Summer microalgae report, Sept 2014

Growing C. vulgaris in mixotrophic conditions and high light intensity; UPRA
Observed Growth Rate of Chlorella Vulgaris under
Mixotrophic Conditions –
Observed Growth Rate of C.v. under Hight Light Intensity in
Autotrophic Conditions
Undergraduate Investigation
QUIM 4999
Commenced: July 8, 2014
Ended: August 22, 2014
Student: Joseph A. Barnes (440-13-0709)
Laboratory Supervisor: Dr. Hirohito Torres
University of Puerto Rico at Arecibo
Abstract
Green microalgae can serve as a source for biodiesel production, providing a means of
generating renewable energy, which is eco-friendly and comparatively simple to manage.
This experimental study has been partitioned into two phases. The first phase of this
study was to evaluate the effects of subjecting Chlorella vulgaris, a species of green
microalgae, to mixotrophic conditions. Three groups, each in duplicate, were observed
over a period of eight days in order to analyze the effects of feeding C. vulgaris simple
and complex sugars (heterotrophic conditions). The autotrophic effects were satisfied by
a steady light-source regulated to a 12:12 hour light-dark cycle. The experimental study
was evaluated on the basis of observed changes in cell-concentration and light
absorbance. Initial findings demonstrated a failure of the algae to successfully thrive
under heterotrophic effects (sugar supplementation plus aeration). The second phase of
this study was to evaluate the outcomes of subjecting the remaining batches of C.
vulgaris to a highly intensified light source, with wavelengths of blue and red, optimal for
photosynthetic activity. Initial findings revealed a positive correlation between cell
concentration and light intensity, although without a substantial gain in cell biomass.
1

Introduction
The topic of microalgae has been
gaining considerable attention in recent
years, due to its capacity of serving as a
source of renewable energy and as a
means of finding economical
independence from costly oil
importation. Green microalgae,
including the species of Chlorella
vulgaris, the principal specimen of this
experimental study, is capable of
autotrophic growth, absorbing sunlight
and carbon dioxide in order to produce
sugars for the purpose of sustaining and
reproducing itself. Once collected and
dried, fatty acids and triglycerides can be
extracted from the algae, subjected to the
process of transesterification, and
converted into biodiesel.
This method of producing
biodiesel possesses clear advantages
over other implemented techniques. First,
green microalgae can be grown
relatively quickly in shallow bodies of
water or in photobioreactors, without the
use of the excessive square-miles of
surface terrain as in the case of other oil-
producing crops (e.g. coconut trees, corn,
soybeans, etc.) (Hirohito, 2013;
Scarsella et al., 2010). In other words, a
good crop of green microalgae can
generate a much higher quantity of
biodiesel per square-mile of land than
any other oil crop. Second, green
microalgae absorb vast quantities of
carbon-dioxide. Producing large masses
of microalgae means sequestering even
larger amounts of carbon-dioxide from
the atmosphere, which signifies that
growing microalgae can be a carbon-
neutral process, that is, the carbon
dioxide emitted by the burning of
biodiesel is reabsorbed by the
microalgae. This cycle may inhibit any
long-term accumulation of carbon-
dioxide in the upper atmosphere from
the combustion of biodiesel. In fact, so
potential is the algae’s property of
sequestering carbon-dioxide, that it is the
subject of research as a potential means
of reducing overall carbon-dioxide
emissions in the environment (Sahoo et
al., 2012). A third advantage of
microalgae is its ability to utilize
heterotrophic metabolism in order to
absorb sugars and other organic-carbon
substances (Debjani et al., 2012; Xiaoyu
et al., 2014; Leesing and Kookkhuntod,
2011; Scarsella et al., 2010). This makes
a two-fold benefit. For one, by being
able to grow and reproduce under
heterotrophic conditions, the farming of
microalgae will not be inextricably
linked to the environment. Some
measures can be taken to maintain high
microalgae production in the absence of
sufficient sunlight and environmental
carbon dioxide. And for the other,
microalgae can prove a viable means for
removing industrial waste and unwanted
byproducts, which substances would
serve as the principal sources of organic
carbon for the algae (Debjani et al.,
2012; Xiaoyu et al., 2014).
The study was divided into two
phases, wherein the first was focused on
observing the effects of growing
Chlorella vulgaris under mixotrophic
conditions as opposed to just autotrophic.
In order to satisfy these mixotrophic
conditions, the microalgae were fed a
simple sugar (dextrose) and a mixture of
simple and complex sugars (molasses).
The autotrophic metabolism was to be
maintained by exposure to a steady
source of light regulated to a 12:12 hour
light-dark cycle. The effects were
evaluated on the basis of cell-
concentration and light absorbance. The
results were observed over a period of
eight days.
2

Following the sugar
supplementation in the first phase, the
study was carried to a second phase
aimed at analyzing the effects of a stark
increase in the light intensity sustaining
the autotrophic conditions. The
parameters observed were cell
concentration and light absorbance.
Materials and Method
Phase 1
The microalgae Chlorella
vulgaris seed culture was purchased
from the UTEX culture collection in
Texas. The growth medium used in this
study consisted of a solution of distilled
water with a 0.75 g/L concentration of a
standard brand-X 20-20-20 all-purpose
fertilizer, which provided 0.15 g/L
concentrations of nitrogen, phosphoric
acid and potassium, essential nutrients
necessary for promoting and sustaining
the growth of autotrophs (table 2). Six 1-
liter flasks were prepared with 630 mL
of growth medium in each. Next, each
flask was inoculated using 70 mL of
seed culture, establishing a 9:1 ratio of
growth medium to cell volume. Two
flasks were set aside for the control
group (Control Group 1 & 2); two other
flasks were supplemented with dextrose
at a concentration of 10 g/L (Dextrose
Group 1 & 2); the final two flasks were
supplemented with molasses at a
concentration of 10.0 g/L (Molasses
Group 1 & 2) (photo a).
All six flasks were placed upon
an industrial mixer (Environ Shaker),
which was set to shake the solutions at a
speed of 125 rpm. The light source
consisted of four regular household
fluorescent light bulbs, providing an
average light intensity of 1,400 lux. The
bulbs were set on a timer which
regulated the exposure of light to a 12:12
hour light/dark cycle.
All six flasks were aerated via
two pumps with the air hoses arranged in
series, in order to supplement the media
with carbon-dioxide drawn from the
surrounding air. The average rate of
aeration was over 660 ml/minute.
Phase 2
One of the four standard
fluorescent light-bulbs was replaced with
a highly efficient LED light device that
emits high intensity light at blue and red
wavelengths. Measured light intensity
climbed to an average of 8,770 lux. The
remaining flasks from phase 1 (CG 1 &
2) were subjected to the new light
intensity.
Analysis Procedures
Changes in cell concentration
were measured directly by cell-counting
using a hemacytometer and a light-
microscope.
Light absorbance was measured
with a spectrophotometer at red
wavelengths (690 nm).
Dry biomass was measured in
triplicate using 15 mL samples from
each unit and applying centrifugation to
form pellets. The samples were diluted
and subjected to a second centrifugation
to remove salts; samples were placed in
an incubator at 80°C for 24 hours to
remove humidity.
Results and Discussion
Phase 1
As the graph provided will
demonstrate (figure 1), the dextrose and
molasses groups experienced a
stagnation coupled with a steady decline
in algae cell concentrations. After the
3

eighth day, both sugar groups were
discarded and phase 1 was terminated.
The sugars and aeration had promoted
the growth of bacteria and protozoa,
which presence was easily detectable by
the microscope as well as by
macroscopic growths (photo b). In a
short while, large concentrations of
bacteria had developed which had stifled
and significantly reduced the cell
concentration of C. vulgaris. The
absorbance tests with regards to the
sugar supplemented groups do not
correlate with the cell-concentration of
algae. The pattern evinced is irregular
due to the nature of contamination by
bacteria and other microbes (figure 2).
We can gather from our studies
that in the presence of initial high sugar
concentrations and aeration, the C.
vulgaris is quickly supplanted by
bacteria and protozoa. Other measures
need to be taken (e.g. sterilization) in
order to sustain algae growth under these
particular mixotrophic conditions.
Phase 2
The LED light was installed on
the 13th
day of the experiment, and the
effects observed until the 28th
day, a
duration of 15 days. As the graphs will
demonstrate, the initial spike in light
intensity (from 1,400 lux to 8,770 lux) is
correlated to a steep climb in cell
concentration and absorbance (figure 3
and figure 4). By the end of the phase,
the cell concentrations and absorbance
were still climbing, albeit at a lesser rate.
Increase in oxygen output was also
visibly noticeable by foaming at the
surface. The main reactor containing the
seed culture experienced certain changes
in color and other properties, for reasons
not conclusively established (photo c
and d). The change in biomass as
calculated by day 28 (table 1), although
not significant, is still perceptible and
warrants consideration. For this section,
a very rough estimate for the specific
growth rate (u) was given, using
biomass concentration calculated at day
8, as the biomass concentration at the
beginning of the exponential growth
phase (Xo). The specific growth rates for
control groups 1 and 2 were very low,
falling far short of given expectations
(less than 0.07 d^-1) (table 1). It must be
noted though, that these specific growth
rates were measured in an interval of
time which included a brief period
before the LED light was installed. We
can gather from these results that an
improvement in the autotrophic
conditions (higher light intensity in red
and blue light spectra) has positive
effects on the cell concentration and
absorbance of the C. vulgaris. However,
it remains to be seen by how much the
algae can be affected by these new
conditions (i.e. maximum output). As
shown here, there is potential in
amplifying autotrophic effects (e.g.
supplying higher light intensity and
carbon dioxide supplementation) in
order to improve microalgae biomass.
Conclusion and Recommendations
Unless we undertake measures to
sterilize the samples, such as by
autoclaving, high initial concentrations
of sugars combined with aeration will
favor bacterial and protozoan growth,
which is debilitating to the microalgae.
Steps to be considered in order to avoid
sterilization, may include using reduced
concentrations of sugar, avoiding
aeration, and fortifying the conditions
which are favorable for photosynthesis
(e.g. more light and more carbon-
dioxide). Here listed are a few
propositions to consider:
4

5
1. Growth of algae solely by
autotrophic means, with a given
high light intensity and graduated
boosts of carbon-dioxide
supplementation.
2. Growth of algae initially under
only autotrophic conditions, but
then after reaching the maximum
yield of cell-concentration,
follow with a supplementation of
sugar at a low concentration (e.g.
2 g/L).
It is certainly recommended that further
research be done to study more
accurately the effects of light intensity
on C. vulgaris. According to earlier
investigations (Cheirsilp and Salwa,
2012), high yields of biomass were
attained for Chlorella sp. at ideal light
intensities of 3000 to 5000 lux. The
intensity from sunlight on a clear day at
noontime (12:00 pm) measured up to
104,000 lux in the Arecibo area of
Puerto Rico in mid-August (19/8/2014).
However, it should be noted that very
high light intensities do not necessarily
correlate positively with biomass yields,
but in fact could actually limit cell
growth (Cheirsilp and Salwa, 2012).

Figures, Tables, and Images
Figure 1
Cell Concentration versus Time (Phase 1)
0
50
100
150
200
250
300
350
400
450
1 3 5 7 9
Time (days)
Cellconcentration[10^4]
(cells/mL)
Control Group 1 Control Group 2 Dextrose Group 1
Dextrose Group 2 Molasses Group 1 Molasses Group 2
Sugar groups were discarded on day 8 due to contamination by microbes other than algae.
Figure 2
Absorbance versus Time (Phase 1)
0
0.2
0.4
0.6
0.8
1
1.2
1 3 5 7 9
Time (days)
Absorbance(A)[690nm]
Control Group 1 Control Group 2 Dextrose Group 1
Dextrose Group 2 Molasses Group 1 Molasses Group 2
Absorbance tests were made at 100% dilution (cell volume was diluted with an equal
volume of distilled water; 0.75mL cell volume + 0.75mL distilled water).
6

Figure 3
Cell Concentration versus Time (Phase 2)
0
500
1000
1500
2000
2500
3000
3500
4000
13 15 17 19 21 23
Time (days)
Cellconcentration[10^4]
(cells/mL)
Control Group 1 Control Group 2
CG 2 received a higher intensity of light due to uneven distribution of LED light.
Figure 4
Absorbance versus Time (Phase 2)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
13 18 23 28
Time (days)
Absorbance(A)[690nm]
Control Group 1 Control Group 2
Absorbance tests were made at 100% dilution (cell volume was diluted with an equal
volume of distilled water; 0.75mL cell volume + 0.75mL distilled water).
7

Table 1
Dry biomass per liter of media
Sample Source Day 1 Day 8 Day 28 u (days^-1)
Main Reactor 0.28 g/L - 0.20 g/L -
Control Group 1 - 0.033 g/L 0.33 g/L 0.0607 d^-1
Control Group 2 - 0.053 g/L 0.39 g/L 0.0544 d^-1
Note: specific growth rate (u) was calculated according to the Monod model, wherein, u
= ln(Xt – Xo)/t, where Xt is biomass concentration at time t and Xo is biomass
concentration at the beginning of the exponential growth phase. Biomass at day 8 will be
regarded as the value at the beginning of exponential growth phase, thus t = 20.
Table 2
Contents of 20-20-20 All Purpose Fertilizer Percent of mass
Nitrate nitrogen (NO2)
Ammoniacal nitrogen (NH3)
Water soluble organic nitrogen
Total available nitrogen
6.09%
3.85%
10.06%
20%
Available phosphoric acid (P2O5) 20%
Water soluble potassium (K2O) 20%
Note: a 0.75 g/L concentration of 20-20-20 all-purpose fertilizer would yield a
concentration of nitrogen of 0.15 g/L; phosphoric acid concentration would be at 0.15 g/L
and potassium concentration would be at 0.15 g/L.
Data Table for the Figures 1, 2, 3 and 4
Time Cell concentration [10^4] (cells/mL) – Sugar Groups
Days DG 1 DG 2 MG 1 MG 2
1
3
6
8
52.7
32.0
0
0.67
47.3
64.7
5.33
4.67
21.0
39.3
42.6
34.0
28.3
39.0
21.3
14.7
Time Cell concentration [10^4] (cell/mL) – Control Groups
Days CG 1 CG 2
1
3
6
8
10
13
16
22
24
41.3
100
250.3
297.4
330.6
513.9
2119
2606
3078
48.7
125
213
356
406.6
513.9
2244
3374
3766
8

Time Absorbance (A) at 690 nm – Sugar Groups
Days DG 1 DG 2 MG 1 MG 2
1
3
6
8
0.053
0.502
0.493
1.132
0.053
0.505
0.415
1.004
0.141
0.612
0.253
0.643
0.179
0.935
0.254
0.252
Time Absorbance (A) at 690 nm – Control Groups
Days CG 1 CG 2
1
3
6
8
10
13
16
22
24
28
0.037
0.052
0.084
0.079
0.111
0.148
0.308
0.518
0.564
0.627
0.044
0.065
0.084
0.104
0.127
0.170
0.337
0.616
0.674
0.714
Photos a, b, c, and d
(a) (b)
(c) (d)
9

10
References
Cheirsilp, B., Salwa, T., 2012. Enhanced growth and lipid production of microalgae
under mixotrophic culture condition: effect of light intensity, glucose
concentration and fed-batch cultivation. Bioresource Technology 110, 510-516
Debjani, M., van Leeuwen, J.H., Lamsal, B., 2012 Heterotrophic/mixotrophic cultivation
of oleaginous Chlorella vulgaris on industrial co-products. Algal Research 1, 40-
48.
Leesing, R., Kookkhunthod, S., 2011. Heterotrophic growth of Chlorella sp. kku-s2 for
lipid production using molasses as a carbon substrate. Internat. Conf. on Food
Engin. and Biotech. IPCBEE vol. 9
Sahoo, D., Elangbam, G., Devi, S.S., 2012. Using algae for carbon dioxide capture and
bio-fuel production to combat climate change. Phykos 42 (1), 32-38.
Scarsella, M., Belotti, G., De Filippis, P., Bravi, M., 2010. Study on the optimal growing
conditions of Chlorella vulgaris in bubble column photobioreactors. Paper
prepared by the Dept. of Chem. Engin. Mater. Environ., Sapienza Uni. of Roma.
Torres, H., 2013. On the growth of Chlorella vulgaris for lipid production. Poster
presentation at the University of Puerto Rico.
Xiaoyu F., Walker, T.H., Bridges W.C., Thornton, C., Gopalakrishnan, K., 2014.
Biomass and lipid production of Chlorella protothecoides under heterotrophic
cultivation on a mixed waste substrate of brewer fermentation and crude glycerol.
Bioresource Technology 166, 17-23.

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