The study investigated using recycled glycerol from biodiesel production as an alternative carbon source to glucose for cultivating Chlorella protothecoides microalgae. Different ratios of glycerol to glucose were tested, with the optimal ratio found to be 25% glycerol and 75% glucose. Biomass production was also highest under nitrogen-deficient conditions with a carbon to nitrogen ratio of 80:1. The 25% glycerol culture exhibited higher productivity and resistance to contamination compared to glucose-only cultures.

1. The Green Solution
Optimizing productivity of bi-substrate microalgae cultures
Leng,M., Ibarra-Vidal,M., De la Hoz Siegler, H.
Department of Chemical and Petroleum Engineering
Background
Goal
Results
Contact Information: maggiejleng@gmail.com
Materials and Methods
Conclusions
Future Work
References
Acknowledgements
Glycerol is a waste generated from biodiesel
production. We can capitalize on its low-cost
and availability by utilizing it as a carbon source
for cultivating Chlorella Protothecoides, an
algae strain known for thriving under
heterotrophic conditions. Microalgae is often
employed for CO2 capture, biofuel production
and most importantly, it can be processed for
antioxidants and lipids. These products are
often found in health supplements and food
additives, with a current market value of
US$2.1 billion.
Determine:
• Feasibility of recycled biodiesel glycerol as an
alternative carbon substrate to glucose in
microalgal biomass production
• The effect of changing glycerol to glucose ratios
in achieving an optimal condition for
maximizing biomass production (100:0, 25:75,
50:50, 25:75, 0:100)
• Interplay of nitrogen deficiency on optimal
glycerol to glucose ratio (10:1, 40:1,
80:1,120:1)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
A B C D E
Biomass(g/100ml)
Flask
Biomass
A B C D E
Figure 3: Absorbance profile for nitrogen-deficient culture.
D (25:75) exhibits high productivity during exponential phase only
Figure 4: Preferential uptake of substrate, 50:50 Glycerol to Glucose
Figure 6:Biomass 25:75, C to N 80:1 Trial
• Evaluate Antioxidant, Chlorophyll-A,
Chlorophyll-B and lipid compositions of
extractable biomass
• Characterize and create Kinetic model
of optimal ratio between substrates
and nitrogen
• Additional trials to confirm finding and
recalibrate results
[1]. Heredia-Arroyo, T., Wei, W., & Hu, B. (2010). Oil accumulation via heterotrophic/mixotrophic Chlorella
protothecoides. Applied biochemistry and biotechnology, 162(7), 1978-1995.
[2]. Shi, X. M., Zhang, X. W., & Chen, F. (2000). Heterotrophic production of biomass and lutein by Chlorella protothecoides on
various nitrogen sources. Enzyme and Microbial Technology, 27(3), 312-318.
[3]. Shi, X. M., Liu, H. J., Zhang, X. W., & Chen, F. (1999). Production of biomass and lutein by Chlorella protothecoides at various
glucose concentrations in heterotrophic cultures. Process biochemistry, 34(4), 341-347.
[4]. O’Grady, J., & Morgan, J. A. (2011). Heterotrophic growth and lipid production of Chlorella protothecoides on
glycerol. Bioprocess and Biosystems engineering, 34(1), 121-125.
[5]. Chen, Y. H., & Walker, T. H. (2011). Biomass and lipid production of heterotrophic microalgae Chlorella protothecoides by
using biodiesel-derived crude glycerol. Biotechnology letters, 33(10), 1973-1983.
[6]. Cerón-García, M. C., Macías-Sánchez, M. D., Sánchez-Mirón, A., García-Camacho, F., & Molina-Grima, E. (2013). A process for
biodiesel production involving the heterotrophic fermentation of Chlorella protothecoides with glycerol as the carbon
source. Applied Energy, 103, 341-349
Substrate Consumption
Figure 5: Liquid Chromatograph of medium supernatant
• Optimal ratios are 25:75 Glycerol to
Glucose, 10:1 Carbon to Nitrogen
• 25:75 Glycerol to Glucose exhibits
highest productivity rate
• Nitrogen deficiency results in Biomass
reduction
• Glycerol maintains Chlorophyll pigment
within Chlorella protothecoides
• Glycerol rich cultures are more resistant
to contaminationFigure 9: (Top) Healthy culture vs.
(Bottom) Contaminated culture
Growth Rate
Biomass and
Antioxidant
1.189956
2.632350.405836
0.517534
1.936099
2.52036
0
1
2
3
4
5
6
B1 B2
µmolT/g
Flasks
Antioxidant Content
Hexane Ethyl Acetate Water
Figure 2: Absorbance profile for nitrogen-rich cultures. D
(25:75) Culture exhibits high productivity during exponential
phase and stagnation phase
.
Figure 7: Antioxidant content for Glucose/Glycerol 25:75, C to N 80:1 Trial I would like to thank everyone in the
Bioprocess Development lab for their
insight and guidance.
Optimal
Culture
Figure 1: Chlorella protothecoides
Figure 8: Top (Day 3), Bottom (Day 9)
Variation in Chlorophyll content as a function of Glycerol to
Glucose ratio.
100 : 0
Glycerol:Glucose
0:100
0
5
10
15
20
25
0
2
4
6
8
10
12
14
16
0 1 2 3 4 5 6 7 8 9
GLYCCEROL-GLUCOSECONSUMPTION(G/L)
DRYWEIGTH(GR/LT)
TIME (DAYS)
Biomass and Glycerol/Glucose Consumption
V.S Time
Biomass C GLY C GLU
Glycerol
Glucose
0
2
4
6
8
10
12
14
16
18
0 1 2 3 4 5 6 7 8 9 10
ABSORBANCE
TIME (DAYS)
ABSORBANCE V.S TIME
0
2
4
6
8
10
12
14
16
18
0 1 2 3 4 5 6 7 8 9 10
ABSORBANCCE
TIME (DAYS)
ABSORBANCE V.S TIME
NITROGEN RICH NITROGEN DEFICIENT
Medium
Preparation
Algal
Cultivation
Inoculum
Sample
Absorbance
Centrifugation
Biomass
Dry Weight
HPLC Analysis
Sterilization
Supernatant
Filter
Dry Oven