Growth of Chlorella Vulgaris
at high carbon dioxide
levels in Swedish light
conditions
Laboratory testing and development of methods
By: Ann Edberg, 2010
Master of Science Thesis in
Energy Engineering.
Umeå Institute of
Technology
Abstract
Petroleum fuels are a finite source of energy and an environmental problem since carbon
dioxide is one of the products from burning them. It is therefore important to find renewable
alternatives to petroleum fuels. Biofuels derived from terrestrial crops, such as sugarcane,
sugar beet and corn; affect the world’s food markets negatively, contributing to water shortage
and the devastation of forests. Biofuel from algae is a technically viable alternative energy
resource that handles some of the problems with traditional biofuels. Most attempts at making
biodiesel from algae are done by treating the lipids in algae like other oilseed and thus use
transesterification to produce biodiesel. Except producing lipids, algae can thrive at high
carbon dioxide levels. Temperature, pH, light intensities and carbon dioxide levels are
conditions that need to be controlled to get a high algae growth rate.
The objective of this thesis work was to test how well Chlorella vulgaris grow in Swedish
light conditions and under high carbon dioxide levels. To do this, photobioreactors for
growing microalgae had to be built and tested and an effective method for measuring lipid
content in algae needed to be found.
Light intensities vary over Sweden due to geography and seasons. Swedish light conditions
were investigated and simulated by a weather-o-meter. Photobioreactors were designed and
developed during a stepwise testing process. The main parameters evaluated during this
process were temperature and contamination in the reactors, addition of 10 % carbon dioxide
and algae survival in different light intensities.
Chlorella vulgaris grew well in 10% carbon dioxide and sequestered approximately 1,15
grams of carbon dioxide per gram dry weight of algae. Chlorella vulgaris did not survive in
light conditions corresponding to a sunny summer day in Sweden since the light intensity was
too high. It did grow well in a cloudy summer period even though the dark period is short. It
grew very slowly in winter conditions because of the short light periods.
There are more tests necessary on algae growth in different light conditions, in terms of light
intensities and length of light and dark periods, before making a complete conclusion on how
algae can grow in Swedish light conditions. The research will continue by testing other algae
species in regards of growth in flue gas, carbon dioxide mitigation, lipid production and light
intensities.
2
Table of Content
Acknowledgement................................................................................................................................... 6
1.
Introduction ..................................................................................................................................... 7
1.1 Background .................................................................................................................................... 7
1.2 Choosing algae species .................................................................................................................. 8
1.3 Objective........................................................................................................................................ 8
1.4 Limitations ..................................................................................................................................... 8
2.
Theory.............................................................................................................................................. 9
2.1 Algae and the algae cell ................................................................................................................. 9
2.1.1Photosynthesis ........................................................................................................................ 9
2.1.2 Photorespiration .................................................................................................................. 10
2.1.3 Lipids in algae ....................................................................................................................... 10
2.2 Algae as biofuel ........................................................................................................................... 10
2.3 Properties of algae in general and Chlorella vulgaris in particular ............................................. 13
2.3.1 Growth of Chlorella species.................................................................................................. 13
2.3.2 Lipid content ......................................................................................................................... 14
2.3.3 Carbon dioxide mitigation .................................................................................................... 14
2.3.4 Temperature......................................................................................................................... 15
2.3.5 pH ......................................................................................................................................... 15
2.3.6 Growth medium ................................................................................................................... 15
2.3.7 Light ...................................................................................................................................... 16
2.4 Light ............................................................................................................................................. 16
2.4.1 Light in Sweden .................................................................................................................... 17
2.4.2 Weather-o-meters ................................................................................................................ 19
2.5 Photobioreactors ......................................................................................................................... 20
2.5.1 Lighting ................................................................................................................................. 20
2.5.2 Temperature......................................................................................................................... 21
2.5.3 Gas and mixing ..................................................................................................................... 21
2.5.4 Axenic cultures and sterile environment ............................................................................. 21
2.6 Analyzing the algae ...................................................................................................................... 21
2.6.1 Cell count .............................................................................................................................. 21
3
2.6.2 Biomass ................................................................................................................................ 22
2.6.3 Lipid content ......................................................................................................................... 22
3.
Methods and Development .......................................................................................................... 23
3.1 Preculture of Chlorella vulgaris ................................................................................................... 23
3.2 Determination of testing conditions ........................................................................................... 24
3.3 Building the photobioreactors .................................................................................................... 25
3.3.1 Containers ............................................................................................................................ 25
3.3.2 Plastic ................................................................................................................................... 26
3.3.3 Gas and mixing ..................................................................................................................... 26
3.3.4 Sterile Environment .............................................................................................................. 26
3.3.5 pH ......................................................................................................................................... 27
3.3.6 Assemble the photobioreactors ........................................................................................... 27
3.4 Test and development of algae growth in photobioreactors ..................................................... 27
3.4.1 Test number one- Functions of photobioreactors ............................................................... 27
3.4.2 Test number two- Temperature regulation ......................................................................... 28
3.4.3 Test number three – Stability of temperature and plastic attachment ............................... 28
3.4.4 Test number four – Growth medium ................................................................................... 28
3.4.5 Test number five – Avoiding contamination ........................................................................ 29
3.4.6 Test number six 6 – Periodicity of cooling system and algae growth during sunny summer
intensities ...................................................................................................................................... 29
3.4.7 Test number seven – Algae growth during cloudy summer intensities ............................... 30
3.5 Summer test – Algae growth during cloudy summer.................................................................. 30
3.6 Winter test – Algae growth during winter .................................................................................. 31
3.7 Test and development of analyzing methods ............................................................................. 31
3.7.1 Measuring biomass .............................................................................................................. 31
3.7.2 Cell count .............................................................................................................................. 31
3.7.3 Component analysis ............................................................................................................. 31
3.7.4 Measuring lipid content ....................................................................................................... 31
4.
Results ........................................................................................................................................... 33
4.1 Test and develop the algae growth in photobioreactors ............................................................ 33
4.1.1 Growth medium and contamination .................................................................................... 33
4.1.2 pH ......................................................................................................................................... 34
4.1.3Temperature and cooling system.......................................................................................... 34
4.1.4 Ten percent carbon dioxide ................................................................................................. 34
4
4.1.5 Bubbling and mixing ............................................................................................................. 34
4.1.6 Plastic ................................................................................................................................... 34
4.2 Chlorella vulgaris grown at different light intensities ................................................................. 34
4.2.1 Summer test – Algae growth during cloudy summer........................................................... 35
4.2.2 Winter test ........................................................................................................................... 36
4.3 Analyzing methods ...................................................................................................................... 37
4.3.1 Biomass ................................................................................................................................ 37
4.3.2 Cell count .............................................................................................................................. 37
4.3.3 Component analysis ............................................................................................................. 38
4.3.4 Lipids ..................................................................................................................................... 38
5.
Discussion ...................................................................................................................................... 39
5.1 Test and development of the algae growth in photobioreactors ............................................... 39
5.2 Chlorella vulgaris grown in different light conditions ................................................................. 39
5.3 Analyzing methods ...................................................................................................................... 40
5.4 The continuation of the project .................................................................................................. 40
6.
Conclusions.................................................................................................................................... 42
7.
References ..................................................................................................................................... 43
8.
Appendix A .................................................................................................................................... 45
5
Acknowledgement
This thesis work is a part of a project initiated by SP Technical Research Institute of Sweden
and financed by Värmeforsk. Project manager and also my instructor for this thesis has been
Susanne Ekendahl, SP. I would like to thank her for the opportunity to perform my thesis
work at SP. SP has been a great place to work and I have learned a lot from all the wonderful
people and they have always made me feel welcome and appreciated. I would also like to
thank Susanne for her help and guidance through this entire time and thank her for keeping a
positive attitude in troubling times.
Thanks also to Francesco Gentili at the Department of Plant Physiology, Umeå University
who has provided a great deal of knowledge in the algae-research area.
I owe great thanks to Lena Bengtsson, Kjell-Åke Andersson and Rauno Pyykkö at SP, who
have helped me with the equipment set up and good advice trough out the project.
Niklas Strömberg has earned big thanks by showing patience with my questions and helping
me investigate a method for lipid measuring.
At last I would like to thank my family. They have always supported me in my decisions and
encouraged me to go my own way. Without them I would not be where I am today and for
that I am very thankful.
6
1. Introduction
Petroleum fuels are a finite source of energy and are also an environmental problem since
carbon dioxide is one of the products from burning them. A well documented fact is that
carbon dioxide is one of the green-house gases, and an elevated level in those will impact the
environment. Therefore, it is important to find renewable alternatives to petroleum fuels. Here
biodiesel and bioethanol are two alternatives with good potential.
Problems with renewable biofuels are that they often origin from oil crops or from other crops
like sugar-canes. Those take up a big land area when they are grown and compete with the
growth of food and agricultural products. In the U.S, nearly 61% of all agricultural cropping
land would be needed to produce enough biodiesel to cover their annual need of petroleumderived transport fuel [1]. This is not sustainable since their food production would be greatly
diminished. Other alternatives are sought and a lot of focus has lately been put on algae as a
source of biofuel.
Using algae as feedstock for energy is however not a recent idea. Between 1978 and 1996,
U.S. Department of Energy’s Aquatic Species Program [2] did a lot of research and concluded
that algae oil would not be economically viable at the current prices on crude oil. The same
conclusion was made in Japan after some extensive research on algae grown in closed
systems. Since then, the crude oil price has risen and at the beginning of the twenty-first
century a number of commercial and research efforts were started in the algae as fuel field.
Recently, there have been a further acceleration in activities around the subject [3]. The
research is mainly focused on the possibility to use the lipids in algae for the production of
biodiesel.
Except for producing lipids like other land crops, algae can thrive at high carbon dioxide
levels. A high carbon dioxide level can actually increase the rate of algae growth. Therefore it
is possible to use algae to reduce the carbon dioxide emissions from for example power
plants, while biofuel is produced in return.
1.1 Background
In 2009, SP initiated a project called “Microscopic algae as combined carbon sink and energy
source in Sweden”. The project is financed by Värmeforsk and started in September 2009.
The objective of this project is to answer the question “Can growth of microscopic algae in
photobioreactors work with economically, energy efficiently and climate gain as combined
carbon dioxide sink and energy resource in the Swedish climate?”. Step one was a literature
study done by Susanne Ekendahl [4]. Algae as biofuel and growing algae in photobioreactors
is a new area to SP and a literature study was a first step to gain necessary knowledge to
perform the rest of the project. Step two began with this thesis work with its focus on Swedish
light, carbon dioxide sequestering, biomass, lipids and preparation of laboratory equipment
for growing algae in artificial sunlight, laboratory testing and analysis. The prerequisite for
the laboratory testing is to use a weather-o-meter to simulate the light conditions in Sweden
[4]. The Weather-o-meter is of the model SUNTEST XXL/XXL+ from ATLAS Material
Testing Technology GmbH, Germany. The results of this thesis will be used for further
experiments where other growth conditions will be investigated.
7
1.2 Choosing algae species
When choosing the algae species for an algae production system it is important to know what
characteristics of the algae that are wanted. In plain talk, what are the algae going to be used
for after growing them, and under what conditions are they going to grow. When the purpose
is to use the algae as biofuel, the following characteristics are important: high lipid
productivity, ability to survive shear stress in photobioreactors, high carbon dioxide sinking
capacity, limited nutrient requirements, tolerance to a wide range of temperatures, a fast
productivity cycle and providing valuable co-products [5].
In this project, the original decision was to work with Botryoccoccus braunii, an algae species
with documented high lipid productivity. But since this is quite slow growing it would take a
long time to scale up in a preculture and each test would run for several weeks. That was not
realistic for this thesis work. Instead, Chlorella vulgaris was chosen as the algae to work with.
Chlorella vulgaris is a fast growing algae and it can withstand high carbon dioxide levels.
1.3 Objective
The objective of this thesis work was to test how well Chlorella vulgaris grow in Swedish
light conditions and under high carbon dioxide levels. The algae growth was planned to be
tested in Swedish weather conditions for four different periods, one cloudy and one sunny
summer period and one sunny and one cloudy winter period. To be able to do this,
photobioreactors for growing microalgae had to be built and tested and a method for
measuring lipid content in Chlorella vulgaris and its biomass had to be found. A secondary
objective was to gain necessary knowledge about growing algae in photobioreactors for future
projects.
1.4 Limitations
The project runs over a long time period which calls for equipment that can be spared over
that time period. The experimental setup needed to be adjusted to what kind of equipment that
is obtainable at SP and what can be bought. Since the experiments were to be conducted in a
weather-o-meter the space for the experimental setup was restricted. The size of the reactor
and consequently the volume of the tests, the number of replicates and the number of algae
species that can be tested are affected.
8
2. Theory
2.1 Algae and the algae cell
Microalgae are the simplest and most abundant form of plant life on earth and are responsible
for more than half of the world’s primary production of oxygen. Algae range from singlecelled algae (microalgae) to large seaweeds (macroalgae). The systematic classification of
algae is based primarily on their pigment components and the focus in this thesis lies on green
algae, also called Chlorophyceae [6].
Microalgae are found mostly in water but also in all types of soils. They are generally freeliving even though some microalgae live in symbiotic association with a variety of other
organisms. Microalgae are located all around the world [7].
Green algae are eukaryotes and their cell structure is basically the same as that of general
plant cells. The outer layer of the cell is composed of a cell wall made of a microfibrillar layer
of cellulose. A cell membrane is a thin membrane that bounds the cytoplasm, which contains
the organelles. Among the organelles there are chloroplasts, ribosomes, mitochondria,
vacuoles and a nucleus to name some. Chloroplasts contain altering layers of lipoprotein
membranes and aqueous phases, called the stroma, and it is the center of photosynthesis.
Ribosomes make proteins from amino acids. Mitochondria generate most of the cells´ supply
of ATP, a source of chemical energy. Vacuoles are membrane enclosed compartments
containing water and inorganic and organic compounds. The nucleus contains most of the
cells genetic material [7]. The simple development of algae allows them to adapt to existing
environmental conditions [5].
Algae can grow autotrophically or heterotrophically. Algae that grows autotrophically requires
only inorganic carbon compounds (such as carbon dioxide), salts and light energy to grow,
through photosynthesis. Algae growing heterotrophically require an external source of organic
carbon and nutrients as energy source. Mixotrophic algae can grow both autotrophically and
heterotrophically [5].
2.1.1Photosynthesis
An algae cell is constructed mainly for energy conversion through photosynthesis. Growth
through photosynthesis provides a way to convert and store light energy into chemical energy,
and this is done by the photosynthetic process [5]. This process converts inorganic carbon
compounds and light energy to organic compounds. E.g. carbon dioxide, water and light
convert to carbohydrates and oxygen.
6 CO2 + 6 H2O + light  C6H12O6 + 6 O2
The photosynthesis is divided into two different stages, the light reactions and dark reactions.
In the membranes of the chloroplasts the light reactions take place and light energy is
converted to chemical energy giving the products NADPH2 and ATP. NADPH2 is a
biochemical reductant and ATP is a high energy compound. The dark reactions take place in
the stroma and in this reaction NADPH2 and ATP is used in the reduction of carbon dioxide to
carbohydrates. [5]
9
2.1.2 Photorespiration
In the photorespiration organic carbon is converted into carbon dioxide. Whether
photosynthesis or photorespiration will occur depends on the relative concentration of oxygen
and carbon dioxide. A high O2/CO2 ratio stimulates the photorespiration and the opposite
favor the photosynthesis. Different photosynthetic organisms have different rates of
photorespiration. In microalgae mass cultures it is desired to keep photorespiration rates low
since high productivity is wanted. This can be achieved by CO2 enrichment and effective
removal of O2 and therefore, microalgae mass cultures are often grown in higher CO2/O2
ratios than found in air. [7]
2.1.3 Lipids in algae
Lipids are fatty acids and their derivatives. Substances related biosynthetically or functionally
to these compounds also counts as lipids. Macroalgae produce only small amounts of lipids
that are mostly components of the cell membrane, and they produce carbohydrates as their
primary energy storage compound. In contrast, many microalgae species produce lipids as
their primary energy storage molecule. Microalgae contain lipids and fatty acids as membrane
components, metabolites and energy storage products. The lipids are mostly located as
droplets in the cytoplasm and often appear close to the mitochondrion. Algae lipids are
primarily triglycerides (tri-alkyl glycerides). Triglycerides are made of three long chains of
fatty acids attached to a glycerol backbone. Algae lipids also consist of fractions of
isoprenoids, phospholipids, glycolipids and hydrocarbons. Algae oil contain more oxygen and
are more viscous than crude petroleum [3].
2.2 Algae as biofuel
Alternative energy resources such as biofuels have had some controversies in the past. The
first generation biofuels derived from terrestrial crops, such as sugarcane, sugar beet and corn;
affect the world’s food markets negatively, contributing to water shortage and the devastation
of forests. Second generation biofuels from lignocellulosic agriculture and forest residues
address some of these problems but the technology to convert those to liquid biofuel has not
yet reached the scales for commercial exploitation. Algae belong to a group of biofuels that is
called the third generation biofuels. Third generation biofuels are a technically viable
alternative energy resource that handle some of the problems with the first and second
generation biofuels [5].
The oil content of for example soybeans are 20% and for sunflowers up to 55%. Algae have a
original lipid content varying from 2-40%. This is not an extraordinary large number. The
promising facts in algae biofuel is their high yields per area unit, over 50 times more than
soybeans and sunflower are reported. This high yield is explained by a high photosynthetic
efficiency of algae, the fact that algae grow faster in grams per day than other oil crops and
that the simple structure of the algae makes it capable to use far more of its biomass for
energy production than land plants are able to [3].
In recent years it has been reported that about 1 percent (14 million hectares) of the world’s
available arable land is used for biofuel production and that this provides 1 percent of the
worlds global transport fuels [5]. If an algae species with an oil content of 30 % of its biomass
10
produce 1,535 kg biomass per cubic metres per day, it is possible to create enough biodiesel to
cover the U.S annual need of petroleum- derived transport fuel on only 3% of U.S cropping
area [1].
Oil productivity of microalgae cultures exceeds the yield of the best oilseed crop. For
microalgae grown in open pond systems the productivity can be 12 000 liters per ha and for
rapeseed you can get 1190 liters per ha [8].
Relying on physical laws and well-known values, Weyer et al. [9] calculated a theoretical
maximum value, working as an absolute upper limit of algal oil production. They found this
maximum to be 354 000 litres per hectare and year. They also calculated a best case scenario,
using more realistic but optimistic efficiencies, to provide a goal to algae producers. The best
case scenario was calculated to be 40 700-53 200 litres per hectare and year.
Biodiesel is a biodegradable renewable and non-toxic fuel. It emits less gaseous pollutants
than fossil diesel and it does not contribute to the net amount of carbon dioxide or sulphur in
the atmosphere [10]. The heating value of algae oil is very similar to that of fossil diesel fuel,
around 40-45 MJ per kilogram [3]. Biodiesel consist of fatty acid methyl esters (lipids)
originating from vegetable oils and animal fats. Most attempts at making biodiesel from algae
are done by first extracting the algae oil (lipids) and then treating the algae oil similar to any
other oilseed and thus use the same transesterification process to produce biodiesel.
Transesterification is the process where the triglycerides from vegetable oils and animal oils
are converted to fatty acid alkyl esters, and glycerol, using an alcohol. The reaction requires
heat and a strong base catalyst. Free fatty acids can also be converted to biodiesel using an
alcohol and a strong acid catalyst. This is a well understood way used to produce biodiesel
from vegetable oils and works equally well for algae oil [3].
Theoretically it is possible to produce microalgae biodiesel in a carbon neutral way thanks to
the possibility to use the algae oil for powering the production facility, or to let algae residue
after the oil extraction undergo anaerobic digestion and produce methane gas, which can be
used as an energy source for the production. Then the carbon dioxide produced from burning
the methane gas or algae oil go back into the process feeding the algae. This closed loop is
showed in figure 1.
11
Algal biomass
production
• Light, water, CO2 and
nutrients added
Power generation
Biomass recovery
• CO2 produced
• Water and nutrients
recovered
Anaerobic digestion
of rest product
• Biogas produced
Biomass extraction
• Algal oil produced
• Rest products
Figure 1: A carbon neutral way to produce algae biomass
An article by Brennan and Owende [5] listed ten advantages with using algae as biofuel
1. Microalgae cultures are capable of all year round production.
2. Even though microalgae grow in aqueous medium they need less water than terrestrial
crops
3. Microalgae production does not compete with food and other crop production since
they can be cultivated on non-arable land.
4. Microalgae have rapid growth potential and often high oil content.
5. Microalgae biomass production can affect biofixation of waste CO2.
6. Nutrients necessary for microalgae growth can be obtained from wastewater and
thereby algae work as a treatment of organic waste in waste water.
7. No herbicides or pesticides are necessary
8. After extracting the oil there are useful products left from the microalgae.
9. The oil yield of microalgae can be enhanced by altering the growth conditions.
10. Microalgae are capable of photobiological production of biohydrogen.
Looking at these advantages in using microalgae as biofuel it is clear that it has some great
potential of success, especially since fuel production can be combined with carbon dioxide
fixation, bio-treatment of waste water and is non-competitive to other crops. A system over
the use of algae is shown in figure 2.
12
Selection of
Micro Algae
species
Growth of Micro
Algae
Harvesting of
Micro Algae
Residual Micro
Algae
Extraction of Oil
from Micro
Algae
Extraction of
Protein for
Human food
Aqua feed,
Animal feed
Oil for
Processing inte
Biofuel
Waste Liquor
Recovery of
Valuable
Nutrients
BIODIESEL
Figure 2 : A detailed process of biodiesel from algae [3]
The development of microalgae for biofuel- production has some challenges and these are
addressed by Brennan and Owende [5] as well.
1. The species of microalgae must balance requirements for biofuel production and
extraction of valuable co-products.
2. A need to attain higher photosynthetic efficiencies by developing the production
systems.
3. Develop the techniques for the cultivation conditions to avoid evaporation, carbon
dioxide diffusion losses and other problems encountered.
4. Make sure there is not a negative energy balance after accounting for energy
requirements in water pumping, harvesting and extraction.
5. There are very few commercial plants in operation and therefore data and experience
of large scale plants are lacking.
There are clearly some steps that need to be taken and research that needs to be done before
algae oil can be produced in large scale but it is also clear that the potential for using algae as
biofuel in the future is hopeful.
2.3 Properties of algae in general and Chlorella
vulgaris in particular
There are a number of properties of an algae species that needs to be known before it can be
determined if it is suitable for biofuel production. Some of these properties are described here,
with a clear focus on Chlorella vulgaris.
2.3.1 Growth of Chlorella species
Species that belong to the genus Chlorella can be found in both fresh water as well as marine
water habitats. Chlorella sp. has a simple life cycle and simple nutritional requirements. It can
grow both photoautotrophically and heterotrophically, and is thereby a mixotrophic alga [7].
13
Mixotrophic mass cultivation was introduced from the initial stage of commercial production
of Chlorella in 1964, replacing photoautotrophic cultivation. Acetic acid is preferably used as
carbon source in outdoor mixotrophic cultivation [7]. A major problem with this kind of
cultivation is bacterial contamination in the system.
There are not many studies on the relationship between organic carbon assimilation and light
energy utilization. But it has been reported that the growth in mixotrophic cultivation was the
sum of heterotrophic growth and the growth induced by incident light, autotrophic growth.
Another study showed that the photosynthetic mechanism and the heterotrophic growth
mechanism seem to function independently in Chlorella vulgaris [7].
The first pilot plant for mass cultivation of Chlorella was tested in Boston, USA. Israel,
Japan, Germany and Czechoslovakia followed successively, each plant with different ideas
considering mass cultivation. The first plant built to cultivate Chlorella for commercial
purposes was in 1961 in Japan. The total culture area was about 4000 m2 and the plant was
constructed mainly for research and development of mass cultivation, with the focus on
growing Chlorella. Later, Chlorella was mass cultivated commercially for health food and
mariculture feed [7].
Chlorella vulgaris growing in 10 percent carbon dioxide was tested by Yoo et. al. [11] Under
the specified conditions Chlorella vulgaris produced about 105 mg dry weight per litre and
day. This is more than other algae, such as Botryococcus braunii but lower than for example
Scenedesmus sp.
2.3.2 Lipid content
Yoo et. al. [11]found that Chlorella vulgaris produced a total lipid content of about eleven
percent of the dry biomass, in an environment with 10 percent CO2. This was significantly
lower than in B. braunii that produced about 25 percent lipids compared to the total dry
weight[11]. Widjaja et. al. [10] showed that Chlorella vulgaris, growing under added carbon
dioxide, after 20 days of growth produced approximately 30 percent of its dry biomass as
lipids. They also showed that by growing Chlorella vulgaris in high carbon dioxide levels and
under nitrogen depletion the lipid content can reach over 50 percent of the algae dry weight
[10].
2.3.3 Carbon dioxide mitigation
In plants growing on land, the atmospheric carbon dioxide is enough to satisfy the carbon
requirement. In contrast to land plants, an addition of carbon dioxide greatly increase
autotrophic algae production.
Chlorella vulgaris ability to grow in high concentrations of carbon dioxide has been
demonstrated in multiple studies [11-12]. It has been showed that Chlorella vulgaris is a very
effective organism in sequestering carbon dioxide and that one kilogram of dry algae biomass
utilize about 1.83 kilogram of carbon dioxide [1]. Chlorella vulgaris grown on waste water
discharge from a steel plant sequestered 0.624 g CO2 per liter and day [13].
It has been showed that the total lipid content in Chlorella vulgaris can be affected by the
carbon dioxide concentration added. This is most probably caused by the fact that the growth
14
is much affected by carbon dioxide concentrations and therefore affects the productivity of
lipids [10].
2.3.4 Temperature
Different algae have different optimal growth temperatures. Chlorella vulgaris has a decreased
growth rate if the medium is above 25 degrees Celsius. They do still grow in temperatures up
to at least 40 degrees Celsius but not at all at the same rate as at 25 degrees C. [14]
2.3.5 pH
The pH of the growth medium affects the growth of Chlorella vulgaris. At pH 2 it is unable to
grow at all. At pH 4 the cell number is decreased. At pH 6 the growth is more rapid than at pH
8, but after 18 days the same levels of cell number were obtained in both pH 6 and 8. At pH
10 and 12 growth was reduced compared to pH 8. Even though Chlorella can grow in a wide
range of pH, it is desired to strive for a pH at approximately 6 [15].
2.3.6 Growth medium
To get a good algae production it is important to choose a growth medium that contains all
essential nutrients that the algae needs. The biggest contributor to the production of algae
biomass is carbon. Carbon can be added in organic compounds such as peptone or acetic acid
or as inorganic compounds such as carbon dioxide [7].
After carbon, nitrogen is the second most important nutrient contributing to the production of
algae biomass. Over 10% of the biomass content may consist of nitrogen. A lack of nitrogen
can cause a decrease in chlorophylls, an increase in carotenoids and accumulation of organic
carbon compounds such as polysaccharides and certain oils [7]. Widjaja et al. [10] found that
Chlorella vulgaris produced more lipids when exposed to nitrogen starvation. They also
found a gradual change in the lipid composition from free fatty acids-rich lipids to lipids
mostly containing triacylglycerides. Nitrogen is most commonly added to the medium as
nitrate (NO3-) [7].
Phosphorus is needed in many cellular processes for growth like energy transfer and
biosynthesis of DNA. The best way to supply phosphorus to algae is as orthophosphate (PO42)
in the medium. Lipid content and carbohydrates are affected by phosphorus supplies. [7]
Many other nutrients like S, K, Na, Fe, Mg and Ca are important as algae nutrition. Trace
elements such as B, Cu, Mn, Zn and Mo are also important and many of them are used in
enzyme reactions [7].
The water used in making growth medium is often distilled water, filtered water or de-ionized
water. This is not applicable in large scale facilities and in those, ground water or available
domestic water is used [7].
There are different ideas concerning what kind of medium that should be used when growing
Chlorella vulgaris. A common choice of medium is BG-11 [11, 16-17]. In BG-11, NaNO3 is
the nutrient in the largest amount, followed by K2PO4 [18]. This gives the algae enough
nitrogen and phosphorus to grow. Other combinations of nutrients are also frequently used
and they all have nitrogen and phosphorous in enough amounts [10, 12].
15
2.3.7 Light
Chlorella vulgaris has shown low saturation light intensity and inhibition at light intensities
lower than compared to other algae species such as Chlorella pyrenoidosa and
Chlamydomonas reinhardtii [19].
Using a strain of Chlorella vulgaris, Wijanarko et al. [20] showed that under a
photoperiodicity of eight or nine hours per day, the cell density was approximately 40 percent
higher than that under continuous illumination and they got a final biomass density of 1.43
g/dm3 at nine hours of light per day.
2.4 Light
Light, e.g. electromagnetic radiation is the energy source of the photosynthesis. The
wavelengths of light lies between 10-3 and 10-8 meters and based on the wavelength, light
can be divided into several groups. For example radio waves, gamma and X-rays, ultra violet
radiation and visible light [7]. UV-radiation is divided into different categories depending on
wavelengths, UVC (100-280 nm), UVB (280-315 nm) and UVA (315-400 nm). The visible
light is the radiation with wavelengths from 400 nm (violet light) to 750 nm (red light). It is
only the visible light that is utilized in the photosynthesis [21].
The fraction of light energy that is fixed as chemical energy during photoautrophic growth is
called the photosynthetic efficiency. As an average, light photons contain 218 kJ per mole. A
least eight moles is required to generate one mole of base carbohydrate (CH20), therefore
1744 kJ is used to create one mole of CH2O. Energy contained in one mole of CH2O is
approximately 467 kJ. This gives a solar-to-chemical energy conversion efficiency of
approximately 27 percent. Since only the visible light is used in photosynthesis, only 42,3
percent of the total energy from the light spectrum can be captured trough photosynthesis.
This gives a maximum photosynthetic efficiency on approximately 11,3 percent [5].
11,3 percent is a theoretical maximal value on photosynthetic efficiency and the actual value
is often much less. Terrestrial plants have photosynthetic efficiency levels at 1-2 percent [5].
Microalgae have higher values than terrestrial plants on the photosynthetic efficiencies thanks
to their simple structure. For example, a photosynthetic efficiency for Chlorella sp. on 6,56
percent has been reported [22].
The driving force of photosynthesis is light but it is also a limiting force. The light energy
received by the algae is a function of the photon flux density reaching the culture surface. The
cells only absorb a fraction of the photon flux, depending on multiple variables such as optical
properties of the cell and rate of culture mixing. The photons that are not absorbed are either
dissipated as heat or reflected. Microalgae mass cultures reflect only a small fraction of the
photons reaching a culture surface. Cell density will continue to increase exponentially until
all photosynthetically available photons are absorbed. Once the cell density are so high that
all photons are absorbed the cell mass accumulates at a constant, linear rate until light per cell
becomes overly low or if the level of some nutrient in the growth medium becomes low. [7]
The photosynthetic photon flux density required to saturate the photosynthetic units in the cell
is often only one fifth or one tenth of the photosynthetic photon flux density coming from the
light in midday. Even a relatively short exposure with a light dose much above saturation may
damage the photosynthetic units in the cells and reduce the productivity [7].
16
The higher the cell density, the shorter the light can penetrate into the culture. The zones
where light reaches and can support photosynthesis is called the outer illuminated volume and
the volume where photosynthesis cannot occur is called the dark volume. If the depth of the
culture is large it becomes complex to evaluate the light requirements for the culture.
Achieving an even distribution with a optimal light dose per cell from the light available may
also be difficult.
If there is a dark volume in the culture, the cells can be exposed to shading inside the reactor,
and the cells experience cycles of light and dark. This can be very short cycles, of
milliseconds or a few seconds long depending on how far down in the culture the light
penetrates and the amount of turbulence on the culture. How much light the cells are exposed
to at any given moment depend on the ratio between dark and light cycles and the frequency
of the cycle. With a higher frequency of the cycle it is possible to use strong light more
effectively. [7]
2.4.1 Light in Sweden
It is important to use free available sunlight to keep the cost as low as possible when growing
algae. The production is then affected by both daily and seasonal variations in light intensity
and the number of hours the light is available [1]. In Sweden the light intensity and the
number of hours that the sun is over the horizon varies geographically, especially since the far
south is located at latitude 54°37'48"N (Smygehuk) and the far north is located at latitude
69°03'36"N (Treriksröset) [23] giving a distance of approximately 1 572 kilometers.
The variation in the number of hours the sun is shining in Sweden in June and December is
showed in figure 3. The Swedish Meteorological and Hydrological Institute [21], defines a
sunny day as when solar radiation exceeds 120 W/m2.
17
Figure 3: Hours of sun per month, June to the left and December to the right. The numbers are mean values for
the years 1961-1990 [21].
During June the hours of sunshine in Sweden varies between 180 to 320 hours per month. In
December the variation is between 0 to 60 hours per month.
Figure 4 shows the variation in radiation in June and December in Sweden.
18
Figure 4: Mean sun intensity in kWh/m2 per month, June to the left and December to the right. The numbers are
a mean value for the years 1961-1990 [21].
For June the variation of mean radiation lies between 140 and 200 kWh/m2 depending on
geography. For December the variation of mean radiation is between 0 and 20 kWh/m2. Based
on numbers from SMHI [21] in hours of sun and light intensity in kWh/m2, the variation in
Sweden in terms of radiation in W/m2 is calculated and is approximately 100 W/m2 in June
and 25 W/m2 in December.
2.4.2 Weather-o-meters
Weather-o-meters are able to simulate sunlight. A SUNTEST XXL/XXL+ from ATLAS can
simulate sunlight at different intensities by using xenon arc lamps. The radiation cannot be
varied within one test run but dark cycles can be programmed. It is also possible to simulate
sunlight trough a window glass, as in green houses.
19
2.5 Photobioreactors
For over fifty years, algae cultures have been grown in open pond production systems. This is
the most natural way of growing algae and by adding circulation, in so called raceway ponds,
the algae growth can increase drastically. The major drawbacks with open pond systems are
the risk of contamination affecting the algae growth, the weather influences like rain and
evaporation and variation in light intensity and temperature. [5]
To overcome some of these problems the technique of growing algae in closed systems
developed. The equipments used to grow algae this way are called photobioreactors and they
have a better controlled environment than open ponds. Both the temperature and the gas
transfer can be controlled and the evaporation losses and the risk of contamination are
minimized. [5]
The equipment costs of open ponds are generally lower than the costs of photobioreactors, but
with respect to biomass productivity closed photobioreactors are better alternatives than open
ponds [3, 5].
Three different systems for closed photobioreactors are often discussed, tubular, flat plate and
column photobioreactors. The flat plate photobioreactors was of the earliest model and a lot of
research has been performed. Their main advantages are the large surface area exposed to
illumination and high cell density. The reactors are made of transparent material and a thin
layer of dense culture flows across the plate. They have a low accumulation of dissolved
oxygen and high photosynthetic efficiency compared to tubular versions [5].
Tubular photobioreactors have a large surface area exposed to sunlight and therefore is
suitable for outdoor cultivation. But the possible lengths of the tubes are limited and therefore
cannot be scaled up indefinitely. To create a large scale production system, multiple units need
to be integrated with each other. [5]
Column photobioreactors are made of vertical columns that are aerated from the bottom and
illuminated through transparent walls. They are the most effective when it comes to mixing
the culture; they have the highest volumetric mass transfer rates and the best controllable
growth conditions [5].
In a photobioreactor with continuous culture, fresh medium is added in the same rate and
quantity as the microalgae suspension is removed, or harvested [1]. A batch culture is when a
closed system of algae culture, in a fixed volume of medium, grows up to a certain density
and are then harvested and processed in a batch [7].
It is important to be clear on the purpose of the photobioreactors before designing and
building them. But regardless of what the purpose is, there are some basic principles to
consider.
2.5.1 Lighting
Ideally the maximum growth rate of microalgae would be the same as the maximum rate of
the photosynthesis. When growing algae in a photobioreactor, a vessel with specific
dimensions are used and a finite amount of solar radiation can reach into the reactor. And light
cannot always penetrate the culture all the way through. This may lead to overexposure of
20
light for the algae in the outer layers and insufficient light in the inner volume [7]. The kind of
light, sunlight or artificial light that is to be used affect the design of photobioreactors. It is
also necessary to plan for a dark period and consider how long it should be.
2.5.2 Temperature
The temperature needs to be in the range of what the algae species can survive. When the
algae suspension in the reactor is exposed to sunlight the temperature may rapidly go up and
cooling during daylight is often essential to keep a good growth temperature [1].
2.5.3 Gas and mixing
Sufficient mixing in the culture is important to get the algae to grow well and to prevent the
biomass from sedimenting. Highly turbulent flow in the tubes is often created by a pump or
by bubbling with the added air or gas.
If a continuous light source is to be used and a high productivity is sought, a way to get the
cells to move from the dark volume to the illuminated volume is to make sure that sufficient
stirring moves the cells from one volume to the other. A high mixing rate is in that way
essential to achieve a high productivity [7].
2.5.4 Axenic cultures and sterile environment
Contamination should be avoided to prevent the algae growth to be affected by other algae,
bacteria or viruses. Axenic cultures are cultures containing only one species. Ergo, only one
type of algae and no bacteria are present [7]. The easiest way to make sure to start with an
axenic culture is to order an already isolated alga from a lab or a company. To keep a culture
axenic it is needed to avoid contamination by keeping the equipment and working area as
sterile as possible.
A good way to keep surfaces free from bacteria is to clean them with 70 percent ethanol.
Filters with membranes with very small pores, which capture cells, are a good way to keep
flowing gas or fluid sterile. By autoclaving equipment, the high temperature and pressure will
kill any livings cells. The high temperature in an open flame can also be used to make things
sterile. Bacterial cells cannot survive a long time exposure to UV light. Thanks to this,
equipment can be put under a UV-lamp to make them sterile [24].
Many of the techniques are not realistic for large scale production plants, but it is always
important to keep equipment and areas as clean as possible and realistic for every individual
case.
2.6 Analyzing the algae
2.6.1 Cell count
To perform a cell count successfully it is often required that cells are separated individually
and it is often necessary to dilute the sample to be able to use a microscope to count the cells
in a counting chamber [7]. Counting chambers are precision measuring instruments used to
count cells or other particles in suspensions under a microscope [25].
21
2.6.2 Biomass
The biomass of an algae culture is most often measured by weighing the dried biomass. It is
possible to measure the wet biomass but it is far less accurate than measuring the dry biomass.
Dry mass is measured by first separating the algae cells from the medium by either membrane
filtration or centrifugation. The filters or centrifuge tubes should be pre-weighed before
separating the algae. Some cleaning of the algae cells with distilled water is preferable before
drying them. Relatively low drying temperatures (60-100 degrees C) are often used to prevent
loss of volatile components. After drying, the filters should be weighed in the same room
temperature and moisture climate as they were pre-weighed in, to avoid moisture
accumulation in the filters [7]. It is important to make sure the filters have small enough pores
to prevent algae cells to slip trough. The biomass of Chlorella vulgaris has been measured
gravimetrically by Yeoung-Sang Yun et al. [26] who filtered 5 ml algal suspension through
pre-weighed 0,45µm filterpapers. They were dried over night at 90 degrees and then weighed.
2.6.3 Lipid content
Many different methods on how to measure lipid content are available in the literature. Bligh
and Dyer [27] created a method in 1959, where the lipid content is determined gravimetrically
after extraction using chloroform and methanol. Since then, scientists have developed this
method to find a more time effective way to determine lipid content of cells. Lee et al. [17]
compared different methods for cell disruption before extracting the lipids, but the matter of
time was still there. If lipid content is to be measured often and in many replicates it is
required that the method is time efficient.
A fluorescence method using Nile red to dye the living cells is a way to measure lipid content
without having to extract the lipids. If the goal is to find the quantitative amount of lipids in
the cells and not to extract them for further use, such a method is suitable. Methods like these
have been described in many research projects. In 2007, Elsey et al. [28] pointed out the
importance of daily calibration of the fluorimeter and reported modifications to the Nile red
assay that improved its´ ability to screen for neutral lipids in different cell types. Alonzo et al.
[29] showed a linear relationship between fluorescence intensity and lipid concentration. They
also showed the importance to use specific lipids as standards to get absolute quantification of
neutral and polar lipids, and not use commercial lipid standards or lipids obtained from other
species since they gave large errors.
A Nile red method used for quantitative measurements of lipids in green microalgae was
evaluated and modified by Chen et al. [16]. A previously used method applied to microalgae
was evaluated for nine species of microalgae. It was discovered that the green algae strains
were not giving a strong fluorescent signal. Modification to the method was made and an
optimized way to first stain microalgae and then measure the fluorescence to determine the
amount of lipids in the cells was found. The algae chosen by Chen et al. to develop this
method was Chlorella vulgaris.
22
3. Methods and Development
3.1 Preculture of Chlorella vulgaris
Chlorella vulgaris, strain # 1803 was ordered from UTEX [18], (Austin, Texas) and was
delivered on proteose agar medium. The algae were grown as a pre-culture, in flasks
containing liquid proteose medium. The algae were successively grown to a larger cell density
and continuously inoculated to keep them alive.
Due to bacterial growth in the photobioreactors the medium was changed to Bristol medium.
Bristol medium, e.g. proteose medium without peptone, was enriched with inorganic carbon
by bubbling air.
To test how Chlorella vulgaris grew in Bristol medium, an algae culture was bubbled with 10
percent carbon dioxide. This showed that Chlorella vulgaris grew well under this condition.
For about one hour, every weekday, the flasks in the preculture were bubbled with ten percent
carbon dioxide instead of air. This was done to increase the carbon supply to the algae and
increase their growth rate.
The growth rate was much slower in Bristol medium than in proteose medium so the air
bubbled was necessary to make the growth relatively fast. The gas was filtered before entering
the flasks to minimize bacterial contamination. BG-11 medium [18] would have been another
option but at that time, all the chemicals were not available to mix and use BG-11. Since the
algae grew well in the Bristol medium this was kept as growth medium.
The light source to the pre-culture was regular 18W fluorescent lamps (OSRAM L 18w/77),
set to a light and dark cycle of 16:8 hours and the culture were kept in room temperature,
approximately 20 degrees C.
Figure 5: Preculture of algae, bubbled with air.
23
3.2 Determination of testing conditions
To investigate algae growth during summer and winter in Sweden, it is important to know
what kind of light intensities are representative at that time. June was chosen as a
representative summer period and December was chosen as winter period.
Figure 4 shows the variation in radiation over Sweden. The radiation within Sweden differs
with in fact only about 60 kWh/m2 in June and with about 20 kWh/m2 in December. With this
in mind the decision to take radiation data from only one site in Sweden was made. The light
intensity conditions were investigated in Umeå. Umeå is located in the northern part of
Sweden at latitude: 63° 50’ North and longitude 20° 15’ East.
Umeå University has a weather station with data on incoming light intensity in watts per
square meters. The intensity is measured with a pyranometer sensitive to light from 400 nm,
ergo measuring in the visible light spectrum. This data is used to evaluate radiation conditions
in Umeå. The mean value of radiation, over the hours the sun is above the horizon, is
calculated for the first to last of June 2001, 2005 and 2009. In Umeå, the sun is above the
horizon on average 20 hours per day in June and five hours per day in December [30].
Since the definition of sunny weather is when the radiation is above 120 W/m2 a mean value
on days with a mean radiation over 120 W/m2 were calculated to 293,71 W/m2. The same was
done on days with radiation below 120 W/m2 and this became 76,32 W/m2.
Data from December 2001, 2005 and 2009 was investigated in the same way as for June. The
mean value never exceeded 120 W/m2 on a day in Umeå in December those years. By
definition [21], there are no sunny days in December. The mean value over all winter days
was 9,36 W/m2.
Year
2001, 2005 and 2009
2001, 2005 and 2009
2001, 2005 and 2009
2001, 2005 and 2009
Month
June
June
December
December
Sun*
Cloudy**
Sun
Cloudy
Average solar radiation
293.71 W/m2
76.32 W/m2
9,36 W/m2
Table 1: Light intensities in Umeå, *radiation above 120W/m2, ** radiation below 120W/m2
Based on this data, it was decided to simulate a sunny period during the summer with the
weather-o-meter set to 300 W/m2 and a cloudy summer period by setting the weather-o-meter
to 100 W/m2 in the visible spectrum. Radiation as low as 100 W/m2 was not possible to obtain
from the weather-o-meter. Therefore, a piece of off-white cotton fabric was used as filter to
obtain this radiation level.
The radiation in the weather-o-meter was measured with a spectroradiometer (Optronic
laboratories Inc.). The weather-o-meter was set to simulate sunlight through a window glass,
representing growth in greenhouses. The window glass filters the UVC and UVB radiation
but UVA radiation and all visible light was let trough. When the intensity was 300 W/m2
visible light about 40 W/m2 in the UV spectrum reach the reactors. At 100 W/m2 visible light
about 3 W/m2 in the UV spectrum reached the reactors.
Since the data showed that, by definition, there was no sunny day in December it was decided
that only one period should be tested for winter. And the period should have the light intensity
24
at approximately 10 W/m2. This cannot be simulated by the weather-o-meter so instead two
fluorescent lamps were used. Their light intensity was measured to 9,6 W/m2 with zero UV
radiation. Instead of a sunny period during winter, it was decided to growth under added
artificial light during winter to get an idea of if and how much added light is necessary during
winter.
3.3 Building the photobioreactors
One of the challenges in building the photobioreactor was that it had to fit in the weather-ometer. The weather-o-meter demanded that the containers cannot be higher than 5 centimetres
since the light distribution in the weather-o-meter are affected if the reactors are higher than
that.
Figure 6: The weather-o-meter used in all laboratory testing
Algae should be grown in two separate containers making duplicate samples for the analytical
testing. The photobioreactor was planned to simulate algae cultures grown in plastic bags,
since this is a common material used for photobioreactors. Also, it is simulated that the plastic
bags are located in a green house since the outdoor temperature in Sweden often are too low
for algae cultivation otherwise.
3.3.1 Containers
Two containers were designed for growing algae and they were prepared by the workshop on
SP. Four entries and exits were created to be able to connect gas tubes to the containers. One
extra exit was made to be able to measure gas out of the reactors. An edge was formed at the
25
top of each side to have something to attach a plastic cover to. The containers were made in
stainless steel since this tolerates water and the salts in the medium.
Gas flow out
Edge for plastic attachment
Gas flow in
Figure 7: Containers in stainless steel for cultivation of algae.
3.3.2 Plastic
The plastic on top of the containers needed to let light trough. Two kinds of plastics were
available at SP, both of polyethylene, one with process stabilizers and one without. They were
tested in terms of transmittance (see Appendix A).
The difference was neglectable and the polythene with process stabilizers was chosen so it
might last longer in strong light.
The plastic filters some of the light from the weather-o-meter. When it was set to 300 W/m2
visual light, the algae suspension was exposed to 268 W/m2 visual light and 30 W/m2
ultraviolet light. When the weather-o-meter was set to 100 W/m2 visual light, the algae
suspension was exposed to 65 W/m2 and 3 W/m2 ultraviolet light.
3.3.3 Gas and mixing
The concentration of carbon dioxide was set to 10 percent since this is close to the
concentrations found in flue gases (Vattenfall, Uppsala). Carbon dioxide, hundred percent,
was diluted with pressurized air to achieve the desired carbon dioxide level. The dilution
devise (Signal, Series 850 GAS BLENDER) used to mix the gases has maximum possible
flow rate at 2,636 litres per minute. The gas flow rate was measured by a flow meter (Gilian
Gilibrator-2). The flow rates were determined partly on how much mixing the suspension
should have and how much carbon the algae should be supplied with. Maximum flow rate,
2.363 litres per minute, was chosen to create as much mixing as possible in the reactors.
3.3.4 Sterile Environment
To avoid contamination in the reactors different techniques were successively tested to keep
them sterile. Before starting the first test, everything belonging to the reactors was cleaned
26
with 70 percent ethanol and the growth medium was autoclaved. Other techniques were tested
as the work proceeded.
3.3.5 pH
The pH of the algae suspension in the reactors was measured several times at each test with
indicator paper to see if there was any change due to algae growth or other changes in the
reactor.
3.3.6 Assemble the photobioreactors
The gas mixture was drawn to the weather-o-meter by Teflon tubes and connected to the
containers by Teflon connectors. Teflon is gas-proof and will not be affected by the light
exposure or the chemicals in the growth medium. The plastic was attached to the steel edge
with a butyl rubber lining.
Figure 8: Reactors mounted and connected in the weather-o-meter.
3.4 Test and development of algae growth in
photobioreactors
To develop a way to successfully cultivate the algae in the photobioreactors, a stepwise
approach was used.
3.4.1 Test number one- Functions of photobioreactors
The first things that were tested is how the bubbling works, if the plastic was sufficiently
attached to the reactor, if the environment was sterile enough to keep bacteria growth low and
to see if and how the algae grows.
The reactors and everything belonging to them was wiped clean with alcohol, the weather-ometer as well. The reactors were installed in the weather-o-meter and it was set to 300 W/m2.
The reactors were filled with 2.5 litres of proteose medium and 40 ml of algae suspension.
After only one day the temperature reached almost 50 degrees C in the reactors. The radiation
entered the reactors causing heat to accumulate and the algae suspension in the reactor
became too warm for growing Chlorella vulgaris. The heat made the plastic bubbly and the
moisture caused the butyl butyl rubber to let go of the plastic.
27
Cooling coils needed to be installed into the reactors and the plastic attachment needed to be
investigated after this.
3.4.2 Test number two- Temperature regulation
A system for temperature regulation inside the reactors was installed and tested.
Two of the gas connections were used to install a Teflon tube into the reactor. When cold
water flows through the tube it works as a cooling coil. The cooling coils were connected to
an immersion cooler so the temperature of the cooling water could be regulated depending on
the light intensity. The installed cooling system was tested over night. The reactors were filled
with deionised water and covered with plastic. The weather-o-meter was set to 300 W/m2 and
a thermocouple was mounted in one of the reactors to measure temperature.
The temperature in the reactors was stable at 25 to 27 degrees Celsius when the cooling water
was set to 12 degrees Celsius.
In further tests the cooling temperature was set to 12 degrees C when the radiation was 300
W/m2.
3.4.3 Test number three – Stability of temperature and plastic
attachment
Next, it was tested how algae survived in the reactors, if the temperature would stay stable at
a good growth temperature and if a new way to attach the plastic was needed.
The reactors were cleaned with alcohol and 40 ml of algae suspension was added to 2.5 litres
of proteose medium. In one of the reactors butyl rubber was attached both on top and under
the metal edge, supported with some metal clamps. The other reactor was set up as before.
The weather-o-meter was set to 300 W/m2. The temperature was logged. A dark period was
not used in this test since the cooling system is not get built and programmed to follow the
light going on and off. The test was run for six days.
The temperature was stable around 27 degrees Celsius the whole time. The algae did not grow
in this test but there were a lot of bacterial growth. There was no apparent difference in the
two reactors when it came to the plastic attachment in the reactors.
A probable cause to the lack of algae growth is the fact that there was no dark period. The
bacteria growth may also have affected the algae growth negatively. It seemed difficult to
keep the environment sterile enough to grow algae in proteose medium. Bristol medium was
therefore tested to try to avoid bacteria growth. The old, less complex way to attach plastic
seemed enough when the temperature in the reactors was not too high.
3.4.4 Test number four – Growth medium
In this test, it was tested how Bristol medium would work, compared to proteose medium, in
the reactors.
One reactor was filled with 2.5 litres Bristol medium and one with 2.5 litres Proteose medium.
The algae added suspension come from proteose medium since there was not get enough
algae growth in the Bristol medium in the preculture. 150 ml of suspension was used this time
28
to make sure that the algae suspension was not too small. A fluorescent lamp, as those used
for the preculture of algae, was used as light source to eliminate the light intensity as an
uncertainty factor. The lamp was on for 16 hours and off for 8 hours and the test was run for
three days.
In both reactors bacteria growth occurred. In the reactor with proteose medium a lot of
bacteria were found and the algae cells had not survived. In the reactor with Bristol medium
some bacteria growth was found but a lot less than in the proteose medium. The algae were
alive but no apparent growth was observed.
The bacteria growth could be explained by the fact that both reactors contained some proteose
medium from the inoculums. This would explain the lesser growth of bacteria in the Bristol
medium. Proteose medium should not be used in the reactors.
3.4.5 Test number five – Avoiding contamination
The focus was still on finding a way to eliminate bacteria growth. Ten percent carbon dioxide
was added to the preculture as described in “Preculturing of Chlorella vulgaris”
To further improve the sterile environment of the reactors they were put in UV light for two
hours before they were installed in the weather-o-meter. The reactors were covered with
sterile aluminium foil until the reactors were covered with plastic. A filter was put on the
incoming gas. Ampicillin was added in one (100mg/l, SIGMA) reactor to see if it made any
difference for the bacteria growth. 150 ml of algae suspension was added in 2,5 litre Bristol
medium. The algae suspension was still from proteose medium since there were still not
enough algae grown in Bristol medium in the pre- culture. The light source was still a
fluorescent lamp.
The growth of algae was apparent and visible but some bacteria were still growing. No
difference between the reactor with ampicillin and in the one without was observed. The
bacteria growth was less than in the last try with Bristol medium.
The bacteria growth could once again be explained mainly by the occurrence of proteose
medium in the algae inoculum. The bacteria growth did not take over and kill the algae. It
seemed to be a more sterile environment with the filter and UV treatment since the bacteria
growth was less than in the reactor with Bristol medium in the last try. These steps was added
in upcoming tests.
3.4.6 Test number six 6 – Periodicity of cooling system and algae
growth during sunny summer intensities
At this point it had been tested and proved that the algae survived and grew in Bristol
medium, in 10% CO2 and in the stainless steel containers. A way to keep the reactors sterile
enough for the algae to survive had also been found. The next step was to see how the algae
could survive a light intensity on 300 W/m2 (sunny weather) and therefore a cooling system
that could be regulated with the dark and light periods, were created.
The light intensity was set to 300 W/m2 in the weather-o-meter and the light period was set to
16 hours followed by 8 hours of dark. The cooling system was developed so that the
temperature of the cooling water was set to the same periodicity as the light. One hour before
29
the light turned on; the immersion cooler was set to bring the cooling water to 12 °C. When
the light was off, the immersion cooler turned off and the cooling water rose to 25 °C. The
temperature of the medium was logged. 100 ml of algae suspension from Bristol medium was
added.
The weather-o-meter stopped after only two hours of light because the detector that regulates
the conditions in it was not positioned right, with the consequence that it shut down. No
bacteria were seen in the medium. 100 ml new algae suspensions were added to each of the
reactors and they were started again. After three days there was still no growth observed so
another 150 ml of algae suspension was added to each of the reactors and left for another
three days. Growth was still not observed. When disconnecting and emptying the reactors it
was seen that some living algae was hidden under the cooling coils. The temperature of the
medium in the reactors was always between 22-30 degrees Celsius.
The reason to the algae growth under the cooling coils was most likely an effect of the
shading provided by the coils. The lower temperature close to the coils should not be the
reason to the hidden algae, since the temperature of the medium was in the range of what
Chlorella vulgaris should be able to survive. The algae did not survive at a light intensity
exposure of 300 W/m2.
3.4.7 Test number seven – Algae growth during cloudy summer
intensities
A light intensity of 100 W/m2 representing cloudy summer was tested next.
A sheet of fabric was put on top of the plastic to bring down the light intensity to 100 W/m2.
There was still a light and dark period of 16:8 and the test was run for nine days.
On day 6, an apparent growth in the reactors was observed. The reactors were opened
completely and the algae were mixed to get homogenous algae suspension. The gas had a
hard time to produce bubbles since the algae had sealed the bubbling holes. 100 ml algae
suspension from each reactor was taken out to be analysed. The plastic was reattached to the
reactors. There were some problems to get the plastic to reattach to the reactor since the butyl
butyl rubber became wet. The biomass was measured and a cell count was done from each of
the two reactors. This was done the following 4 days before the test was ended. The biomass
reached almost 0.7 grams per litre on the last day of testing.
The problems with the plastic that were discovered were not big enough to spend time on
adjusting. The gas coils needed to be cleaned at the same time as the manual mixing was
done, to guarantee a good gas flow. The biomass yield was good and a successful way to grow
algae in the photobioreactors inside the weather-o-meter had been found.
3.5 Summer test – Algae growth during cloudy
summer
The method development had lead to a point where the photobioreactors no longer needs
improvements or alterations to work properly. A test with the conditions of a sunny summer
day was not tested further since the algae did not survive in those light intensities. Instead,
this test simulated a cloudy summer period in Umeå. A light intensity on 100 W/m2 was set,
with four hours dark period and 20 hours light period. 150 ml of algae suspension was added
30
to 2,5 litres of Bristol medium and the number of cells added in the suspension was
calculated. The biomass was measured on day 4 to 7 and 10 to 13. 50 ml of algae suspension
was taken from each reactor and frozen at minus 20 °C for future lipid content investigations.
Fresh medium was added in the same amount as what was taken from the reactors.
3.6 Winter test – Algae growth during winter
This test was going to simulate a winter period in Umeå. A light intensity of 9,6 W/m2 was
simulated by two fluorescent lamps and they were set on 19 hours dark period and five hours
light period. The reactors were set up as in the cloudy summer test. The biomass was
measured only on day 13 since the algae growth was too low to get a result. The winter test
with added artificial lighting was never conducted since there was no time for it.
3.7 Test and development of analyzing methods
While developing the photobioreactors some analyzing methods were tested and developed to
be able to analyze the alga growth properly.
3.7.1 Measuring biomass
Measurements of the biomass in the algae cultures were done gravimetrically. Filters
(Whatmann GF/C 1.2 µm, 55 mm in diameters) were stored in a room with controlled
temperature (23±2°C) and moisture content (50±5%) and were pre-weighed. The algae
suspensions were filtered through the filters. Some deionised water was run through the filters
to rinse them from any residues from the growth medium. The filters were then dried over
night in 100 °C. The filters were again stored in a room with controlled climate conditions
and are weighed when they had reached constant weight. The biomass was measured in
triplicates. Taken into consideration the filtration time and standard deviation of samples it
was found that filtering 30 ml of algae suspension in each test was appropriate for a biomass
test.
3.7.2 Cell count
The number of algae cells was counted using a Bürker counting chamber [25]. The numbers
of cells was counted in 25 squares. The mean value of number of cells per square was
multiplied with 4*106 to get the number of cells per ml suspension.
3.7.3 Component analysis
When the summer test was completed a component analysis was done on the algae
suspension. This gave the inorganic and organic carbon content as well as the nitrogen and
hydrogen content. From these values it is possible to calculate a approximate value on the
carbon dioxide uptake by the algae. The effective heating value was also analyzed.
3.7.4 Measuring lipid content
Since Chlorella vulgaris was the algae chosen for this thesis, the method described by Chen
et. al. [16] was chosen. The article describes an optimum procedure how to dye and prepare
the algae before measuring the lipidcontent.
31
Nile red dye was created by mixing Nile red powder with DMSO (dimethyl sulfoxide). It was
important to mix the Nile red powder with 100 percent DMSO first, before diluting the
DMSO to 25 percent aqua solution. If the powder was mixed with 25% percent DMSO it
would not dissolve.
It needed to be investigated if the TLC- apparatus (CAMAG, TLC Scanner 3) own by SP
could replace the Varian 96-plate spectrofluorometer used in the described method, to
measure the fluorescence.
Since a microtiterplate did not fit in the TLC-apparatus it was tested if the lid of the plate
could be used due to its slimmer shape. A small drop of the sample was put on the plate. The
lid did fit, but the bubble-shaped sample disturbs the signal so much that it was not useful.
The main problem causing this interruption was the fact that the light comes from above in
this machine and not from the bottom as in other fluorescent-measurement apparatus. That
causes the signal to read the sample in different distances instead of a flat sample (see fig 9).
Instead, filter papers were used to keep the samples. This way a droplet shaped sample was
avoided.
Figure 9: Light signal in TLC- apparatus was disturbed by the uneven shape of the sample
Nile red was mixed with Triolein as a lipid standard to create a fluorescent mix. Triolein was
mixed with DMSO to create solutions containing different amount of lipids. As described by
Chen et. al. [16] six solutions in the following concentrations, 20; 17; 14; 11; 8; 5; 2 µg/ml
was made. A lipid stock solution of 20,0 µg per ml was made by weighing 0.002 g triolein
and mixing it with 100 ml 25%- DMSO.
The lipid standard mixtures were dripped on the filter papers and dried before measuring
fluorescence. These standards did not give any significant fluorescent signals, so instead lipid
solutions with corn oil in much larger concentrations were used. Some fluorescent signals
were found from these lipid solutions but the signal strength did not correspond to the lipid
concentrations. Since there is a long way from finding a florescent signal to actually having
an operating method for quantifying lipids in algae cells, it was decided that the method was
not viable for this thesis work.
32
4. Results
4.1 Test and develop the algae growth in
photobioreactors
All tests preformed to develop the photobioreactors are summarized in table 2 below. The
variables tested each time, what the results was and what kind of conclusions were made, are
presented.
Test
Variables tested
Results
Conclusions made
1



Aeration and mixing
Plastic attachment
Sterile environment with alcohol
cleaning



2

Cooling system

3



New plastic attachment
Temperature stability
Algae growth




No problem with plastic
Stable temperature
No algal growth
High bacteria growth
4

Bristol medium compared to proteose
medium.
Fluorescent lighting

Elimination of bacteria growth by gas
filter, UV treatment.
Ampicillin compared to no
ampicillin.
Algae growth
Growth in 10% CO2 in preculture

A cooling system that follows the
light and dark cycle.
Algae growth in 300W/m2

Algae growth in 100W/m2 light and a
dark period of 16:8

Bacteria growth is present in
both media but much less in
Bristol
Some algae alive in this
lighting
Some bacteria growth but
less than before.
No difference with
ampicillin
Apparent algae growth.
Algae grew in 10% CO2 in
the preculture
The temperature in the
reactors varied between 2230°C.
The algae survived under the
cooling coils, not in direct
exposure to 300W/m2
Apparent algae growth, up to
0.7g/l on day 13.
Algae hindered the gas to
flow freely

5




6


7








High temperature (50°C),
Problem with plastic
attachment
Test too short to conclude
sterility
Stable at 25-27°C with
cooling temp 12°C
Cooling system needs to be
installed to create a good
growth temperature.

Plastic attachment was affected
by the high temperature.

12°C in cooling water creates a
good growing temperature for
Ch.v.

The original way to attach
plastic was enough.

Algae need a dark period.

Bristol medium should be
tested to avoid bacteria growth
All growth of Ch. v. should be done
in Bristol medium









Use UV treatment and filters
Don’t use ampicillin.
The material in the reactors do
not affect the algae growth
The cooling system works.
Algae do not survive in direct
light exposure at 300W/m2 in
these reactors
Algae grow well in 100W/m2.
Suspension needs to be
manually mixed before
sampling.
Gas tubes needs to be cleaned
when algae growth is high.
Biomass and cell count can be
tested.
Table 2: A summary of the results of the development of the photobioreactors.
4.1.1 Growth medium and contamination
Both proteose medium and Bristol medium worked for the growth of Chlorella vulgaris but
Proteose medium had a higher growth rate. This was visibly observed in precultures. When
enhancing Bristol medium with carbon dioxide the growth rate was observed to be higher
than without. The proteose medium had a much higher sensitivity to bacterial contamination
33
due to its organic carbon content. Bristol medium in combination with UV-light exposure and
alcohol cleaning of the equipment and a filter on the incoming gas was enough to keep the
bacteria growth as low as necessary not to compete with the algae growth.
4.1.2 pH
The pH was stable between 5 and 6 at all measurements preformed.
4.1.3Temperature and cooling system
A cooling system in the photobioreactors was necessary to keep the culture at a good growth
temperature. This cooling system had to be able to adapt to the strength of light. A simple
system with temperate water running through Teflon coils worked well to keep the suspension
at good growth temperature. An immersion cooler programmed after the number of light
hours kept the temperature stable regardless of light intensity.
4.1.4 Ten percent carbon dioxide
Chlorella vulgaris were able to survive and grew well in ten percent carbon dioxide.
4.1.5 Bubbling and mixing
Adding the gas worked well from the beginning. When the algae growth was high, the tubes
needed to be cleaned so that the gas could flow through them. The gas was supposed to work
as mixing of the algae suspension but this did not work well. The algae dropped to the bottom
of the reactors and manual mixing was needed to achieve a homogeneous suspension during
sampling. A higher gas flow would not fix this problem.
4.1.6 Plastic
The plastic withstood the light exposure well during the short time periods used. The
attachment worked as long as the reactors were not opened. When opened the butyl butyl
rubber became wet and the plastic did not completely stick to the containers.
4.2 Chlorella vulgaris grown at different light
intensities
During 300 W/m2, representing light intensity in sunny summer period with eight hours of
dark period, the algae did not grow except under the cooling coils. The light intensity was too
high.
34
Figure 10: Algae growth under cooling coils
4.2.1 Summer test – Algae growth during cloudy summer
With a light intensity of 100 W/m2, representing a cloudy summer day, the biomass analysis
showed algae production up to 1.2 grams per liter. The biomass was measured from day 4 to
13 and the growth is displayed in figure 11 and table 3.
1,4
1,2
Biomass (g/l)
1
0,8
0,6
Reactor one
0,4
Reactor two
0,2
0
1
3
5
7
9
11
13
Number of days
Figure 11: Biomass measured on day 4, 5, 6, 7, 10, 11, 12 and 13.
35
Reactor one
Biomass (g/l)
Mean of triplicates:
Reactor two
Biomass (g/l)
Mean of triplicates:
4
0,031111
0,010184
5
0,086667
0,011547
Standard
deviation
0,003849
0,017778
0,006667
0,033333
6
0,175556
0,015753
0,081111
0,005092
7
0,223333
0,005774
0,143333
0,011547
8
-
-
-
-
9
-
-
-
-
10
0,517778
0,062391
0,603333
0,012019
11
0,694444
0,020367
0,624444
0,410884
12
0,76
0,024037
0,752222
0,040046
Day:
Standard
deviation
13*
1
1,2
Table 3: Biomass and the standard deviations from the summer test. *on day 13 only one biomass test was done.
When running the reactors for 13 days a lot of evaporation water losses were observed.
Almost one deciliter of growth medium per day evaporated and should have been replaced by
new medium. Taking the concentrating effect of the evaporation into account, the average
algae growth in grams per liters and day and grams per day in the reactors are presented in
table 4.
Average growth rate
g/l*day
g/day in reactor
Reactor one
0,076923
0,107692
Reactor two
0,092308
0,129231
Table 4: Average growth rate in g/l*day and g/day in the reactors, in summer test
4.2.2 Winter test
Algae did grow 10 W/m2 and five hours of light, representing winter conditions, but the
growth was very slow. When the light period was 16 hours at equivalent light intensities in
precultures, the algae growth was visibly higher. The growth was so low that biomass testing
was not practically possible to perform. Only when emptying the reactors on day 13 it was
possible to do a biomass test. It showed that the biomass was 0.012 grams per liter in reactor
one and 0.02 grams per liter in reactor two.
Day
Reactor one
Reactor two
Biomass (g/l)
Standard Biomass (g/l)
Standard
Mean of triplicates: deviation Mean of triplicates: deviation
0,012222 0,008389
0,02 0,016667
13
Table 5: Biomass and the standard deviation from winter test.
During the winter condition the evaporation losses were much smaller, approximately 0,2
deciliters per day. This was not replaced with new medium and taking the concentrating effect
of the evaporation into account, the average algae growth in grams per liters and day and
grams per day in the reactors are presented in table 6.
36
Average growth rate
Reactor one
0,00094
0,002106
g/l*day
g/day
Reactor two
0,001538
0,003446
Table 6: Average growth rate in g/l*day and g/day in the reactors, in summer test.
4.3 Analyzing methods
4.3.1 Biomass
The standard deviation on three 30 ml samples from each reactor was ±0,045 g per liter. The
source of error when measuring biomass lies in the weighing of the filters since the weights of
them are low and they are measured in 4 decimals and in the sampling of the algae since the
suspension was hard to get homogenous.
4.3.2 Cell count
By counting the cells at the same time as measuring the biomass in the summer test a
correlation between the two is found.
1,4
1,2
y = 8E-10x + 0,0445
Biomass (g/l)
1
y = 7E-10x + 0,0593
0,8
Reactor one
0,6
Reactor two
0,4
0,2
0
0
500 000 000
1 000 000 000
Number of cells
Figure 12: Correlation between number of cells and biomass.
1 500 000 000
The correlation gives equations that can be used to calculate an approximate value on grams
per cell. Extrapolation of equation from reactor two; y=8*10-10*x+0,0445 were y is the
biomass and x is the number of cells, give the initial biomass in the reactors. The number of
cells in the initial inoculums were 6529811,32 cells per ml medium in the reactor and this
gives a biomass on approximately 0,050 g per liter. This is a non realistic value since this is
the gravimetric analysis of the biomass showed that 0,05 grams per liter was achieved around
day ten.
37
4.3.3 Component analysis
Result on dry sample
Reactor one
Reactor two
Dry weight (g/l)
1,0
1,2
C, weight-%
31,5
29,2
H, weight -%
4,7
4,3
N, weight -%
3,6
3,9
Total organic Carbon, weight -%
31,4
28,9
Higher heating value (MJ/kg)
13,57
12,40
Lower heating value (MJ/kg)
12,54
11,44
Table 7: Result of component analysis from cloudy summer test after 13 days.
From data presented in table 7, it is calculated that one gram of dry algae biomass sequestered
1,15 grams of carbon dioxide in reactor one and 1,06 grams of carbon dioxide in reactor two.
4.3.4 Lipids
The fluorescent method for measuring the lipid content in Chlorella vulgaris did not work
with the equipment and time available.
38
5. Discussion
5.1 Test and development of the algae growth in
photobioreactors
Each test done using growing algae in batch culture are very time consuming since every
change requires time for algae to grow before it can be evaluated.
The change of growth medium done was necessary to avoid bacterial contamination. The
preculture was grown in proteose medium and it took considerable time to phase it out it. If
growth is to be conducted in environments with risk of contamination a medium free from
organic carbon is important. Also, if the goal is to sequester carbon dioxide a medium without
organic carbon is preferred to make sure that the algae only grow autotrophically. Chlorella
vulgaris grew well under ten percent carbon dioxide. This makes it an interesting species for
algae growth in flue gases in the future, to investigate if it can be used as carbon dioxide
sequestering from for example power plants. Bristol medium might not be optimal for the
growth of Chlorella vulgaris since it does not contain trace metals located in for example BG11. So if the purpose is to gain as high an algae growth as possible it might be good to try out
another growth medium than Bristol.
Cooling of the algae suspension is necessary when growing algae at high light intensities
since there was heat accumulated in the reactors. When growing algae in photobioreactors
with the sun as the light source, you need to have a system for temperature regulation that
follows the variation in light. Cooling systems makes the reactors more complex and demand
energy to work. Therefore it is important to design a system that is energy effective so the net
gain of energy from the algae is as high as possible.
In order to make sure that the gas flow out of the reactors can be measured and to minimize
the evaporation losses, another way to attach the plastic should be tried out. It is important to
replace evaporated medium to make sure that the algae have enough nutrients. The reactors
also need to be redesigned if the growth rate is to be optimized, to achieve sufficient mixing
of the algae suspension. This is difficult as long as they are located inside the weather-ometer. Also, an optimal growth rate might not be the main goal in tests conducted in the
weather-o-meter. Another design should be tested if any tests are run outside it. For example
could a column photobioreactor described in Theory give better mixing of the algae
suspension.
5.2 Chlorella vulgaris grown in different light
conditions
The light intensity occurring during a sunny summer day was too high for the growth of
Chlorella vulgaris in the photobioreactors designed. The UV radiation let trough to the algae
suspension is in the UVA area and therefore it should not damage the cells. Since there was no
growth when the reactor had eight hours of darkness, the real summer conditions with only 4
hours of darkness was never tested. To avoid a high light intensity when growing algae the
photobioreactors and its surroundings can be constructed so the light intensity is brought
39
down. This can for example be done by shadowing the reactors during sunny days and having
a high cell density with intensive mixing of the algae culture. Other algae species might
withstand higher light intensities so it is important to take that into consideration when
choosing algae species.
With the light intensity of 100 W/m2 and a dark period of 4 hours the algae grew well. This is
an indication that algae can be grown during cloudy summers even though the dark periods
are short during some months during the summer in Sweden.
The biomass achieved is not very high compared to levels reported [11]. Therefore one can
think that there are many factors that can be further optimized before growing algae for any
kind of commercial purpose.
The algae growth under winter conditions was very slow. The light intensity produced by
fluorescent lamps is enough to achieve a good growth rate of Chlorella vulgaris in a 16:8
hour light cycle, seen in the preculture. Therefore it seems to be the length of the light period
that is the problem. The geographic placing will therefore affect the alga production since the
number of hours that the sun is up varies over Sweden. In December the sun is up for about
three hours longer in Malmö in the far south in Sweden compared to Umeå. In June the sun is
up for about seven hours more in Umeå than in Malmö (stjärnhimlen.se). To have a high
production rate of algae during winter in Sweden an external source of light during the short
winter days would be necessary. Unfortunately, an addition of light during winter was not
tested due to limitations in time.
5.3 Analyzing methods
The gravimetric method for biomass chosen and evaluated was an easy and relatively quick
method with a relatively small standard deviation. The method worked well as long as the
biomass was not too low. It was not realistic to use extrapolation of a correlation curve
between measured biomass and number of cells. So if a gravimetric method is not possible to
use it is better only to calculate cells to analyze algae growth. There were no technical
problems with counting the cells. Since the algae have a tendency to stick together it was
important to dilute and shake the samples if the cell density was high. The composition
analysis gave the means to calculate the carbon dioxide uptake by the algae. The uptake is
lower than what have been reported in literature and one possible explanation could be that
the lipid production is low. This would also explain the low heating values. It will be
interesting later on to compare carbon dioxide uptake, the heating value on algae and the lipid
content in algae.
5.4 The continuation of the project
There are other algae species than Chlorella vulgaris that might be interesting to test in terms
of lipid production, higher biomass production and tolerance to light intensities. As another
thesis work, as a part of SP’s project, the plan is to use both Botryoccoccus braunii and
Pheodactylum tricornutum in different growth conditions. Since one of the areas considered
having big potential in algae biofuel research is sequestering of carbon dioxide from point
sources it is going to be tested how algae grow in flue gases. There are also several different
light conditions, in terms of light intensities and length of light and dark periods, which
40
should be tested to be able to make a complete conclusion on how algae can grow in Swedish
light conditions.
Since there was no method for lipid measuring successfully developed in this thesis work, the
suggestion is to make it into a separate thesis work. During the continuance of SP’s project
different lipid measurement methods are going to be tested and for this future work, samples
of the algae from the tests performed here have been frozen.
41
6. Conclusions
The weather-o-meter caused some restrictions in the photobioreactor design leading to
difficulties with mixing and sampling. Improvements to the photobioreactor might be able to
solve some of the problems encountered. To avoid bacterial contamination it is important to
use a growth medium free from organic carbon. It is also important to have a temperature
regulation system in the reactors that can keep the temperature stable regardless of light
intensity.
Chlorella vulgaris was not able to grow in light intensities representing a sunny summer
period in Sweden. Some kind of shading of the algae culture in combination with intensive
mixing will be necessary if the light intensity is high. In light conditions representing a cloudy
summer period Chlorella vulgaris grew well. The short summer days provide enough dark
hours for algae cultivation. During winter conditions the algae growth was very slow mainly
due to the short light period. An external light source will be necessary for algae cultivation
during winter in Sweden. The algae biomass produced was lower than what has been reported
in literature. Likewise the carbon dioxide sequestering rate is low compared to literature. This
is an indication that there are many factors that can be further optimized before growing algae
for any kind of commercial purpose.
A method for analysing the lipid content in algae was not found in this thesis and it is
recommended that this is done as a separate thesis work since it is a very time consuming
assignment.
SP will continue the research in this field by testing other algae species in regards of growth,
carbon dioxide mitigation, lipid production and light intensities. More testing on algae growth
in different light conditions, in terms of light intensities and length of light and dark periods,
should be done before making a complete conclusion in how algae can grow in Swedish light
conditions.
42
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Stjärnhimlen. Available from: http://stjarnhimlen.se/2006/umea.html.
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8. Appendix A
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