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The effect of the initial nitrogen concentration in the medium (mol/L) on the growth rate

(cells/mm2/day) of Chlorella vulgaris

Lisa Shen
Mr. Persaud
SBI3UB
February 22, 2016

Research Question
What is the effect of the initial nitrogen concentration in the medium (mol/L) on the growth rate
(cells/mm2/day) of Chlorella vulgaris?
Introduction
Climate change has become a dangerous threat to the earth. Fossil fuel emissions have increased the
concentration of greenhouse gases in the atmosphere, resulting in alarmingly high global
temperatures. One solution to this problem is the development of alternative vehicular fuels, such as
biodiesel. The primary sources of oil for biodiesel production are food crops, such as wheat, corn,
and beets. High demands for fuel render it impractical for biodiesel to be derived from these
resources alone (Al-lwayzy, Yusaf, & Al-Juboori, 2014). Therefore, there has been increasing
interest in the possibility of biofuels derived from non-food sources of biomass, such as microalgae.
Microalgae are unicellular, eukaryotic organisms that undergo photosynthesis using light energy and
carbon dioxide (Al-lwayzy et al., 2014). They are an efficient source of biomass for biodiesel
production because they fix carbon dioxide from the atmosphere, replicate quickly, and require
relatively little nutrients to grow (Nigam, Rai, & Sharma, 2011). Chlorella vulgaris is the strand of
microalgae that is best suited for biodiesel production. It has the highest lipid production, a
particularly fast growth rate, and can be easily cultivated (Al-lwayzy et al., 2014). The main concern
regarding biodiesel made from microalgae is the efficiency of the production process. In order for
biodiesel made from C. vulgaris to become commercially available, both the biomass and lipid
content of the C. vulgaris must be increased. These two attributes are closely related to the
composition of the medium in which the C. vulgaris is grown (Castellanos, 2013). Microalgae grow
faster in nutrient rich media, where nitrogen and phosphorus are the main limiting nutrients
(Castellanos, 2013). Other key factors affecting the growth rate of C. vulgaris are light exposure,
carbon dioxide concentration, and temperature (Castellanos, 2013). On a whole, researchers in this
field aim to discover a method of maximizing both the lipid content and biomass yield of C. vulgaris.
For instance, a study published in 2013 investigated how different mixing speeds, media
compositions, and photo-bioreactor configurations affected the growth of C. vulgaris cultures
(Castellanos, 2013). Another study in 2009 investigated the effect of the temperature and nitrogen
concentration on the lipid content of C. vulgaris (Converti, Casazza, Ortiz, Perego, & Borghi, 2009).
This experiment investigated whether increasing the initial concentration of nitrogen in the medium
could accelerate the growth rate of C. vulgaris. The initial concentration of nitrogen in the medium
refers to the nitrogen concentration in the water (mol/L) when the C. vulgaris cultures were
inoculated. The stock culture used to inoculate the trial cultures was kindly supplied by Heather
Roshon from the Waterloo Canadian Physiological Culture Centre. The stock culture was grown
under dim lighting in a Bolds Basal Medium adjusted to a pH of 6.8, with a 12h light:12h dark
photoperiod and continuous agitation of 70 (rpm). The newly inoculated cultures were grown in
separate beakers in phototrophic conditions for 10 days. The beakers were covered with plastic wrap
and secured with elastic bands to reduce fungal and bacterial contamination. The method of data
collection used was direct microscopic count. The initial and final cell densities was determined by
counting the number of cells in the microscopic field at 400X magnification. The data collected was
used to calculate the change in cell density per microscopic field area, and extrapolated to estimate
the change in cell density per (mm2). It is possible to extrapolate the cell density of a small sample
of C. vulgaris to estimate the cell density of an entire culture because C. vulgaris cultures are a
relatively homogenous solution (Giri & Ramadoss, 1979). Direct microscopic count is often used in
experiments on microalgae, and has been used in numerous experiments on strains of Chlorella (Kao

et al., 2012). It is a relatively quick and simple method of determining cell densities that requires
minimal equipment.
There were three safety concerns in this experiment. Firstly, sodium nitrate and dipotassium
phosphate can cause adverse effects if handled improperly. Ingesting sodium nitrate can result in
dizziness, pain in the abdomen, nausea, headaches, confusion, difficulty breathing, convulsions,
diarrhoea, and unconsciousness (National Institute for Occupational Safety and Health [NIOSH],
2014). Ingestion or inhaling dipotassium phosphate may result in irritation of the digestive tract and
respiratory system (International Organization for Standardization [ISO] 2000). If sodium nitrate or
dipotassium phosphate come in contact with the skin, they will cause redness and irritation (NIOSH,
2014). Contact with the eyes will result in redness and pain (NIOSH, 2014). In order to prevent this,
gloves and safety goggles were worn while preparing the growth media and working with the C.
vulgaris cultures. Secondly, some of the growth media evaporated during the growth period, and the
vapour was trapped inside the beakers. Removing the covering over a beaker allowed the vapour to
escape from the beaker and into the surrounding air. If the vapour came in contact with the eyes, it
would have caused redness, watering, and pain (ISO, 2000). Therefore, safety goggles were worn
while manually agitating the cultures. Thirdly, the growth media were disposed of in an appropriate
manner after the completion of the experiment. The media contained dissolved sodium nitrate and
dipotassium phosphate. Sodium nitrate is a strong oxidizer, and dipotassium phosphate is an acid
(NIOSH, 2014). Therefore, the media were disposed of in the acid and oxidizers waste container.

Hypothesis
If the initial nitrogen concentration in the water (mol/L) increases, the growth rate of Chlorella
vulgaris (cells/mm2/day) will increase. Nitrogen is one of the main limiting factors of algae growth
because it is a key component of DNA, RNA, enzymes, chlorophyll, and ATP (Andrews, Raven, &
Lea, 2013).
Independent
variable
Initial nitrogen
concentration in
the medium
(mol/L)

Dependant
variable
Cell density of
the C. vulgaris
cultures
(cells/0.16mm2
0.05)

Relevance
The initial nitrogen concentrations used were 0.00100 (mol/L), 0.00300 (mol/L),
0.00500 (mol/L), 0.00700 (mol/L), and 0.00900 (mol/L). Within this range, the
growth of C. vulgaris is limited by the nitrogen concentration (Converti et al., 2009).
The conditions were varied by diluting a 0.100 (mol/L) stock solution of sodium
nitrate to create media with the correct nitrogen concentration for each trial. C.
vulgaris cultures were then inoculated and grown in each medium.
Relevance
The initial and final cell densities of each inoculated culture were measured using
direct microscopic count. The initial cell density (cells/0.16mm2 0.05) of each
culture was determined by counting the initial number of cells in the microscopic
field at a magnification of 400X. After 10 days, the final cell density (cells/0.16mm2
0.05) was determined by counting the final number of cells in the microscopic field
at a magnification of 400X. The prepared microscope slides were left still until the
cells stopped moving around. This reduced the risk of counting a cell twice. The
data collected was used to calculate the change in cell density (cells/0.16mm2
0.1/day), and extrapolated to estimate the change in cell density in (cells/mm2
0.1/day) of each culture.

Control variable
1) Inoculation
ratio:
The ratio of the
volume of C.
vulgaris stock
culture to the
volume of the
medium was
1:50 in each
trial.
2) Nitrogen

source:
Sodium nitrate
was used.

3) Phosphorus
concentration:
(mol/L)
The phosphorus
concentration in
each medium
was 0.003
(mol/L)

4) Type of
water:
Tap water was
used.

Relevance
The ratio of the volume of C. vulgaris stock culture to the volume of medium in each
trial was 1:50. This was achieved by pipetting 2 (ml) of stock C. vulgaris culture into
100 (ml) of medium when inoculating each new culture. C. vulgaris undergoes rapid
cell division, so only a small volume of stock culture was required to start each new
culture (Al-lwayzy et al., 2014). It was also predicted that this inoculation ratio
would result in an initial cell density of about 20 (cells/0.16mm2). This cell density
could be accurately quantified using direct cell count. The inoculation ratio was kept
constant because it also has an effect of the growth rate of cultures. Using a higher
volumes of stock culture to inoculate the new cultures would have resulted in higher
initial cell densities. This could have caused a faster decrease in growth rates due to
self-shading in higher density cultures (Luangpipat, 2013).
Sodium nitrate was used to alter the nitrogen concentrations of the growth media.
Sodium nitrate is the nitrogen compound used in the Bolds Basal Medium, an
enriched nutrient solution that is commonly used to grow algae (Converti et al.,
2009). The source of nitrogen had to be the same for all trials. Ionic compounds
disassociate into ions in water, and different ions have different effects on growth rate
of C. vulgaris. Some metal ions are nutrients that will enhance growth, while others
may inhibit growth (Chia, Lombardi, & Melao, 2013). Using the same nitrogen
compound ensured that the same ions were present in each medium. Therefore, the
initial nitrogen concentration was the only factor altering the cultures growth rates.
Dipotassium phosphate was added to the media. This provided phosphorus to sustain
the C. vulgariss growth. Phosphorus was a nutrient that had to be present in each
medium because it is the other limiting nutrient for C. vulgaris growth (Luangpipat,
2013). The phosphorus concentration in each medium was 0.003 (mol/L). This
concentration of phosphorus was established by dissolving 0.05 (g) of dipotassium
phosphate in each 150 (ml) beaker prior to inoculating each new C. vulgaris culture.
A phosphorus concentration of 0.003 (mol/L) resulted in the media being
hypereutrophic. This meant that they were expected to have a high level of biological
productivity (Castellanos, 2013). The amount of phosphorus in the media was also
higher than the maximum amount the C. vulgaris cultures could intake (Castellanos,
2013). This eliminated the possibility of the phosphorus concentration limiting the C.
vulgariss growth. Nitrogen was the only limiting nutrient. Hence, the effect of the
initial nitrogen concentration on the C. vulgariss growth rate could be accurately
investigated.
Tap water was used as the main component of the media. Tap water contains
dissolved minerals such as copper, iron, and zinc that are essential for the metabolism
of C. vulgaris (Chia et al., 2013). It has a similar composition to freshwater, in which
C. vulgaris grows best (Luangpipat, 2013). However, the chlorine gas dissolved in
tap water is harmful to C. vulgaris (Peterson, Hrudey, Cantin, Perley, & Kenefick,
1995). The tap water was allowed to sit undisturbed in large Erlenmeyer flasks for 24
hours before it was used to prepare the growth media. The majority of the chlorine in
the water evaporated (Soo-Voon et al., 2001). The type of water was kept the same
because water was the largest component of the media, so its composition had a large
influence on the growth rate of C. vulgaris (Castellanos, 2013). Using a consistent
type of water ensured that the initial nitrogen concentration in the medium was the
only factor changing the growth rates of the cultures.

Control variable
5) Volume of
medium:
100 (ml) of
medium was
used in each
trial.

6) Photoperiod:
The C. vulgaris
cultures were
grown under
continuous
lighting.
7) Agitation:
The C. vulgaris
cultures were
manually
agitated once
every 24 hours.

8) Vessel:
Identical 150
(ml) beakers
were used as
vessels in all
trials.

Relevance
The volume of medium in each vessel was 100 (ml). This volume of medium is often
used in experiments on the growth and lipid production of C. vulgaris (Castellanos,
2013). Using 100 (ml) of medium in a 150 (ml) beaker also created an ideal depth for
algae growth because it allowed for sufficient light penetration (Krzemiska,
Piasecka, Nosalewicz, Simionato, & Wawrzykowski, 2015). The volume of medium
had to be the same in all trials because it also has an influence on growth rate of C.
vulgaris. Changing the volume of medium would have changed the amount of
nitrogen available to the C. vulgaris cultures. This could have altered their growth
rates from the true values. Using a constant volume of medium increased the
accuracy of the data collected.
The C. vulgaris cultures were grown under continuous lighting. Continuous lighting
was chosen in order to maximize the growth rates of the cultures (Pipes,
Koutsoyannis, 1961). The duration of light exposure has a significant effect on the
growth rate of C. vulgaris because light is required for photosynthesis (Krzemiska et
al., 2015). A constant duration of light exposure was used to ensure that the initial
nitrogen concentration was the only factor altering the growth rates of the C. vulgaris
cultures.
The C. vulgaris cultures were manually agitated once every 24 hours. Each medium
was gently stirred with a stirring rod until the culture was evenly dispersed throughout
the medium. The agitation was slow and gentle to prevent damaging the cells
(Castellanos, 2013). It was crucial for the cultures to have access to all the nitrogen
dissolved in their medium, because the initial nitrogen concentration was the
independent variable. Agitating the C. vulgaris cultures prevented them from
constantly remaining at the bottom of the beakers. The cultures were able to access
more of the nitrogen available in their medium, increasing the accuracy of the data
collected (Castellanos, 2013).
All the C. vulgaris cultures were grown in identical 150 (ml) beakers. This provided
a large enough environment with 50 (ml) of headspace. This way, the vessel size did
not limit the growth of the cultures. The beakers were also clear, to allow for
sufficient light penetration. This was necessary because C. vulgaris undergoes
photosynthesis and requires light to grow (Al-lwayzy et al., 2014). Using clear
beakers resulted in equal light penetration for all trials. Therefore, the initial nitrogen
concentration in the medium was the only factor changing the growth rates of the
cultures, increasing the accuracy of the data collected.

Figure 1: Table of independent, dependant, and control variables.

Materials
- 3000 (ml) tap water
- 50 (ml) C. vulgaris stock culture
- 2.12 (g) sodium nitrate powder
- 1.25 (g) potassium phosphate powder
- 25 x 150 (ml) Pyrex beakers
- 1 x 100 (ml) Pyrex beaker
- 1 x 2000 (ml) Pyrex Erlenmeyer flask (ml) 100
- 1 x 1000 (ml) Pyrex Erlenmeyer flask (ml) 25
- 1 x 100 (ml) Pyrex volumetric flask (ml) 0.08
- 1 x 250 (ml) Pyrex volumetric flask (ml) 0.12
- 1 x 1 (ml) Pyrex volumetric pipette (ml) 0.006
- 1 x 2 (ml) Pyrex volumetric pipette (ml) 0.006
- 1 x 100 (ml) Pyrex graduated cylinder (ml) 0.5
- 1 x Ohaus Scout Pro Balance electronic weigh scale (g) 0.005
- 1 x Micromaster Fisher Model E scientific microscope
- 1 x FisherfinestTM Premium Plain Glass Microscope Slide
- 1 x Fisher ScientificTM plastic cover slip
- 1 x Floralight plant grow light
- 1 x sheet of weigh paper
- 1 x scoopula
- 1 x digital timer
- 1 x stirring rod
- 1 x eye dropper
- 25 x elastic bands
- 1 x roll of plastic wrap
- 1 x safety goggles
- 1 x pair of gloves
Growth Media Preparation
1)
2)
3)
4)
5)

Put on gloves and safety goggles


Fill a 2000 (ml) Erlenmeyer flask with 2000 (ml) of tap water.
Fill a 1000 (ml) Erlenmeyer flask with 1000 (ml) of tap water.
Allow the 2000 (ml) and 1000 (ml) Erlenmeyer flasks to sit uncovered for 24 hours.
Make 250 (ml) of a 0.100 (mol/L) stock solution of sodium nitrate in a 250 (ml) volumetric flask.
Use sodium nitrate powder and water from the Erlenmeyer flasks.
6) Make 100 (ml) of a 0.00100 (mol/L) sodium nitrate solution in a 100(ml) volumetric flask by
diluting the stock solution. Pour the solution into a 150 (ml) beaker.
7) Repeat step 6 four more times for the 0.00100 (mol/L) nitrogen concentration.
8) Repeat steps 6-7 for the 0.00300 (mol/L), 0.00500 (mol/L), 0.00700 (mol/L), and 0.00900
(mol/L) nitrogen concentrations.
9) Place a piece of weigh paper on an electronic weigh scale and tare the scale.
10) Measure out 0.05 (g) of dipotassium phosphate onto the weigh paper using a scoopula.
11) Pour the dipotassium phosphate into one of the 150 (ml) beakers.
12) Stir the mixture using a stirring rod to dissolve the dipotassium phosphate.
13) Repeat steps 9-12 twenty-four more times for the remaining 150 (ml) beakers.

Procedure
1) Pour 50 (ml) of C. vulgaris stock culture into a 100 (ml) beaker.
2) Stir the C. vulgaris stock culture using a stirring rod.
3) Measure out 2 (ml) of the C. vulgaris stock culture using a 2 (ml) volumetric pipette, and insert
the culture into one of the 150 (ml) beakers.
4) Repeat step 2-3 twenty-four more times for the remaining trials.
5) Stir the C. vulgaris culture in the first trial of a 0.00100 (mol/L) nitrogen concentration using a
stirring rod until it is evenly dispersed throughout the medium.
6) Place one drop of the C. vulgaris culture on a microscope slide using an eye dropper.
7) Place a coverslip at a 45() angle with the slide, and gently lower it over the drop of C. vulgaris
culture.
8) Place the slide under a Micromaster Fisher Model E scientific microscope.
9) Bring the image to focus under 400X magnification.
10) Wait until the C. vulgaris cells are still.
11) Count the number of C. vulgaris cells in the microscopic field. Record the number of cells in a
data table as the initial cell density for the first trial of the 0.00100 (mol/L) nitrogen
concentration.
12) Repeat steps 5-11 for the remaining 24 trial cultures.
13) Tear a piece of plastic wrap large enough to cover the opening of a 150 (ml) beaker.
14) Place the piece of plastic wrap over the opening of one of the 150 (ml) beakers and secure it with
an elastic band.
15) Repeat steps 13-14 twenty-four more times for the remaining 150 (ml) beakers.
16) Place all the 150 (ml) beakers under a Floralight plant grow light.
17) Set a digital timer for 240 hours.
18) Once every 24 hours, remove the plastic covering over each of the 150 (ml) beakers.
19) Stir each culture with a stirring rod until it is evenly distributed throughout the medium.
20) Replace and secure the plastic covering over each 150 (ml) beaker.
21) Place the beakers back under the Floralight plant grow light.
22) After 240 hours, take all the 150 (ml) beakers out from under the Floralight plant grow light.
23) Remove the plastic covering over the first trial of the 0.00100 (mol/L) nitrogen concentration.
24) Stir the C. vulgaris culture using a stirring rod until it is evenly dispersed throughout the medium.
25) Place one drop of the C. vulgaris culture on a microscope slide using an eye dropper.
26) Place a coverslip at a 45() angle with the slide, and gently lower it over the drop of C. vulgaris
culture.
27) Place the slide under a Micromaster Fisher Model E scientific microscope.
28) Bring the image to focus under 400X magnification.
29) Wait until the C. vulgaris cells are still.
30) Count the number of C. vulgaris cells in the microscopic field. Record the number of cells in the
data table as the final cell density for the first trial of the 0.00100 (mol/L) nitrogen concentration.
31) Repeat steps 23-30 twenty-four more times for the remaining trial cultures.
32) Dispose of the growth media in the acid and oxidizers waste container.

Sample calculations
The following calculation was done to determine the mass of sodium nitrate powder (g) required to
make 250 (ml) of a 0.100 (mol/L) sodium nitrate stock solution.
number of moles = molarity x volume
number of moles = 0.100 (mol/L) x 0.250 (L)
number of moles = 0.025 (n)
mass of NaNO3 = number of moles x molar mass of NaNO3
mass of NaNO3 = 0.025 (n) x 84.9947 (g/mol)
mass of NaNO3 2.12 (g)
The following calculation was done to determine the volume of 0.100 (mol/L) sodium nitrate stock
solution required to make 100 (ml) of media with nitrogen concentrations of 0.00100 (mol/L),
0.00300 (mol/L), 0.00500 (mol/L), 0.00700 (mol/L), and 0.00900 (mol/L).
Vstock solution = Cfinal solution x Vfinal solution Cstock solution
Vstock solution = 0.00100 (mol/L) x 100. (ml) 0.100 (mol/L)
Vstock solution = 1.00 (ml)
The following calculation was done to determine the mass of dipotassium phosphate powder (g) to
dissolve in each medium to create a phosphorus concentration of 0.003 (mol/L).
moles of K2HPO4 = molarity x volume
moles of K2HPO4 = 0.003 (mol/L) x 0.100 (L)
moles of K2HPO4 = 0.0003 (n)
mass of K2HPO4 = moles x molar mass
mass of K2HPO4 = 0.0003 (n) x 174.2 (g/mol)
mass of K2HPO4 0.05 (g)
Cell densities (cells/0.16mm2 0.05) of C. vulgaris cultures grown in media with initial nitrogen
concentrations of 0.00100 (mol/L), 0.00300 (mol/L), 0.00500 (mol/L), 0.00700 (mol/L), and 0.00900
(mol/L)
Initial nitrogen
concentration
(mol/L)
0.00100
0.00300
0.00500
0.00700
0.00900

Initial cell density on day 1


(cells/0.16mm2 0.05)
Trial Trial Trial Trial Trial
1
2
3
4
5
22
8
14
7
26
12
9
9
18
31
26
9
10.
5
6
8
35
10.
6
6
14
18
27
16
28

Final cell density on day 10


(cells/0.16mm2 0.05)
Trial Trial Trial Trial Trial
1
2
3
4
5
38
14
6
8
37
31
32
27
37
50.
57
21
20.
12
26
18
30.
49
79
29
120.
54
114
54
97

Figure 2: Raw data table

The following calculation was used to determine the field diameter (mm) at 400X magnification.
field diameter = field diameter at 40X magnification 10
field diameter = [4.5 (mm) 0.5] 10
field diameter = 0.45 (mm) 0.05

The following calculation was used to determine the area (mm2) of the microscopic field at 400X
magnification.
A = (d2)2
A = [(0.45 (mm) 0.05) 2]2
A = [0.225 (mm) 0.05]2
A = [0.050625 (mm) 0.05]
A 0.16 (mm) 0.05
Qualitative observations of C. vulgaris cultures
C. vulgaris
stock culture
- dark green
colour
- fresh smell
- cells were
small, round,
light green, and
in motion when
observed under
a microscope at
400X
magnification
- the cells
moved across
the microscope
slide with a
constant
velocity

Inoculated C. vulgaris cultures

C. vulgaris cultures viewed at 400X


magnification after 10 days of growth
- the cultures sunk to the bottom of the beakers
- the cells were initially moving across
after 24 hours
the microscope slide, making it
- the beakers felt warm after 24 hours
difficult to count the number of cells
- there was condensation on the plastic covering - after the microscope slide was left
and the sides of the beakers each day
still for over three minutes, the cells
- on the 6th day of the experiment, media with
ceased to move across the slide
higher nitrogen concentrations were a noticeably - many of the cells were different sizes
darker shade of green
- the cells in the majority of the
- on the 8th day of the experiment, unidentified
cultures were healthy, round, small,
white particles were seen floating at the surfaces and light green
of some media
- the 2nd, 3rd, and 4th trials for the
- in general, media with lower nitrogen
0.00100 (mol/L) nitrogen
concentrations had more white particles and less concentration were extremely small
C. vulgaris growth
and odd shaped
- when a medium was stirred with a stirring rod, - a couple cells were seen were
most of the C. vulgaris detached from the
moving around the slide in a quick,
bottom of the beaker and was dispersed evenly
unpredictable pattern, and changing
throughout the medium
directions
- some C. vulgaris remained stuck to the bottom
of each beaker, particularly in the trials with
higher cell densities

Figure 3: Qualitative observations table

Growth rates of C. vulgaris cultures (cells/mm2 0.1/day) grown in media with initial nitrogen
concentrations of 0.00100 (mol/L), 0.00300 (mol/L), 0.00500 (mol/L), 0.00700 (mol/L), and 0.00900
(mol/L)
Initial nitrogen
concentration
(mol/L)
0.00100
0.00300
0.00500
0.00700
0.00900

Growth rate (cells/mm2 0.1/day)


Trial 1

Trial 2

Trial 3

Trial 4

Trial 5

10.
12
20.
6
66

4
10
8
-3.1
22

-5
10
6.2
24
54

0.6
12
4
50
24

6.9
12
12
10
43

Figure 4: Processed data table

Average growth
rate (cells/mm2
0.1/day)
3
11
10
17
42

Standard
deviation
(cells/mm2/day)
6
1
6
21
19

The following calculation was done to determine the growth rate (cells/mm2 0.1/day) of each C.
vulgaris culture.
growth rate =

initial density (cells/0.16mm2 0.05) final density (cells/0.16mm2 0.05)


6.25
10 ()

growth rate =

38 (cells/0.16mm2 0.05) 22 (cells/0.16mm2 0.05)


6.25
10 ()

growth rate =

16 (cells/0.16mm2 0.1)
6.25
10 ()

growth rate = 1.6 (cells/0.16mm2 0.1/day) 6.25


growth rate = 10. (cells/mm2 0.1/day)

Average growth rates (cells/0.16mm2 0.1/day) of C. vulgaris cultures grown in media with initial
nitrogen concentrations of 0.00100 (mol/L), 0.00300 (mol/L), 0.00500 (mol/L), 0.00700 (mol/L),
and 0.00900 (mol/L)

Average growth rate


(cells/mm2 0.1/day)

70
60

R = 0.7646

50
40
30
20
10
0
-10 0

0.002

0.004

0.006

0.008

0.01

Initial nitrogen concentration (mol/L)

Figure 5: Scatter plot of average growth rates (cells/mm2 0.1/day) of C. vulgaris cultures

Conclusion
The results of the experiment support the hypothesis. The growth rates of the C. vulgaris cultures
increased as the initial nitrogen concentration in the media increased. The average growth rate for a
nitrogen concentration of 0.00100 (mol/L) was 3 (cells/mm2 0.1/day) 6. With the exception of
the 0.00500 (mol/L) nitrogen concentration trials, the average growth rate increased at higher
nitrogen concentrations. The average growth rates for the 0.00300 (mol/L), 0.00700 (mol/L), and
0.00900 (mol/L) nitrogen concentrations were 11 (cells/mm2 0.1/day) 1, 17 (cells/mm2 0.1/day)
21, and 42 (cells/mm2 0.1/day) 19, respectively. As seen in Figure 5, the data appears to
follow a linear pattern. The correlation coefficient of the linear regression line of the data is 0.7646.
This indicates that there is a high positive correlation between the initial nitrogen concentration in
the medium and the growth rate of C. vulgaris (Mukaka, 2012). Therefore, it can be concluded that
the growth rate of C. vulgaris is directly proportional to the initial nitrogen concentration in the
medium when all other factors are kept constant.

The results of this experiment coincide with the known scientific data. A similar study done by
Nigam et al. in 2011 concluded that increasing the concentration of nitrogen in the medium increased
the rate of biomass production of C. vulgaris (Nigam et al, 2011). The recorded biomass production
rates at nitrogen concentrations of 0.0005 (mol/L), 0.001 (mol/L), 0.002 (mol/L), and 0.004 (mol/L)
were 0.127 (g/L), 0.246 (g/L), 0.296 (g/L), and 0.315 (g/L), respectively (Nigam et al, 2011).
However, the trend is not linear. A change in nitrogen concentration from 0.0005 (mol/L) to 0.001
(mol/L) nearly doubled the biomass production rate (Rotich, 2014). However, a change in nitrogen
concentration from 0.002 (mol/L) to 0.004 (mol/L) only increased the biomass production rate by
0.019 (g/L). Another observation was that cells grown in a medium devoid of nitrogen appeared
damaged (Nigam et al, 2011). Similarly, the second, third, and fourth trials for the lowest nitrogen
concentration of 0.00100 (mol/L) in this experiment were extremely small and odd shaped. A low
nitrogen supply may have inhibited the cultures from synthesizing crucial proteins, chlorophyll, and
ATP (Andrews et al, 2013). In addition, a study done in 2014 investigated the effect of the urea
concentration in the medium on the biomass production of Chlamydomonas reinhardtii, another
species of microalgae (Rotich, 2014). The urea was used as a nitrogen source (Rotich, 2014). The
average biomass yields at urea concentrations of 0.020 (mol/L), 0.030 (mol/L), and 0.040 (g/L) were
42 (g/L), 70 (g/L), and 98 (g/L), respectively (Rotich, 2014). The biomass yield increased linearly as
the urea concentration increased. Consequently, the results of previous studies support the
conclusion that an increased nitrogen concentration in the medium results in a higher growth rate.
The results of this experiment can be applied to the commercial production of C. vulgaris for the
purpose of biodiesel production. C. vulgaris is the strand of microalgae that is best suited for
biodiesel production because of its naturally high lipid content and particularly fast growth rate (Allwayzy et al., 2014). This experiment indicates that an effective method of increasing the growth
rate would be growing the C. vulgaris in nitrogen rich media. Yet, increasing the nitrogen
concentration also significantly decreases the lipid content of C. vulgaris (Gouveia and Oliveira,
2009). Only using nitrogen rich media would not maximize the efficiency of the production process.
However, a study in 2012 discovered that the lipid content of C. vulgaris increases after a change
from high nitrogen to low nitrogen conditions (Mujtabaa, Choi, C. Lee, & K. Lee, 2012). C. vulgaris
can be grown in a nitrogen rich medium, inducing rapid cell growth, then transferred to a medium
with an extremely low nitrogen concentration, resulting in an increased lipid content (Mujtabaa et al.,
2012). Furthermore, the results of this experiment can be applied to the eutrophication of the Great
Lakes. The current consensus is that higher nitrogen concentrations in the Great Lakes is one of the
leading causes of toxic cyanobacteria blooms (Heisler et al, 2008). Cyanobacteria is a type of bluegreen algae with similar biological properties as C. vulgaris (Heisler et al, 2008). The results of this
experiment support the current initiatives to reduce the nitrogen concentration in the Great Lakes and
decrease the number of toxic blooms. Lastly, C. vulgaris can also be used in aquaculture (Converti
et al., 2009). Over 1000 tons of microalgae are produced each year to be used as food for rotifers,
marine fish, and certain invertebrates (Converti et al., 2009). Maximizing the growth rate of C.
vulgaris grown for aquaculture would increase the efficiency of the production process.
Evaluation
There were several outliers in this experiment. Firstly, the third trial of the 0.00100 (mol/L) nitrogen
concentration and the second trial for the 0.00700 (mol/L) nitrogen concentration had negative
growth rates. The final cell densities of these two trials were lower than the initial cell densities.

This can only occur if the final number of live cells is lower than the initial number of live cells.
Since all cells seen under the microscope were counted regardless of whether they appeared live or
dead, a negative growth rate should be impossible. The negative growth rates of these trials reduced
the slope of the regression line and resulted in higher standard deviations for the 0.00100 (mol/L)
and 0.00700 (mol/L) nitrogen concentrations. Secondly, the average growth rate for the 0.00500
(mol/L) nitrogen concentration was 1 (cells/mm2 0.1/day) lower than that of the 0.00300 (mol/L)
nitrogen concentration. This outlier did not follow the general trend in the data, and reduced the
slope of the regression line. Thirdly, the first trial for the 0.00700 (mol/L) nitrogen concentration
was also an outlier. The growth rate for the culture was only 6 (cells/mm2 0.1/day), significantly
lower than the growth rates of the other trials for this nitrogen concentration. On a whole, the
occurrence of negative growth rates and the abundance of outliers indicate that the results of the
experiment are not entirely reliable. There is a significant magnitude of absolute uncertainty (mm2
0.1) for the microscopic field area. The standard deviations are also quite high, revealing that the
data is not precise. In particular, the growth rates for the 0.00700 (mol/L) and 0.00900 (mol/L)
nitrogen concentrations have high standard deviations of 21 (cells/mm2/day) and 19 (cells/mm2/day).
In Figure 5, the consistent overlapping of error bars indicates that the overall trend in the data is not
statistically significant. More trials are required to strengthen the statistical significance of the data.
Error
Insufficient
and
contaminative
method of
agitation.

Inaccurate
method of
determining
cell count

Significance
The C. vulgaris cultures were manually agitated only once a day. Due to this discontinuous
agitation, the cultures regularly sank to the bottom of the beakers where they remained for
several hours each day. The cultures were not always evenly dispersed throughout the
media, and did not always have equal access to all the nitrogen in the media (Castellanos,
2013). This may have resulted in a reduced nitrogen intake that decreased the cultures
growth rates from their true values. Insufficient agitation also resulted in the cultures being
more heterogeneous when samples were taken to determine the cell densities.
Consequently, the cell density of a sample did not accurately represent the cell density of
the entire culture. Furthermore, the covering over the vessels had to be removed in order to
manually agitate the cultures using a stirring rod. This resulted in an open system for
several minutes each day. The main issue with an open culture system is the risk of
contamination by other algae species, algal pathogens, or rotifers that feed on C. vulgaris
(Luangpipat, 2013). In this case, it is likely that contamination of certain cultures inhibited
them from growing at their full capacity. This effect was notably observed in the second,
third, and fourth trials of the 0.00100 (mol/L) nitrogen concentration. These trials had high
amounts of white contaminant floating at the surfaces of the media, and significantly less C.
vulgaris growth. Exposing the C. vulgaris cultures to the outside environment decreased
the growth rates of contaminated cultures, lowering the accuracy of results.
The initial and final cell densities per microscopic field were determined using direct
microscopic count. The first problem with this method is that the C. vulgaris samples were
viewed on a basic microscope slide. The depth of the samples were unknown, so their
precise volumes could not be calculated. As a result, the cell densities had to be measured
in a unit of area, (cells/0.16mm2 0.05), and could not be converted to a cell density by
volume. This was an improper representation of the cell densities because C. vulgaris cells
are three dimensional, not two dimensional. Moreover, the cells were viewed at too low of
a magnification. At a magnification of 400X, some cells were still too small to see clearly
with the naked eye, and many may have been missed entirely. This affected the accuracy of
the data because it decreased the recorded cell densities from their true values.

Error
No method to
distinguish
between live
and dead cells.

Significance
There was no method to clearly distinguish between live and dead cells when counting
cells under the microscope to determine the cell densities. All visible cells were counted
as live cells, even odd shaped cells. If cells were dead, they should not have been
included in the cell count. The lack of a method to distinguish between live and dead cells
decreased the accuracy of the recorded cell densities, and hence the accuracy of the
calculated growth rates.

Figure 6: Table of sources of error

Improvement
Use stirred tank
photobioreactors
as vessels for the
C. vulgaris
cultures.

Significance
Stirred tank photobioreactors are often used to grow algae for experimental purposes
(Luangpipat, 2013). An agitator located in the centre of each unit would enable
continuous mechanical agitation of each culture (Luangpipat, 2013). A mixing speed of
around 100 (rpm) would be used, to avoid cell damage (Castellanos, 2013). Constant
agitation would prevent the C. vulgaris cultures from sinking to bottom of the vessel,
allowing them to access all nutrients in the medium at all times. In addition, each culture
would be more homogenous when a sample was taken to determine the cell density. The
cell density of a sample would be closer to the cell density of the entire culture, resulting
in more accurate extrapolations. Furthermore, using stirred tank photobioreactors would
ensure that the C. vulgaris cultures are grown in a closed environment. This would
prevent other microorganisms from contaminating the cultures and interfering with the
growth of the C. vulgaris (Luangpipat, 2013). With sufficient agitation and no
contamination, the cultures would be able to grow at their full capacity, limited only by
the amount of nitrogen available. This would enable a more accurate investigation of the
effect of the initial nitrogen concentration on the growth rate of C. vulgaris.
Use a Neubauer A Neubauer hemocytometer is a counting chamber used to accurately determine cell
hemocytometer
counts at a high magnification (Bastidas, 2015). Its counting grid is subdivided into
under a high
increasingly smaller squares with known areas (Bastidas, 2015). A glass cover would be
magnification to placed on top of the chamber at a distance of 0.1 (mm) from the bottom of the chamber
determine cell
(Bastidas, 2015). A micropipette would be used to insert 10 (L) of a C. vulgaris culture
density.
into the chamber (Bastidas, 2015). The number of cells within a square could then be
counted and extrapolated to accurately calculate the cell density of the entire culture.
Furthermore, the hemocytometer could be viewed using an optical microscope capable of
displaying a magnification of 800X. All C. vulgaris cells would be visible at this
magnification (Castellanos, 2013). This would eliminate the possibility of some cells not
being counted because they are too small. This would increase the accuracy of the results
because the recorded cell densities would be closer to their true values.
Stain the C.
The C. vulgaris cultures could be stained with Trypan blue before a sample is taken and
vulgaris cultures counted under a microscope. Trypan blue uses the differences between membrane
with Trypan blue permeability to contrast living and dead cells (Zetsche & Meysman, 2012). Dead cells
before counting have permeable membranes, so they would intake the dye and turn blue (Zetsche &
cells.
Meysman, 2012). Live cells would remain light green because they have intact
membranes that prevent the dye from entering (Zetsche & Meysman, 2012). When
counting cells, only light green cells would be counted. This would increase the accuracy
of results because the recorded cell densities would reflect the correct number of live
cells.
Figure 7: Table of improvements

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