) for reactive phosphorus
(orthophosphate), high range (1-100 mg/L PO4-3), were used to analyze the concentration
of phosphates in the filtered sample. Maximum theoretical and expected phosphate
concentration was calculated and the filtered sample was diluted as needed. Method 8114
of the DR2800 spectrophotometer procedures manual was followed.
31
Chapter 4
Results and Discussions
4.1
Batch Cultivation in 4L Flasks
First, the growth of Chlorella vulgaris in regular Bolds media and in Bolds media with
added sodium bicarbonate at a concentration of 1.2 g/L was investigated. The total
working volume for each culture was 3.5L with 20% of that being inoculum. The reason
for this experiment was to confirm a previously reported study on the effect of NaHCO3
on the growth of newly isolated strain of Chlorella vulgaris, this study showed a
beneficial effect with a concentration of 1.2 g/L of NaHCO3 in an artificial wastewater
media (Yeh et al., 2010). It was hypothesized that the addition of NaHCO3 may serve as
an additional supply of carbon for the microalgae hence eliminating the need to supply
the air with gaseous CO2.
4.1.1
Modified Bolds Media with NaHCO3
It can be observed in Figure 4.1, that both cultures have a close biomass concentration
during the first couple of days, the culture with added NaHCO3 grows more rapidly at the
beginning but then its growth decreased as the controls (Bolds media) continued to
increase. At the end of the experiment, the controls biomass concentration (0.23 g/L) was
higher than the one for Bolds media with added NaHCO3 (0.15 g/L). The culture with
NaHCO3 presented an initial growth rate of 0.345 d-1 (td = 2.01 d) but as mentioned
before, growth decreases at around the seventh day and even biomass concentration
decreases, the control had a lower but steadier growth rate of 0.125 d-1 (td = 5.53 d). High
flocculation of the microalgae was observed in the culture with added NaHCO3.
32
0.30
Biomass Concentration (g/L)
0.25
0.20
0.15
0.10
Control
NaHCO3
0.05
0.00
0
10
12
14
16
Days
Note: Error bars represent standard deviation from the average biomass.
Figure 4.1: Biomass concentration of C. vulgaris in 4L flasks.

Bolds media (Control) vs. Bolds with added NaHCO3 (1.2 g/L)
Figure 4.2 shows that the pH increased drastically in the culture with 1.2g/L NaHCO3,
which reaches a value above 10.0, the pH of the control culture did not go above a value
of 8.0 throughout the whole experiment. The pH of the NaHCO3 culture was way above
the range which most fresh water microalgae prefer (5-7), the pH values reached also
contradict the expected effect of added bicarbonate ions acting as buffers (Andersen,
2005). The decrease in growth rate and the flocculation of cells observed could be due to
the change in pH. Generally, pH is expected to increase due to depletion of NO3- anions
from the media and if it reaches values above 9, it can cause precipitation of various salts
which can lead to nutrient deficiency for the microalgae (Becker, 1994). The study done
by (Yeh et al., 2010) did find a positive effect of NaHCO3 on Chlorella vulgaris growth
but they also found that higher concentrations of NaHCO3 caused a higher final pH
(pH=11) which resulted in a detrimental effect on biomass concentration. It is possible
that the strain used in our study has different sensitivity levels to NaHCO3 than the one
used in the study by (Yeh et al., 2010).
33
11
10
pH
Control
NaHCO3
6
0
10
12
14
16
Days
Figure 4.2: Change of pH with the addition of NaHCO3
4.2
Batch Cultivation in Tubular PBR.
4.2.1
Batch Cultivation in C30 and PCS-2 Media
The tubular PBR configuration previously mentioned and shown in Appendix A was
used. For the experiment with C30 media, the total working volume was of 3.5L with an
inoculum concentration of 8.7%. The reactors were placed by the window so sunlight
was the main light source and the air was enriched with 5% CO2 (v/v). C30 media has
been used to grow Chlorella sp (Perez-Garcia et al., 2011). It was expected that Chlorella
vulgaris would grow well in both C30 and PCS-2 media. In the case of C30 media,
because of the relatively high nitrate content (5g/L of KNO3) and the enriched air with
5% CO2 (v/v), since it has been proven that nitrates is a preferred form of nitrogen by
microalgae and appear to be significant for adequate growth (Lam and Lee, 2012; Li et
al., 2008). The results with PCS-2 media were also expected to be quite favorable due to
the similar formulation as Bolds media and with added source of nitrogen as ammonium
chloride (200mg/L of NH4Cl) since microalgae seem to prefer nitrogen in the form of
ammonia and have also been successfully used to remove ammonia from artificial an real
34
wastewater (Aslan and Kapdan, 2006; Chandhok, 2010; Gonzlez-Fernndez et al., 2012;
Park and Craggs, 2011; Xiao et al., 2011). There was an initial increase of the cell number
but after just a couple days, the count steadily decreased (Figure 4.3, A). The experiment
was stopped after 7 days, with an overall negative growth rate. It appears that C30 was
not an appropriate media for this strain of Chlorella vulgaris.
0.13
0.7
Biom ass Concentration (g/L)
0.6
0.12
0.11
0.10
0.5
0.4
0.3
0.2
PCS-2
Bold's
0.1
0.0
0.09
0
Days
(A)
10
12
14
Days
(B)
Figure 4.3: Chlorella vulgaris growth in C30 (A) and PCS-2 (B) media.
The stunted cell growth in C30 media could be due to different reasons, inhibition by CO2
is quite possible, it has been found that CO2 inhibition levels can vary depending on the
microalgae specie (Lee and Hing, 1989; Rados et al., 1975) and (Westerhoff et al., 2010)
has found that Chlorella sp. and Scenedesmus sp., from a Sonoran desert mineral spring
(Mexico), can tolerate a CO2 concentration of up to 20% (in gas). It has also been found
that the addition of iron, in the form of FeCl3/EDTA, increased the final cell density and
lipid production, in comparison with the control (no iron added), of Chlorella vulgaris
cultures (Liu et al., 2008), so in the case of C30 media, the source of iron (FeSO47H2O)
could be not as biologically available for the algae, due to the lack of a chelator as EDTA,
or the concentration could be too low. The relatively low inoculum concentration, the
stage of growth the inoculum was in, could have also been a factor affecting the growth
(Mattos et al., 2012). No real improvement was noticed with PCS-2 media when
compared even with the slowest growing microalgae experiment in Bolds media (B6)
which contained only nitrate as a source of nitrogen, a comparison of the change in
35
biomass concentration with time can be seen in Figure 4.3, B. In both the C30 and PCS-2
experiments a lower average pH value can be seen, at 5.54 and 5.33 respectively, than the
Bolds media experiment values (pH=6.0-7.5). (Mayo, 1997) found that Chlorella
vulgaris can tolerate a pH as low as 3.0 but biomass and lipid productivities in Chlorella
sp. and other microalgae have been found to be higher around pH values of 7.0
(Moheimani, 2012; Yu et al., 2011).
4.2.2
Batch Cultivation in Bolds Media
Multiple experiments were conducted, in the tubular photo-bioreactor set-up, with various
experimental specifications. Experiments with Bolds media in a tubular PBR were
labeled from B1 to B6. The specifications and a compilation of results are shown in
Figure 4.5, Figure 4.6 and Table 4.1. Experiment B1, B2 and B3 all were aerated, at the
same flow rate, with CO2 enriched air, experiment B1 was performed in April (early
spring), experiment B2 and B3 were performed in June and July (late spring/summer)
respectively. Experiment B2 and B3 had a higher inoculum percentage than exp. B1but
exp. B1 had a higher initial biomass concentration. Experiments B4, B5 and B6 were
supplied with ambient air, a similar flow rates, with no additional CO2. In Figure 4.5 it
can be observed that experiments aerated with CO2 enriched air reached their maximum
biomass concentration after 6-9 days of the start of the experiments with the highest
biomass concentration at 0.547 g/L for experiment B1. Experiments with no added CO2
reached their maximum biomass concentration after 14-16 days, the highest biomass
concentration was 0.717 g/L for experiment B6.
Despite all the differences in experimental conditions, experiments B1, B2 and B3 all had
very close specific growth rates () values, from 0.29 d-1 to 0.32 d-1, which are higher
than those values for experiments B4, B5 and B6 (from 0.15 d-1 to 0.2 d-1) which were
not supplemented with CO2 enriched air. This results are in line with what is normally
seen an expected in experiments where photosynthetic organisms are involved, since the
normal concentration of CO2 in air is very low at around 0.033% (Andersen, 2005), it is
commonly seen that in microalgae cultures, that have an added source of carbon (organic
or inorganic) there will be a positive effect on cell growth (Gonzlez-Lpez et al., 2012;
Westerhoff et al., 2010; Yeh et al., 2010). The lower specific growth rate within the
36
tubular photo-bioreactor experiments with Bolds media, was that of experiment B6, this
could possibly be due to inhibition by longer light periods usual in the time of year when
the experiment was performed, it has been found that different light/dark periods do have
an effect on microalgae growth, nutrient removal and oil productivity (Amini Khoeyi et
al., 2012; Jacob-Lopes et al., 2009; Lee and Lee, 2001)
Table 4.1: Comparison of Chlorella vulgaris growth in Tubular PBR with Modified
Bolds Media
Experiment Number
B1
B2
B3
B4
B5
B6
Avg. Temperature (C)
21.8
21.8
21.0
22.3
23
24.1
Avg. pH
6.00
6.10
6.54
7.54
7.40
7.30
Avg. light intensity (lux)
2680
2350
1860
---
4160
3800
Month of exp.
Jun
Jul
Sep
Jul
Jan
May
Working Volume (L)
3.7
6.3
6.0
16.8
10
10
Inoculum Concentration (%)
20
38
30
21
20
20
Additional Light Source
No
Yes
Yes
No
Yes
Yes
% CO2
2.5
No
No
No
1.5
1.5
Specific Growth Rate, (d )
0.307
0.298
0.32
0.202
0.233
0.154
Doubling time (days)
2.26
2.32
2.17
3.42
2.98
4.50
Max. Biomass Conc. (g/l)
0.547
0.444
0.445
0.680
0.550
0.717
Air Flow Rate (L/min)

-1
37
0.35
5
CO2
CO2
0.30
0.25
0.20
0.15
0.10
No CO2
Doubling Time (days)
Specific Growth Rate ()
No CO2
1
0.05
0.00
0
B1
B2
B3
B4
Experiment
(A)
B5
B6
B1
B2
B3
B4
Experiment
(B)
*See Table 4.1 for more details.
Figure 4.4: Comparison of (A) specific growth rate and (B) doubling time.
For experiments performed in a tubular photo-bioreactor with Bolds media.
B5
B6
38
0.8
CO2
0.4
0.5
0.3
0.2
Exp. B1
Exp. B2
Exp. B3
0.1
No CO2
0.6
0.4
Exp. B4
Exp. B5
Exp. B6
0.2
0.0
0.0
0
10
Days
Days
(A)
(B)
10
12
14
16
*See Table 4.1 for more details.
Figure 4.5: Comparison of change in biomass concentration of experiments with (A) CO2 enriched and (B) no added
CO2 in air. For experiments performed in a tubular photo-bioreactor with Bolds media.
39
4.3
Batch Cultivation in Stirred Tank Photo-bioreactor.
Bolds media was used for all experiments performed in the CSTR, temperature and pH
were continuously monitored, not only during samples as was the case with the other
experiments. The purpose of the first experiment was to test the effect in biomass
concentration when changing the mixing speed after biomass concentration had reached
steady state. The purpose of the second experiment was to use the best mixing speed
found through the first experiment and test different flow rates. Figure 4.6 and Table 4.2
shows the results and parameters for the exponential growth phase of both previously
mentioned experiments; all values are very close except for the mixing speed and the
resulting specific growth rates and maximum biomass concentration. Both experiments
were run for around 17-18 days.
The specific growth rate was higher, at a value of 0.205 d-1, when the culture was mixed
at 350rpm than when mixed at 150rpm (=0.123 d-1). It is clear that the higher mixing
speed had a positive effect on the growth rate and final biomass concentration. These
results are consistent with what is reported in literature for microorganisms grown in
CSTR, increase of mixing speed improves overall cell growth and productivity (AguirreEzkauriatza et al., 2008; Fenice et al., 2012; Funahashi et al., 1999; Hellebust et al.,
1973). Not much has been researched on the effect of mixing speed on microalgae but
there is a study the effect of mixing speed on two strains of microalgae, Phaedactylum
tricornutum and Porphyridium cruentum, as the rpm was increased during steady state so
did the biomass concentration up to 350rpm and 550rpm for P. tricornutum and P.
cruentum respectively (Sobczuk et al., 2006).
40
Table 4.2: Parameters and results of CSTR experiments.

Chlorella vulgaris in Bolds media.
Experiment (rpm)
CSTR1
(150)
CSTR2
(350)
25
25
Avg. pH
6.62
6.61
7150
7300
Month of exp.
Aug
Oct
Working Volume (L)
10
10
20
20
Yes
Yes
235
148
Specific Growth Rate, (d-1)
0.123
0.205
5.63
3.39
0.470
0.590
% CO2
Avg. CO2 conc. in culture (ppm)
41
0.7
0.6
0.5
0.4
0.3
0.2
150 rpm
350rpm
0.1
0.0
0
10
15
Days
Note: Error bars represent standard deviation from the average biomass concentration in steady state
conditions
Figure 4.6: Effect of mixing speed on Chlorella vulgaris cultivated in a CSTR

Since the flowrate of air remains constant within the two experiments, the positive
changes in biomass concentration are due to the effects of a higher mixing speed which
led to the CO2 in the air being more readily available to microalgae. In Figure 4.7 it can
be seen that at 300 rpm air bubbles are broken up into a smaller size bubbles than the ones
observed when mixing at 150 rpm, which increases the concentration of CO2 in the media
therefore making CO2 more readily available to cells in the culture (Kazim, 2012).
42
150 rpm
300 rpm
Note: Air flowrate = 2 L/min
Figure 4.7: Turbulence inside the stirred tank reactor at different mixing (Kazim,
2012)
4.4
Batch Cultivation in Tubular and Continuously Stirred

Tank Photo-bioreactors
When the results of the CSTR experiments performed are placed side by side (Table 4.3)
with those obtained in the two other configurations, 4L flask and tubular photobioreactor, parameters and results can be compared. Off all the media formulations,
Bolds media with added NaHCO3 showed the highest specific growth rate () of all the
experiments at 0.345 d-1 but the maximum biomass concentration achieved was only
0.177 g/L. A similar value was achieved on experiment B3 at 0.32 d-1 but with a higher
final biomass concentration of 0.445 g/L. Both of these experiments had an additional
source of carbon, one in the form of carbonate (NaHCO3) and the other in the form of
gaseous CO2 mixed in with the bubbled air, and they had the highest specific growth rates
of all the experiments but not the highest biomass concentration.
Both of the CSTR experiments presented no better growth rates or maximum biomass
concentrations than the other experiments even when they were bubbled with 3% CO2
enriched air. A study by (Muoz et al., 2005) found a similar result, they tested various
43
microalgae species, including Chlorella vulgaris and Chlorella sorokiniana, for the
removal of the carcinogenic acetronitrile from wastewater digester sludge on both tubular
and stirred tank reactors. The highest acetonitrile removal rates and biomass density were
shown by Chlorella sorokiniana when cultured in a tubular photo-bioreactor.
Table 4.3: Comparison of Batch Studies with Chlorella vulgaris on different photobioreactor configurations.
Experiment Number/Media
(RPM)
B3
NaHCO3
CSTR1
(150)
CSTR2
(350)
21.0
23.4
25
25
Avg. pH
6.54
9.80
6.62
6.61
860
8500
7150
7300
Month of exp.
Sep
Jun
Aug
Oct
Working Volume (L)
6.0
3.5
10
10
30
15
20
20
Yes
No
Yes
Yes
% CO2
No
---
Specific Growth Rate, (d-1)
0.32
0.345
0.123
0.205
2.17
2.01
5.63
3.39
0.445
0.177
0.470
0.590
44
4.5
Semi-Continuous Cultivation in a Tubular Photobioreactor
For the continuous part of the experiment in the tubular photo-bioreactor (PBR),
temperature and pH were measured; the average was 23C and 7.40 respectively. pH was
controlled and kept in range with a 1N HCl solution.
450
1.0
0.7 d-1
-1
400
0.8
0.5 d-1
350
0.6 d-1
300
0.6
250
Biomass
Nitrate
Phosphate
0.4
200
150
mg/L (NO3 - or PO4-3)
0.75 d
100
0.2
50
0
0.0
0
20
40
60
Days
Figure 4.8: Semi-continuous cultivation and nutrient consumption of C. vulgaris in a

tubular photo-bioreactor.
Figure 4.9 and Table 4.4 summarize the effect that different dilution rates had on the
maximum biomass concentration achieved by Chlorella vulgaris when it reached steady
state. Biomass concentration steadily increased with the dilution rate (3). At a dilution
rate of 0.75 d-1, a decrease of biomass concentration (beginning washout of cells) was
observed. The concentration of nitrates decreased rapidly within the first 20 days to the
point of almost being depleted, it stayed constant through the rest of the experiment.
Phosphates in the media were used sparingly and the concentration remained constant
45
through most of the experiment. Nitrates were reduced by almost 100% and phosphates
only by 67%. The results of a study, of the cyanobacterium Thermosynechoccus,
suggested that the dissolved inorganic phosphate had no effect on the cell mass
production (Su et al., 2012).
The highest biomass concentration reached through this experiment was 0.79 g/L at
3=0.7 d-1, which was slightly higher than the maximum concentration reached during the
batch phase which was 0.68 g/L. Equation (2.10) can be used to find the specific growth
rate (), which equals the dilution rate (3) in steady state therefore =0.7 d-1 in this
experiment which gives a doubling time (td) is 0.99 days, this means that the amount of
microalgae in the culture will double every 0.99 days.
Average Biomass Concentration (g/L)
0.85
0.80
0.75
0.70
0.65
0.60
0.55
0.50
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
Dilution Rate (day-1)

Note: Error bars represent standard deviation from the average biomass concentration in steady
state conditions
Figure 4.9: Steady state behaviour of C. vulgaris.
46
Table 4.4: Steady state biomass concentration at different dilution rates in a tubular
photo-bioreactor
4.6
Dilution Rate, 7
Biomass Concentration
(d-1)
(g/L)
0.5
0.67
0.6
0.73
0.7
0.79
0.75
0.54
CSTR
4.6.1
Effect of Agitation Speed on Cell Growth
A stirred tank reactor was used to test the effect of mixing speed on the biomass
concentration of Chlorella vulgaris. Results are shown on Figure 4.10, the run was started
at a mixing speed of 150 rpm, once the culture has passed the exponential growth phase
and had reached steady state, a dilution rate of 0.4 d-1 was set and the speed was increased
to 250 rpm until the culture reached steady state again then the mixing speed was
increased to 350 rpm, steady state was reached again and speed was increased to 450 rpm.
Biomass concentration increased steadily along with the increase of mixing speed, Table
4.5 shows the maximum biomass concentration reached with the different mixing speeds.
It can also be observed that the concentration of CO2 decreased within the first 20 days of
the experiment until steady state was reached at 150 rpm and then remained constant
through the rest of the experiment.
47
200
180
120
100
150
80
Biomass
Nitrate
Phosphate
CO2
0.2
60
40
-3
140
200
-1
250
0.6
0.4
250
160
mg/L (NO or PO4 )
350
150
ppm CO2
450
0.8
300
100
50
20
0.0
0
0
10
20
30
40
50
Days
Note: numbers on squares is the mixing speed in rpm. Dilution rate, 0.4 d-1
Figure 4.10: Effect of Mixing Speed on Cell Growth and Biomass Concentration
It was expected that at higher mixing speeds there would be visible cell damage but none
was observed through the microscope at the maximum mixing speed tested of 450rpm.
At a constant air flow rate, an increase in agitation speed (rpm) can cause cell damage this
can be mostly due to the air bubbles in the medium undergoing breakup and coalescence
in the impeller stream region that encompasses spinning impellers (Yang and Wang,
1992). (Sobczuk et al., 2006) measured damage to microalgae cells by quantifying the
decrease of biomass concentration after steady state had been achieved at the mixing
speeds tested. They found that the biomass concentration increased with agitation speed
up to 350rpm, in the case of Phaeodactylum tricornotum, and up to 550rpm in the case of
Porphyridium cruentum and found drastic decrease in biomass concentration at higher
speeds.
48
In this experiment, in which the effect of mixing speed on biomass concentration of

Chlorella vulgaris was tested, it was found to have the same initial effect as the study
done by (Sobczuk et al., 2006) but as mixing speed was increased to 450rpm, it was
found that it had little effect in the increase of biomass, going only from 0.637 g/L at the
previous speed of 350rpm, to 0.656 g/L at 450rpm and no visible cell damage was
noticed.
The mixing speed of 350rpm was chosen for further experiments to save on energy and to
avoid possible cell damage due to long periods of high speed mixing at 450rpm.
Table 4.5: Biomass concentration at different mixing speeds in a CSTR
4.6.2
Mixing speed
(rpm)
(g/L)
150
0.467
250
0.574
350
0.637
450
0.656
Effect of Dilution Rate on Cell Growth
In order to investigate the effect of different dilution rates in the growth of Chlorella
vulgaris, the mixing speed was fixed at 350 rpm and changed the dilution rate of the
system once it reached steady state. The results are illustrated in Figure 4.11. Three
dilution rates (3) were tested in this experiment, 0.5 d-1, 0.6 d-1 and 0.75 d-1. As 3 values
increased and the culture reached steady state, biomass concentration values were higher
up until a dilution rate of 0.75 d-1, where a decrease of biomass concentration in the
bioreactor due to the washout of the cells was noticed. Nitrates were reduced by 80% and
phosphates by 20% within the batch phase of the experiment, which lasted about 14 days.
When the first dilution rate of 0.5 d-1 was started, the concentration of both nitrates and
phosphates increased slightly as was expected, since fresh media was being added to the
culture in the bioreactor, but concentration of both nutrients reached a steady value that
remained through the rest of the experiment.
49
The concentration of CO2 in the culture increased the most during the batch phase of the
experiment. The change in dilution rate seemed to have an effect on the concentration of
CO2 which increased slightly with the increasing change in dilution rate.
220
1.0
-1
0.75 d
160
0.6 d-1
200
140
-1
0.5 d
0.6
100
80
Biomass
Nitrate
Phosphate
CO2
0.4
60
180
160
ppm CO2
120
mg/L (NO-1 or PO4-3)
0.8
140
40
0.2
120
20
0.0
0
0
10
20
30
40
100
50
Days
Figure 4.11: Effect of dilution rate on biomass concentration of Chlorella vulgaris in

a CSTR
Figure 4.12 and Table 4.6 show more clearly the effect that different dilution rates have
on the maximum biomass concentration achieved by C. vulgaris in steady state. Biomass
concentration steadily increased with the dilution rate (3). The highest biomass
concentration reached was 0.8 g/L at 3=0.6 d-1, which was 35% higher than the
maximum concentration reached during the batch phase. When the microalgae cell
concentration is constant, as in steady state, equation (2.10) can be used to find the
specific growth rate (), in steady state 3= therefore the dilution rate at which the
highest biomass concentration =0.6 d-1 is found, will have a doubling time of the cells
(td) of 1.15 days, this means that the amount of microalgae in the culture will double
every 1.15 days.
50
Average Biomass Concentration (g/L)
0.85
0.80
0.75
0.70
0.65
0.60
0.55
0.3
0.4
0.5
0.6
0.7
0.8
Dilution Rate (d-1)

Note: Error bars represent standard deviation from the average biomass concentration in steady
state conditions
Figure 4.12: Biomass concentration vs dilution rate in a CSTR
Table 4.6: Biomass concentration at different dilution rates in a CSTR

Dilution Rate, 7
-1
(d )
(g/L)
0.4
0.64
0.5
0.69
0.6
0.8
0.75
0.6
51
Chapter 5
Conclusions and Recommendations
The results obtained through the experiments presented in this thesis lead to a better
understanding of the behavior of the microalgae Chlorella vulgaris in different photobioreactor configurations and growth conditions.
The results of this study show that Chlorella vulgaris UTEX 2714 grows better in tubular
photo-bioreactors than in stirred tank reactors or 4L flasks. The addition of CO2 in air was
beneficial to growth and final biomass concentration, it resulted in some of the highest
growth rates of all the experiments performed except for the case of when C. vulgaris was
grown in C30 media. The negative results with C30 media were most likely due to the
inhibitory effect of the higher CO2 concentration used and the form in which the iron was
added to the media which could have made it biologically unavailable to the algae cells.
Bold`s media with a different source of carbon in the form of NaHCO3 did not have the
positive overall results that were expected. At the beginning, the experiment with added
NaHCO3 had the highest specific growth rate of all the experiments at 0.345 d-1 but the
rapid increase of pH had an adverse effect on the final concentration of biomass. The
results of the different media formulations show that C30 media and Bold`s media with
added NaHCO3 are not suitable for growth of the microalgae strain Chlorella vulgaris
UTEX 2714.
For most applications in engineering, what is of greatest importance is the steady state
behavior of a system. Continuous culture of microalgae allows for maintenance of steady
state over long periods of time, it has its main advantage on allowing for constant
extraction of biomass (and any compounds it might produce) from the system. The best
overall results found in this study were when growing C. vulgaris was grown semicontinuously in a tubular photo-bioreactor and Bold`s media, it reached the highest
biomass concentration of 0.79 g/L when the dilution rate was set at 0.7 d-1. The results of
the continuous culture in CSTR were similar; the highest concentration of biomass
obtained was 0.8 g/L when the dilution rate was set at 0.6 d-1. It could be argued that the
52
biomass concentration is the same so there is no difference but with this result being
obtained at a dilution rate of 0.7 d-1 for a tubular photo-bioreactor, it will mean that
overall it would be possible to obtain a higher volume of culture everyday and hence a
higher biomass concentration than in the case of the CSTR.
CSTR and 4L flask results might have not been the best within all the experiments
performed but both this configurations allow for easier initial investigation of parameters.
In the case of 4L flasks there is the convenience of how easy and fast this type of set-up
can be assembled, also, the smaller volumes of media needed allow for multiple
experiments being run at the same time which saves a lot of time since microalgae grow
slower than other microorganisms like bacteria and fungi. CSTR can be operated at
different volumes, the working volume used for this study was 10L which was the same
volume than the one used in a couple of tubular photo-bioreactor experiments and higher
than the 3.5L working volume that was utilized for the 4L flask configuration. CSTR still
has similar advantages as those of a 4L flask set-up, it is easy to automatically monitor
and control different parameter as the system is usually connected to a computer that
keeps tracks of changes in the system. In this study there was automatic control of
temperature and mixing speed and constant tracking of CO2 concentration and pH. This
easiness of control and tracking allowed for testing the effect of mixing speed and
dilution rate on the growth of Chlorella vulgaris, similar studies for other microorganisms
are reported in literature but few in the case for microalgae and none found for C.
vulgaris.
5.1
Future Work
The importance of microalgae in today`s market is emphasized by the increase need for
all the products that microalgae can be used to obtain (pigments, nutritional and medicinal
supplements) and all the benefits to the environment that this brings (treating and
eliminating harmful chemicals from wastewater, helping in the reduction of CO2
emissions, etc.).
The results of this study show that tubular photo-bioreactors are amongst the best designs
for the mass cultivation of microalgae, Bold`s media showed to be the best media
53
formulation amongst those tested and even though other variations of the formulation did
not show the same kind of results, it gives a starting point for testing other formula
variations and the effects that certain chemical compounds can have on microalgae
growth.
Now that the effect of dilution rate on microalgae growth in tubular photo-bioreactor and
CSTR has been studied, the added effect of other parameters such as different light/dark
cycles, temperature, pH and nutrient media formulations can be studied. Different strains
of algae can be looked into as well as monitoring other properties like oil and cellulose
content or focus on the downstream processing of the biomass obtained.
54
References
Aguirre-Ezkauriatza, E.J., M.G. Galarza-Gonzlez, A.I. Uribe-Bujanda, M. Ros-Licea, F.
Lpez-Pacheco, C.M. Hernndez-Brenes and M.M. Alvarez, "Effect of mixing during
fermentation in yogurt manufacturing," J. Dairy Sci. 91, 4454-4465 (2008).
Ahn, J.-., K. Hwangbo, S.Y. Lee, H.-. Choi, Y.-. Park, J.R. Liu and W.-. Jeong, "A new
Arctic Chlorella species for biodiesel production," Bioresour. Technol. (2012).
Aishvarya, V., N. Pradhan, R.R. Nayak, L.B. Sukla and B.K. Mishra, "Enhanced
inorganic carbon uptake by Chlorella sp. IMMTCC-2 under autotrophic conditions for
lipid production and CO 2 sequestration," J. Appl. Phycol., 1-9 (2012).
Amini Khoeyi, Z., J. Seyfabadi and Z. Ramezanpour, "Effect of light intensity and
photoperiod on biomass and fatty acid composition of the microalgae, Chlorella vulgaris,"
Aquacult. Int. 20, 41-49 (2012).
Andersen, R.A., "Algal culturing techniques," Elsevier/Academic Press, Burlington,
Mass. (2005), pp. 578.
Aslan, S. and I.K. Kapdan, "Batch kinetics of nitrogen and phosphorus removal from
synthetic wastewater by algae," Ecol. Eng. 28, 64-70 (2006).
Bailey, J.E. and D.F. Ollis, "Biochemical engineering fundamentals," McGraw-Hill,
Montreal (1977).
Bajpai, D. and V.K. Tyagi, "Biodiesel: Source, Production, Composition, Properties and
its Benefits" Journal of Oleo Science. 55, 487 (2006).
Becker, E.W., "Microalgae: Biotechnology and Microbiology," Cambridge University
Press, New York (1994).
Cern-Garca, M.C., M.D. Macas-Snchez, A. Snchez-Mirn, F. Garca-Camacho and
E. Molina-Grima, "A process for biodiesel production involving the heterotrophic
55
fermentation of Chlorella protothecoides with glycerol as the carbon source," Appl.

Energy. (2012).
Chandhok, G.S., "Investigation of lipid and biodiesel production from Chlorella vulgaris
(UTEX 2714) cultured in photo- bioreactors," School of Graduate and Postdoctoral
Studies, University of Western Ontario, London, Ont. (2010).
Chisti, Y., "Biodiesel from microalgae," Biotechnol. Adv. 25, 294-306 (2007).
Clement, G., M. Rebeller and C. Zarouk, "Wound treating medicaments containing
algae," La France Medicale. 59, 5279 (1967).
Crdoba-Castro, N.M., A.M. Montenegro-Jaramillo, R.E. Prieto and G.E. GonzlezMario, "Analysis of the effect of the interactions among three processing variables for
the production of exopolysaccharides in the microalgae Scenedesmus obliquus (UTEX
393)," Vitae. 19, 60-69 (2012).
Demirbas, A., "Production of Biodiesel from Algae Oils," Energy Sources Part ARecovery Utilization and Environmental Effects. 31, 163-168 (2009).
Devgoswami, C.R., M.C. Kalita, J. Talukdar, R. Bora and P. Sharma, "Studies on the
growth behavior of chlorella, haematococcus and scenedesmus sp. in culture media with
different concentrations of sodium bicarbonate and carbon dioxide gas," African Journal
of Biotechnology. 10, 13128-13138 (2011).
Feng, P., Z. Deng, L. Fan and Z. Hu, "Lipid accumulation and growth characteristics of
Chlorella zofingiensis under different nitrate and phosphate concentrations," Journal of
Bioscience and Bioengineering. 114, 405-410 (2012).
Feng, Y., C. Li and D. Zhang, "Lipid production of Chlorella vulgaris cultured in
artificial wastewater medium," Bioresour. Technol. 102, 101-105 (2011).
Fenice, M., P. Barghini, L. Selbmann and F. Federici, "Combined effects of agitation and
aeration on the chitinolytic enzymes production by the Antarctic fungus Lecanicillium
muscarium CCFEE 5003," Microbial Cell Factories. 11 (2012).
56
Filali, R., S. Tebbani, D. Dumur, A. Isambert, D. Pareau and F. Lopes, "Growth modeling
of the green microalga Chlorella vulgaris in an air-lift photo-bioreactor," IFAC
Proceedings Volumes (IFAC-PapersOnline). 18, 10603-10608 (2011).
Funahashi, H., Y. Nishiyama, M. Asano and K. Nakao, "Effects of pH shift and agitation
speed on chlorophyll biosynthesis in heterotrophic culture of Chlorella," Kagaku Kogaku
Ronbunshu. 25, 218-219 (1999).
Garca Camacho, F., A. Contreras Gmez, F.G. Acin Fernndez, J. Fernndez Sevilla
and E. Molina Grima, "Use of concentric-tube airlift photo-bioreactors for microalgal
outdoor mass cultures," Enzyme Microb. Technol. 24, 164-172 (1999).
Gellenbeck, K., "Utilization of algal materials for nutraceutical and cosmeceutical
applicationswhat do manufacturers need to know?" J. Appl. Phycol. 24, 309-313
(2012).
Gonzlez-Fernndez, C., B. Sialve, N. Bernet and J.-. Steyer, "Impact of microalgae
characteristics on their conversion to biofuel. Part I: Focus on cultivation and biofuel
production," Biofuels, Bioproducts and Biorefining. 6, 105-113 (2012).
Gonzlez-Lpez, C.V., F.G. Acin Fernndez, J.M. Fernndez-Sevilla, J.F. Snchez
Fernndez and E. Molina Grima, "Development of a process for efficient use of CO 2
from flue gases in the production of photosynthetic microorganisms," Biotechnol. Bioeng.
109, 1637-1650 (2012).
Gouveia, L. and SpringerLink, "Microalgae as a Feedstock for Biofuels," (2011).
Gustafson, K.R., J.H. Cardellina, R.W. Fuller, O.S. Weislow, R.F. Kiser, K.M. Snader
and Patterson, G.M.L. & Boyd, M.R, "AIDS-Antiviral Sulfolipids From Cyanobacteria
(Blue-Green Algae)," Journal of the National Cancer Institute. 81, 1254 (1989).
Heidari, S.A., O. Farhadian and N.M. Soofiani, "Biomass production and ammonia and
nitrite removal from fish farm effluent by scenedesmus quadricauda culture," Journal of
Environmental Studies. 37, 15-28 (2011).
57
Hellebust, J.A., J.S. Craigie, J.R. Stein-Taylor and Phycological Society of America,
"Handbook of phycological methods," Cambridge University Press,
Cambridge,Cambridgeshire (1973).
Henrard, A.A., M.G. de Morais and J.A.V. Costa, "Vertical tubular photo-bioreactor for
semicontinuous culture of Cyanobium sp." Bioresour. Technol. 102, 4897-4900 (2011).
Hodaifa, G., M.E. Martnez, R. rpez and S. Snchez, "Inhibitory effects of industrial
olive-oil mill wastewater on biomass production of Scenedesmus obliquus," Ecol. Eng.
42, 30-34 (2012).
Hulatt, C.J. and D.N. Thomas, "Energy efficiency of an outdoor microalgal photobioreactor sited at mid-temperate latitude," Bioresour. Technol. 102, 6687-6695 (2011).
Jacob-Lopes, E., C.H.G. Scoparo, L.M.C.F. Lacerda and T.T. Franco, "Effect of light
cycles (night/day) on CO2 fixation and biomass production by microalgae in photobioreactors" Chemical Engineering and Processing: Process Intensification. 48, 306-310
(2009).
Kazim, S.A., "Experimental & empirical correlations for the determination of the overall
volumetric mass transfer coefficients of carbon dioxide in stirred tank bioreactors,"
(2012).
Krichnavaruk, S., S. Powtongsook and P. Pavasant, "Enhanced productivity of
Chaetoceros calcitrans in airlift photo-bioreactors," Bioresour. Technol. 98, 2123-2130
(2007).
Kumar, K. and D. Das, "Growth characteristics of Chlorella sorokiniana in airlift and
bubble column photo-bioreactors," Bioresour. Technol. 116, 307-313 (2012).
Lam, M.K. and K.T. Lee, "Potential of using organic fertilizer to cultivate Chlorella
vulgaris for biodiesel production," Appl. Energy. 94, 303-308 (2012).
58
Lang, X., A.K. Dalai, N.N. Bakhshi, M.J. Reaney and P.B. Hertz, "Preparation and
characterization of bio-diesels from various bio-oils" Bioresour. Technol. 80, 53-62
(2001).
Lee, K. and Lee, C. "Effect of light/dark cycles on wastewater treatments by microalgae,"
Biotechnology and Bioprocess Engineering. 6, 194-199 (2001).
Lee, Y. and Hing, H., "Supplying CO2 to photosynthetic algal cultures by diffusion
through gas-permeable membranes" Appl. Microbiol. Biotechnol. 31, 298-301 (1989).
Li, Y., M. Horsman, B. Wang, N. Wu and C.Q. Lan, "Effects of nitrogen sources on cell
growth and lipid accumulation of green alga Neochloris oleoabundans" Appl. Microbiol.
Biotechnol. 81, 629-636 (2008).
Liu, Z.Y., G.C. Wang and B.C. Zhou, "Effect of iron on growth and lipid accumulation in
Chlorella vulgaris" Bioresour. Technol. 99, 4717-4722 (2008).
Mattos, E.R., M. Singh, M.L. Cabrera and K.C. Das, "Effects of Inoculum Physiological
Stage on the Growth Characteristics of Chlorella sorokiniana Cultivated Under Different
CO 2 Concentrations" Appl. Biochem. Biotechnol., 1-12 (2012).
Mayo, A.W., "Effects of temperature and pH on the kinetic growth of unialga Chlorella
vulgaris cultures containing bacteria" Water Environ. Res. 69, 64-72 (1997).
Mazzuca Sobczuk, T. and Y. Chisti, "Potential fuel oils from the microalga Choricystis
minor" J. Chem. Technol. Biotechnol. 85, 100-108 (2010).
McGinn, P.J., K.E. Dickinson, K.C. Park, C.G. Whitney, S.P. MacQuarrie, F.J. Black, J.-.
Frigon, S.R. Guiot and S.J.B. O'Leary, "Assessment of the bioenergy and bioremediation
potentials of the microalga Scenedesmus sp. AMDD cultivated in municipal wastewater
effluent in batch and continuous mode," Algal Research. (2012).
Miao, X. and Q. Wu, "Biodiesel production from heterotrophic microalgal oil,"
Bioresour. Technol. 97, 841-846 (2006).
59
Mitra, D., van Leeuwen J. and B. Lamsal, "Heterotrophic/mixotrophic cultivation of

oleaginous Chlorella vulgaris on industrial co-products" Algal Research. 1, 40-48 (2012).
Moheimani, N.R., "Inorganic carbon and pH effect on growth and lipid productivity of
Tetraselmis suecica and Chlorella sp (Chlorophyta) grown outdoors in bag photobioreactors" J. Appl. Phycol., 1-12 (2012).
Muoz, R. and B. Guieysse, "Algal-bacterial processes for the treatment of hazardous
contaminants: A review" Water Res. 40, 2799-2815 (2006).
Muoz, R., M. Jacinto, B. Guieysse and B. Mattiasson, "Combined carbon and nitrogen
removal from acetonitrile using algalbacterial bioreactors" Appl. Microbiol. Biotechnol.
67, 699-707 (2005).
Ohira, Y., M. Shimadzu and E. Obata, "Leakage of intracellular substances from bluegreen algae, spirulina platensis due to agitation" Kagaku Kogaku Ronbunshu. 38, 57-60
(2012).
Park, J.B.K. and R.J. Craggs, "Nutrient removal in wastewater treatment high rate algal
ponds with carbon dioxide addition" Water Science and Technology : A Journal of the
International Association on Water Pollution Research. 63, 1758-1764 (2011).
Perez-Garcia, O., Y. Bashan and M. Esther Puente, "Organic carbon supplementation of
sterilized municipal wastewater is essential for heterotrophic growth and removing
ammonium by the microalga Chlorella Vulgaris," J. Phycol. 47, 190-199 (2011).
Rados, S., B. Vaclav and D. Frantisek, "CO2 Balance in industrial cultivation of algae"
Archiv Hydrobiologie., 297 (1975).
Richmond, A., "Handbook of Microalgal Culture: Biotechnology and Applied
Phycology" Blackwell Science Ltd., Victoria, Australia (2004).
Richmond, A., S. Karg and S. Boussiba, "Effects of bicarbonate and carbon dioxide on
the competition between chlorella vulgaris and spirulina platensis" Plant and Cell
Physiology. 23, 1411-1417 (1982).
60
Snchez Mirn, A., M.-. Cern Garca, F. Garca Camacho, E. Molina Grima and Y.
Chisti, "Growth and biochemical characterization of microalgal biomass produced in
bubble column and airlift photo-bioreactors: Studies in fed-batch culture" Enzyme
Microb. Technol. 31, 1015-1023 (2002).
Schenk, P.M., S.R. Thomas-Hall, E. Stephens, U.C. Marx, J.H. Mussgnug, C. Posten, O.
Kruse and B. Hankamer, "Second Generation Biofuels: High-Efficiency Microalgae for
Biodiesel Production" BioEnergy Research. 1, 20-43 (2008).
Seidell, A., "Solubilities of inorganic and organic substances: a handbook of the most
reliable quantitative solubility determinations" D. Van Nostrand company, New York
(1907).
Sirisansaneeyakul, S., S. Singhasuwan, W. Choorit, N. Phoopat, J. Garcia and Y. Chisti,
"Photoautotrophic Production of Lipids by Some Chlorella Strains" Mar. Biotechnol. 13,
928-941 (2011).
Sobczuk, T.M., F.G. Camacho, E.M. Grima and Y. Chisti, "Effects of agitation on the
microalgae Phaeodactylum tricornutum and Porphyridium cruentum" Bioprocess and
Biosystems Engineering. 28, 243-250 (2006).
Srivastava, A. and R. Prasad, "Triglycerides-based diesel fuels" Renewable and
Sustainable Energy Reviews. 4, 111-133 (2000).
Su, C.M., H.T. Hsueh, H.H. Chen and H. Chu, "Effects of dissolved inorganic carbon and
nutrient levels on carbon fixation and properties of Thermosynechococcus sp. in a
continuous system" Chemosphere. 88, 706-711 (2012).
Subramaniam, R., S. Dufreche, M. Zappi and R. Bajpai, "Microbial lipids from renewable
resources: Production and characterization" Journal of Industrial Microbiology and
Biotechnology. 37, 1271-1287 (2010).
Sun, L., S. Zhang, C. Wang and L. Shi, "Effects of renewal regime on consumption of
nitrogen and phosphorus, biomass and polysaccharide production by Porphyridium
61
cruentum in semicontinuous culture" 3rd International Conference on Bioinformatics and

Biomedical Engineering, iCBBE 2009. (2009).
Tang, H., M. Chen, K.Y. Simon Ng and S.O. Salley, "Continuous microalgae cultivation
in a photo-bioreactor" Biotechnol. Bioeng. (2012a).
Tang, H., M. Chen, K.Y. Simon Ng and S.O. Salley, "Continuous microalgae cultivation
in a photo-bioreactor" Biotechnol. Bioeng. 109, 2468-2474 (2012b).
Varfolomeev, S.D. and L.A. Wasserman, "Microalgae as source of biofuel, food, fodder,
and medicines" Appl. Biochem. Microbiol. 47, 789-807 (2011).
Vega-Estrada, J., M.C. Montes-Horcasitas, A.R. Domnguez-Bocanegra and R.O.
Caizares-Villanueva, "Haematococcus pluvialis cultivation in split-cylinder internalloop airlift photo-bioreactor under aeration conditions avoiding cell damage" Appl.
Microbiol. Biotechnol. 68, 31-35 (2005).
Wang, B., C.Q. Lan and M. Horsman, "Closed photo-bioreactors for production of
microalgal biomasses" Biotechnol. Adv. 30, 904-912 (2012).
Wang, L., Y. Wang, P. Chen and R. Ruan, "Semi-continuous cultivation of chlorella
vulgaris for treating undigested and digested dairy manures" Appl. Biochem. Biotechnol.
162, 2324-2332 (2010a).
Wang, L., Y. Wang, P. Chen and R. Ruan, "Semi-continuous cultivation of chlorella
vulgaris for treating undigested and digested dairy manures" Appl. Biochem. Biotechnol.
162, 2324-2332 (2010b).
Westerhoff, P., Q. Hu, M. Esparza-Soto and W. Vermaas, "Growth parameters of
microalgae tolerant to high levels of carbon dioxide in batch and continuous-flow photobioreactors" Environ. Technol. 31, 523-532 (2010).
Xiao, R., R. Chen, H.-. Zhang and H. Li, "Microalgae scenedesmus quadricauda grown in
digested wastewater for simultaneous CO 2 fixation and nutrient removal" Journal of
Biobased Materials and Bioenergy. 5, 234-240 (2011).
62
Xu, L., P.J. Weathers, X.-. Xiong and C.-. Liu, "Microalgal bioreactors: Challenges and
opportunities" Engineering in Life Sciences. 9, 178-189 (2009).
Yang, J. and N.S. Wang, "Cell inactivation in the presence of sparging and mechanical
agitation" Biotechnol. Bioeng. 40, 806-816 (1992).
Yeh, K., J. Chang and W. Chen, "Effect of light supply and carbon source on cell growth
and cellular composition of a newly isolated microalga Chlorella vulgaris ESP-31"
Engineering in Life Sciences. 10, 201-208 (2010).
Yu, R., Y. Liu, S. Tian, Y. Yang, W. Li, Y. Cao and D. Qiao, "Isolation and identification
of oil microalgae and optimization of its culture conditions" Chinese Journal of Applied
and Environmental Biology. 17, 897-900 (2011).
Zheng, H., Z. Gao, Q. Zhang, H. Huang, X. Ji, H. Sun and C. Dou, "Effect of inorganic
carbon source on lipid production with autotrophic Chlorella vulgaris" Shengwu
Gongcheng Xuebao/Chinese Journal of Biotechnology. 27, 436-444 (2011).
63
Appendices
Appendix A: Equipment and Materials
(b)
(a)
Figure A-1: Tubular Photo-bioreactor Set-Up.

(a) 10L with lamps, (b) 3-6L no lamps
64
Figure A-2: Sampling device for the tubular photo-bioreactor
Source: ca.vwr.com
Figure A-3: Improved Neubauer hemacytometer.
65
Appendix B: Calibration Curve for CO2

The calibration curve (shown in Figure A-4) for the carbon dioxide probe was created
before the start of experiments in the CSTR. Equation (A-1) was obtained, which was
later converted to Equation(A-2) , to convert the mV values given by the probe into ppm
of CO2.
mV = 21.584*ln(ppm CO2) 95
(A-1)
ppm CO2 = " ((mV+95)/21.548)
(A-2)
80
60
40
mV
20
y = 21.584ln(x)-95
2
R = 0.99
-20
-40
-60
1
10
100
1000
ppm CO2
Figure A-4: Calibration Curve for CO2 Probe
10000
66
Appendix C: Determination of Dry Weight and Biomass Concentration

A calibration curve was prepared to correlate the absorbance of the culture of Chlorella
vulgaris with dry weight. Figure A-5 shows the correlation of dry weight (biomass
concentration) with the cultures absorbance at 686nm, which was chosen due to a
preliminary study showing 686nm to be the wavelength of maximum absorbance for a
culture of the strain of Chlorella used (in Bolds media). Equation (A-3) was obtained.
Biomass (dry weight) = 0.2936*O.D.686nm+0.0007
(A-3)
0.30
0.25
0.20
0.15
0.10
y = 0.2936x+0.0007
R2 = 0.99
0.05
0.00
0.0
0.2
0.4
0.6
0.8
1.0
Absorbance
Figure A-5: Dry Weight Calibration Curve
1.2
67
Appendix D: Sample Calculations

The following is a sample calculation for the specific growth rate () and doubling time
( ):

12 days , 2 days, 0.228 ?/@, 0.065 ?/@

0.2280.065
12 2

0.1255 d
ln 2

ln 2
5.53 days
0.1255
68
Curriculum Vitae
Name:
Post-secondary
Education and
Degrees:
Universidad Autnoma de Sinaloa

Culiacan, Sinaloa, Mexico
Bachelor in Biochemical Engineering
2000 2005
London, Ontario, Canada
M.E.Sc.
2011 2012
Honours and
Awards:
CONACyT Scholarship (Mexico)

2008 - 2010
UAS Doctores Jovenes Scholarship (Mexico)
2008-2011
Related Work
Experience
Teaching Assistant
2008-2012

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BATCH AND CONTINUOUS STUDIES OF Chlorella vulgaris IN PHOTOBIOREACTORS

Claudia Sacasa Castellanos

Graduate Program in Chemical and Biochemical Engineering

A thesis submitted in partial fulfillment

The School of Graduate and Postdoctoral Studies

Claudia Sacasa Castellanos 2013

THE UNIVERSITY OF WESTERN ONTARIO

Claudia Sacasa Castellanos

Batch and Continuous Studies of Chlorella vulgaris in

What are Microalgae ................................................................................... 3

Current Uses of Microalgae ........................................................................ 3

2.2 Factors Affecting Microalgal Growth ..................................................................... 7

Nutrient Composition of Media .................................................................. 7

2.3 Microalgae Cultivation ........................................................................................... 9

Microalgae Growth Kinetics ....................................................................... 9

Photo-bioreactor Design ........................................................................... 12

Tubular Photo-bioreactors ........................................................................ 14

Continuously Stirred Tank Reactors ......................................................... 15

Batch and Continuous Cultivation ............................................................ 17

2.4 Importance of This Study...................................................................................... 18

Bolds Basal Media ................................................................................... 19

Other Media Formulation ......................................................................... 21

3.3 Inoculum and Strain Maintenance ........................................................................ 22

4 L Conical Flasks .................................................................................... 22

Tubular Photo-bioreactor .......................................................................... 23

Continuously Stirred Tank (CSTR) Photo-bioreactor .............................. 25

Air Supply ................................................................................................. 26

3.5 Sampling of Photo-bioreactor ............................................................................... 28

Cell Count ................................................................................................. 28

3.7 Determination of Dry Weight and Biomass Concentration .................................. 28

Modified Bolds Media with NaHCO3 ..................................................... 31

4.2 Batch Cultivation in Tubular PBR. ....................................................................... 33

Batch Cultivation in C30 and PCS-2 Media ............................................. 33

Batch Cultivation in Bolds Media ........................................................... 35

4.3 Batch Cultivation in Stirred Tank Photo-bioreactor. ............................................ 39

Effect of Agitation Speed on Cell Growth ................................................ 46

Effect of Dilution Rate on Cell Growth .................................................... 48

Volumetric flow rate

Cell number, cell or biomass concentration

Initial cell number, cell or biomass

Rate of cell formation

Specific growth rate

Investigate batch and continuous cultivation of Chlorella vulgaris in different

Investigate the effect of different dilution rates on the biomass concentration of

Investigate the effect of different mixing speeds on the biomass concentration of

Investigate the effect of different dilution rates on the biomass concentration of

What are Microalgae

Microalgae are microscopic photosynthetic microorganisms. They are a type of

Figure 2.1:: Microscopic image of Chlorella vulgaris UTEX 2714

Figure 2.2: Various commercial applications of microalgae (Varfolomeev and

Source of Oil for Biodiesel

Land needed for

Algae (10g m day at

Algae (50 g m-2 day-1 at

Factors Affecting Microalgal Growth

Nutrient Composition of Media

Figure 2.3: Carbon dioxide equilibrium in water (Becker, 1994)

Microalgae Growth Kinetics

Figure 2.4: Microalgae batch growth profile

During the exponential phase 

or  can be used to characterize the state of the cell

During the exponential phase

or can be used to characterize the state of the cell

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!" #$ +# ,-%# .%/% &'&"( 0