Chlorella vulgaris is a species of green microalgae capable of generating lipids suitable for conversion into biofuel via the process of transesterification. Viable production of biofuel from green microalgae requires high biomass densities, 1.0 g/L or more. We attempted to enhance cell concentrations and biomass densities of Chlorella vulgaris by growing the microalgae in a fed-batch system. A practical fed-batch system using indoor photobioreactors was designed and modified during the course of the project; commercial-grade plant fertilizers were used for the principle substrates. Additional mineral nutrients, including MgSO4, were also used in order to boost growth rates and the carrying capacity for the closed bioreactors. During the course of the experiment we implemented three different methods. The fed-batch system successfully enhanced the targeted parameters of biomass yield and cell concentration. We reached a maximum biomass density of 0.58 g/L, this was short of our goal but higher than our earlier results in previous projects. We also analyzed the effects of distinctive wavelengths of visible light (colored light versus white light) on cell concentrations. Red light (wavelength of 650 nm) led to the most positive growth, producing a value twice more than that generated using only green light (540 nm). A final variable which we briefly touched was the surface area to volume ratio of the photobioreactor.

1. On the Growth of Chlorella
vulgaris for Biofuel Production
Students:
Barnes, Joseph, A.
Garcia, Cynthia, N.
Perez, Lirey, J.
Advisor: Torres, Hirohito, PhD, PE
Physics and Chemistry Department
Industrial Chemical Processes Technology
(Accredited by ABET)
Twelfth Undergraduate Research Forum
May 22, 2015
UPR - Arecibo

2. Introduction
Renewable Energy from Biofuel
and Why We Should Use
Microalgae to Get it

3. Biofuel Leads to Global
Improvements
 The demand for energy is increasing world
wide as the population grows and
countries undergo technological
development.
 A finite supply of fossil fuel cannot sustain
an infinite demand for energy.
 Producing fuel from biomass is a form of
renewable energy, a means of meeting
ever-increasing demands.

4. Biofuel Leads to National
Improvements
 Independence from the oil market and
protection from its ripple effects.
 A secure energy source brings stronger
national security.
 Switching from importing fuel to exporting
fuel leads to a boon in the domestic
economy.

5. Using Biomass from Microalgae
as Source for Biofuel
 Reaches an optimal harvesting stage within days
instead of months, providing a high yield of biomass
per acre of land.
 Promises a high output of biofuel with minimal use of
arable land, reducing competition between food and
fuel, and thus reducing costs.
 Suitable for establishing and sustaining a carbon
neutral process.
 Can engage a heterotrophic metabolism, consuming
organic substances and serving as a means of
bioremediation, coupling one environmental benefit
with another.

6. Chlorella vulgaris
 Freshwater unicellular algae with chlorophyll pigments
-a and -b, which enable oxygenic photosynthesis.
 Requires only water, CO2, and some minerals for
growth.
 Possesses a lipid content as low as %15 or as high as
40% or higher, depending on stress conditions, such as
available nitrogen.
 Contains other unique sugars and proteins that are
useful to other industries, making C. vulgaris very
desirable for its multifaceted usefulness.

7. Using Biomass from Microalgae
as Source for Biofuel

8. Chlorella vulgaris – Multifaceted
Usefulness
 Industrial uses:
 Bioremediation (via heterotrophic metabolism).
 Production of ethanol from corn and switchgrass
plant (via cellulose degrading enzymes).
 Medicinal uses:
 Removing heavy metals from the body (possesses
chelating agents).
 Helps restore elasticity to the skin, diminishing
wrinkles and stretch marks (contains active
ingredient, dermachlorella D/DP).
 Reduces detrimental effects caused by exposure to
harmful radiation (e.g. UV radiation).

9. Chlorella vulgaris – Multifaceted
Usefulness

10. Objectives
Long-term and Short-term

11. Long-term Objectives
 Attain a biomass density adequate for
lipid extraction and biodiesel production:
biomass density of 1.0 g/L or higher and
specific growth rate of 0.50 day-1
.
 Encourage the establishment and
expansion of microalgal farms to produce
biofuel.
 Buy less foreign fuel, and invest more in the
economy.

12. Short-term Objectives
 Enhance biomass production by
implementing a fed-batch system.
 Enhance biomass production by use of
higher light intensities and mono-colored
light sources (650 nm and 475 nm).
 Explore other means of enhancing
biomass densities and specific growth
rates.

13. Materials and Equipment
set-up

14. Materials and Inorganic
Substrates
 Graduated bottles.
 Erlenmeyer Flasks.
 LED growth lamps.
 Aquarium fluorescent
lamp.
 Commercial-grade
aquarium pumps.
 Glass bright-line
hemocytometer.
 Optical microscope.
 Light meter.
 LED lights with changeable
wavelengths.
 Spectrophotometer
 Light Meter
 Tropical-brand 20-20-20
(Very economical and
cost-efficient fertilizer).
 OrchidPlus-brand 20-14-13
fertilizer (Very economical
and cost-efficient fertilizer).
 MgSO4
 NaCl
 NaNO2

15. Materials and Inorganic
Substrates

16. Materials and Inorganic
Substrates

17. Procedures Implemented
and Results Recorded

18. Challenge
Exploring any potential means of
enhancing the carry capacity of
indoor photobioreactors in order to
improve biomass density.

19. Method 1
 Three groups, two experimental and one control, each in
duplicate.
 Initial substrate concentrations for all photobioreactors:
 Nitrogen (ammoniacal and organic) – 0.15 g/L
 Phosphate – 0.15 g/L
 Potassium (Potash) – 0.15 g/L
 Aeration - ~613 ml/min.
 Light Source – White light; the average light intensity incident on
the surface of the photobioreactors was 3283 lux. (Summer
sunlight >100,000 lux)
 Photoperiod - 12hr/12hr light/dark cycle.
 Duration – 24 days
 Exp. Group I – Light feeding, receiving a total of 0.45 g N, K, P2O5.
 Exp. Group II – Heavy feeding, receiving a total of 0.90 g N, K, P2O5.

20. Results M-1: Maximum Cell
Concentrations & Biomass Density
0.8175
1.344
1.079
0.295
0.24
0.085
0 0.5 1 1.5
Control Group
Exp. Group I
Exp. Group II
Cell Concentration (10^7 cells/ml) Biomass Density (g/L)

21. Method 2
 Three groups, two experimental and one control, each in duplicate.
 Initial substrate concentrations for all photobioreactors:
 Nitrogen (ammoniacal and organic) – 0.20 g/L
 Potassium (potash) – 0.14 g/L
 Phosphate – 0.13 g/L
 MgSO4 – 0.335 g/L
 NaCl – 0.125 g/L
 NaNO2 – 0.250 g/L
 Micronutrients – Trace amounts
 Aeration - ~613 ml/min.
 Light source – 650 nm and 475 nm light; the average light intensity
incident on the surface of the photobioreactors was 3071 lux.
 Photoperiod - 12hr/12hr light/dark cycle.
 CO2 supplementation - 5 to 10% of total volume of gas injected.
 Duration – 21 days.
 Exp I – Light feeding, receiving a total of 0.07 g N, 0.11 g MgSO4.
 Exp II – Heavy feeding, receiving a total of 0.14 g N, 0.23 g MgSO4.

22. Results M-1 & M-2: Maximum Cell
Concentrations & Biomass Density
1.436
2.119
2.373
0.8175
1.344
1.079
0.57
0.52
0.51
0.295
0.24
0.085
0 0.5 1 1.5 2 2.5
M-2: Control
M-2: Exp I
M-2: Exp II
M-I: Control
M-I: Exp. I
M-I: Exp. II
Cell Concentration (10^7 cells/ml) Biomass Density (g/L)

23. Method 3 – Increasing Light
Intensity
 Three groups, two experimental and one control,
each in duplicate.
 Initial substrate concentrations, Aeration,
Photoperiod, CO2 supplementation, Duration,
Feeding portions – Same as Method 2.
 Light source - Same as Method 2, but with higher
intensity at 6217 lux.

24. Results M-1, M-2 & M-3: Maximum
Cell Concentrations & Biomass Density
1.436
2.119
2.373
0.8175
1.344
1.079
0.4
0.35
0.46
0.57
0.52
0.51
0.295
0.24
0.085
0 0.5 1 1.5 2 2.5
M-3: Control
M-3: Exp. I
M-3: Exp. II
M-2: Control
M-2: Exp. I
M-2: Exp. II
M-1: Control
M-1: Exp. I
M-1: Exp. II
Cell Concentration (10^7 cells/ml) Biomass Density (g/L)

25. Results M-3: Alternative explanation
for drop in biomass density
Interval between last dose and
biomass reading:
 M-2: 12 days.
 M-3: 4 days.
 Longer starvation period, more cell
growth, more biomass.

26. Fed-Batch Redemption

27. M-3: Re-evaluated
Left the experiment running.
Analyzed the relationship between
biomass density and time up till last
biomass reading.

28. Results M-3: Biomass density (g/L)
versus Time (days)
0
0.1
0.2
0.3
0.4
0.5
0.6
1 3 5 7 9 11 13 15 17
Time (Days)
BiomassDensity(g/L)
Control
Exp. Gr. I
Exp. Gr. II

29. Results M-3: Calculating Biomass
Density With More Precision
 Starved all groups for 8 days.
 Method relied on the natural
tendency of C. vulgaris to precipitate,
fall out of solution, when in stagnant
phase.
 Weighed entire biomass and divided
by 0.500 L.

30. Results M-3: Biomass density (g/L)
versus Time (days) – Adjusted
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
1 3 5 7 9 11 13 15 17 19 21 23 25
Time (days)
BiomassDensity(g/L)
Control
Exp. Gr. I
Exp. Gr. II

31. Results M-1, M-2 & M-3:
Biomass Density - Adjusted
0.34
0.48
0.58
0.57
0.52
0.51
0.295
0.24
0.085
0 0.2 0.4 0.6 0.8 1
M-3: Control
M-3: Exp. I
M-3: Exp. II
M-2: Control
M-2: Exp. I
M-2: Exp. II
M-1: Control
M-1: Exp. I
M-1: Exp. II
Biomass Density (g/L)

32. Conclusions
 Providing additional nitrogen
(ammoniacal and organic), potassium,
and phosphate alone did not lead to any
substantial increase in biomass density.
 By modifying the method for reading
biomass density, we can see that a
regulated fed-batch system can augment
carry capacity.

33. Recommendations & Future
Plans
 Analyze other specific variables for possible
enhancements:
 Light intensity (Redo)
 Photoperiod (e.g. 16hr/8hr light-dark cycle)
 Surface area to volume ratio.
 Replicate conditions for reaching 0.60 g/L,
and find a consistent result.
Inoculation:
Day 1
Feeding – 18 d Starvation
3 d
Precipitation
5 d
Extraction:
Day 26

34. 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.
All about algae (2015). Algae Biomass Organization. Retrieved May 26, 2015, fromhttp://allaboutalgae.com/why-
algae/
Christi, Yusuf, 2007. Biodiesel from microalgae. Research review paper prepared at the Institute of Tech. and
Engineering, Massey University, New Zealand. Biotechnological Advances 25 (2007) 294-306.

35. Additional Observations: Effects
of mono-color light on Cell Conc.
 Initial substrate concentrations, Aeration,
Photoperiod – Same as Method 1.
 Duration – 7 days
 Light source - Intensity of light incident on the
surface depended on wavelength:
 Red – 638 lux
 Blue – 653 lux
 Green – 977 lux

36. Additional Observations: Effects
of mono-color light on Cell Conc.
7.45
4.36
11.17
0
2
4
6
8
10
12
CellConcentration(10^6
cells/ml)
475
nm
510
nm
650
nm
Absorbance (A) versus
Wavelength (nm)
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
370
410
450
490
530
570
610
650
690
730
Wavelength (nm)
Absorbance(A)

37. Additional Observations: Surface
Area to Volume Ratio.
 Initial substrate concentrations, Aeration, Light
Source, Photoperiod – Same as Method 2 & 3.
 Duration – 14 days.
 Reactor A – S.A: 332.9 cm2
; Vol: 242.1 ml
 Ratio: 1.375 cm2
/ml
 Reactor B – S.A: 572.3 cm2
; Vol: 880.0 ml
 Ratio: 0.650 cm2
/ml

38. Additional Observations: Surface
Area to Volume Ratio.
1.13
0.6328
0.22
0.09
0 0.2 0.4 0.6 0.8 1 1.2
1.375 cm^2/ml
0.650 cm^2/ml
Cell Concentration (10^7 cells/ml) Biomass Density (g/L)

39. Questions?