This is the PowerPoint presentation I prepared for my research project which is attempting to enhance the growth of microalgae for purposes of generated biofuel. This presentation covers my undergraduate research project at the UPRA during the fall semester of 2014 under the department of physics and chemistry. The phase in the research covered by this presentation involves attempting to grow C. vulgaris, a green microalgae, to the density of 1 gram per liter or more by supplementing the growth media with carbon dioxide gas. Unfortunately, although we failed to reach our mark of 1 g/L, we did learn how mixing carbon dioxide gas with aeration is better than directly injecting the gas into the growth media. Also, we were able to improve the biomass density compared to our earlier attempts.

1. Enhancing the growth of Chlorella
vulgaris using indoor bioreactors
supplemented with CO2 gas.
Students:
Barnes, Joseph, A.
Quiñones, Maria
Advisor: Torres, Hirohito, Phd, PE
Physics and Chemistry Department
Industrial Chemical Processes Technology
(Accredited by ABET)
Eleventh Undergraduate Research Forum
December 12, 2014
UPR - Arecibo

2. Introduction: Chlorella vulgaris, a
multipurpose microalgae.
 C. vulgaris is a unicellular green microaglae,
a photoautotroph which generates sugars,
lipids and proteins via oxygenic
photosynthesis.
 It is one of the most investigated microalgae
because of its multiple uses and multifaceted
properties, including its high concentration of
lipids that can be transformed into different
types of biofuels.

3. Multipurpose Microalgae:
Industrial Uses
 Included among its potential industrial uses
are the following:
– Generation of biodiesel (C. vulgaris has a high
lipid content).
– The industrial production of ethanol from corn and
switchgrass plant (it posses cellulose degrading
enzymes).
– As a water bioremediation agent.
– For the production of pigments (it holds a high
content of chlorophyll).

4. Multipurpose Microalgae:
Industrial Uses

5. Multipurpose Microalgae:
Medicinal Uses
 Currently C. vulgaris is also being studied for
medicinal purposes, including:
– As a chelating agent for its ability to remove
heavy metals in the human body.
– As an active ingredient (Dermachlorella) for skin
cream, for restoring the elasticity of the skin (e.g.
remove wrinkles and stretch marks).
– It is used for reducing problems caused by
exposure to harmful radiation, such as UV
radiation.

6. Multipurpose Microalgae:
Medicinal Uses

7. Biochemical Composition of C.
vulgaris
 C. vulgaris has a high
lipid content, up to 40%
or more.
 Lipids can be extracted
and converted into
biodiesel via
transesterification.
Lipid vacuoles or droplets within the cell,
visualized by TEM, transmission electron
microscope.

8. Benefits of Producing Biodiesel Using
Microalgae like C. vulgaris
 Relatively rapid growth rate in shallow bodies of water or in
photobioreactors, without the use of excessive square-miles of
surface terrain as in the case of other oil-producing crops (e.g.
coconut trees, corn, soybeans, etc.)
 Sequestering vast quantities of carbon-dioxide. Growing
microalgae can be a carbon-neutral process, that is, the carbon
dioxide emitted by the burning of biodiesel is reabsorbed by the
microalgae.
 Ability to utilize heterotrophic metabolism in order to absorb
sugars and other organic-carbon substances, thus it proves a
viable means for removing industrial waste and unwanted
byproducts.

9. Long-Term Objectives
 To grow C. vulgaris microalgae in a cost efficient
manner, using the most economic resources
available.
 To reach a growth rate and biomass production
(> 1 g/L) sufficient for lipid extraction.
 To extract the lipid content and convert it to
biodiesel.
 Devise a means to couple CO2 sequestration with
biodiesel production.

10. Short-Term Objectives
 Establish growth conditions for optimal
production of microalgae biomass.
 Enhance microalgae production by
supplementing growth media with CO2 gas.
 Establish optimal input flow for CO2 gas.
 Generate concentrations of microalgae
biomass which exceed 1 g/L.

11. Materials:
Equipment set-up
 1-liter reactor bottles.
 Growth media (Commercial grade fertilizer).
 Aquarium air pumps (average flow rate: ~ 660 ml/min).
 Growth lamp (luminous flux = 660 Lumens) with high-
intensity light emissions at red and blue wavelengths.
 CO2 gas tank with regulator valves.
 Gang valves.
 Hemocytometer.
 Light microscope.

12. Materials:
Equipment set-up

13. Materials:
Equipment set-up

14. Procedures Implemented and Results
Recorded
Hypothesis:
Feeding C. vulgaris, an
unicellular autotroph,
with CO2 gas at a high
flow rate will increase
its cell concentration.

15. Method 1
 The control group and experimental group were prepared in
duplicate, using distilled water supplemented with 0.75 g/L of
20-20-20 commercial grade fertilizer inoculated with a fresh
culture of C. vulgaris.
 The reactors bottles were placed in an Orbital Shaker set to stir
at 125 rpm.
 The growth lamp was regulated to a 12hr/12hr light/dark cycle
by the timer.
 The experimental group was exposed to CO2 gas at a high flow
rate (~ 620 ml/min) for a an exposure rate of 40 min every 1
hour of light.

16. Results Following Method 1:
Final cell concentrations (106
cells/ml)
 After 8 days of growth, we measured cell concentrations via a
direct cell count using the hemocytometer and the light
microscope.
 As seen on the graph, the control group flourished in
comparison to the experimental group.
0
0.2
0.4
0.6
0.8
1
1.2
Method 1: 8 days
Control
Experimental

17. Method 2
 We repeated the procedure used in the
anterior experiment, however, we reduced
the rate of exposure to CO2 gas from
40 min/hr-light to 20 min/2hr-light (8 hr/day to
2 hr/day).
 At the end of 8 days, we measured the final
cell concentrations via direct cell count using
the hemocytometer.

18. Results Following Method 2,
Combined with Those of Method 1:
Final cell concentrations (106
cells/ml)
0
0.2
0.4
0.6
0.8
1
1.2
Method 1:
8 days
Method 2:
8 days
Control Experimental

19. Method 3
 We repeated the procedure used in the 1st
attempt, however, both the control and the
experimental groups were aerated.
 A gas line carry CO2 gas was connected to
the airline which aerated the experimental
group. The air flow was mixed with CO2 gas
(~500 ml/min).
 The exposure rate to the air mixed with CO2
gas was limited to 30 min/2hr-light (3 hr/day).

20. 0
5
10
15
20
25
Method 1: 8
days
Method 2: 8
days
Method 3: 13
days
Control
Experimental
Results Following Method 3,
Combined with Those from Methods 1
and 2:
Final cell concentrations (106
cells/ml)

21. Immediate Observations Regarding
Direct CO2 Supplementation
 Direct injection of CO2
at high flow rates
inhibits cell growth and
reproduction of C.
vulgaris.
 Addition of CO2 gas at
a more regulated rate
(diluted with a strong
airflow) sustains
growth.

22. Method 4:
Determining Optimal CO2 Dilution
Rates
 We prepared 6 bioreactors, arranged into two
groups, control (2 reactors) and experimental (4
reactors). Both groups were well aerated. All
bioreactors had 0.75 g/L of 20-20-20 fertilizer.
 The airflow for each bioreactor composing the
experimental group was mixed with CO2 gas at a
flow rate distinct from the rest.
 Dilution rate determined by measuring the fraction of
total gas volume occupied by CO2 gas:
– CO2 gas flow/ (CO2 gas flow + airflow) x 100%

23. Results Following Method 4:
Final cell concentrations (106
cells/ml)
0
5
10
15
20
25
30
35
40
1 2 1 2 3 4
Control Experimental
Exp.
Units
CO2 gas
flow rate
CO2 %
of total
volume
1 403
ml//min
38%
2 93.6
ml/min
12%
3 36.7
ml/min
5.2%
4 9.36
ml/min
1.4%Cell counts were performed
following 16 days of growth

24. Immediate Observations Following
Method 4
 Higher concentrations of C.
vulgaris were attained at
higher CO2 dilution rates,
with optimal growth where
CO2 is at 5% of total
volume.
 Unable to measure growth
rates.
 Final cell concentrations are
determined by carry
capacity.
 Highest dried biomass
reached was 0.43 g/L.

25. Other Observations:
Observed Growth Curve
 During its observed life cycle, C. vulgaris
undergoes certain patterns of development.
– Inoculum in fresh growth medium undergoes a
growth phase (log phase), generating a dark
green medium with low transmittance.
– Following a given period of time, cells begin to
clump or agglomerate, eventually forming
flocculants which tend to precipate on available
solid surfaces. The medium loses its green color
and remains clear with a high transmittance.

26. Growth Curve in a Static System

27. Other Observations:
Why Excessive CO2 May Inhibit Growth
 Dissolving CO2 in water will generate
carbonic acid (H2CO3), lowering the pH and
increasing the acidity of the solution.
 The pH may drop below the acceptable
range for sustainable growth.
CO2 (aq) + H2O  H2CO3 (aq)

28. Conclusions
 Direct infusion of CO2 gas at a high flow rate inhibits
growth.
 Diluting the CO2 gas in airflow at regulated levels will
enhance the growth C. vulgaris.
 The optimal level of CO2 in the airflow is around 5%.
 Biomass readings fell short of the 1g/L mark, however
there were higher than earlier attempts:
(~30 days) Spring  0.30 g/L; (28 days) Summer  0.39 g/L;
(19 days) Fall  0.43 g/L.
 Feeding CO2 is more complicated than earlier assumed.

29. 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
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.
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.
Torres, H., 2013. On the growth of Chlorella vulgaris for lipid production. Poster presentation at
the University of Puerto Rico.

30. Recommendations and Future Plans
 New ideas to consider:
– Fed batch systems (dynamic rather than static) will enhance
growth and increase yield (e.g. remove old medium and
infuse fresh growth medium).
– Increase the carry capacity will increase maximum yield.
– Applying a buffer to control pH fluctuations will allow higher
flow rates of CO2 gas and thus enhance growth.
– Measure and control growth rates to guard against
overshoots and establish a maximum yield capacity (MYC).

31. Final Words and Acknowledgments
Small steps and small
improvements, taken in
the right direction, will
sooner or later take you
where you need to be.
Special thanks to the all the
members of the
departments of chemistry
and biology who had a
hand to play in this
endeavor.