Maximizing Lipids

Productivity of
Microalgae for
Biofuels Production
and Carbon Recycle
Janice Zhuang, Civil Engineering,
University of Virginia

Katherine Lupariello, Chemical
Engineering,
University of Cincinnati

FM: Dr. Joo-Youp Lee,
University of Cincinnati

GRA: Mr. Jinsoo Kim,
University of Cincinnati
1
SUE REU SUMMER 2013
May 22, 2013
Sponsored by: The National Science Foundation Grant ID No: EEC-0851986
Introduction
2



Microalgae can be
used to make
biodiesel
Neochloris
oleoabundans
strain
Energy demand
&
Fossil fuel supply
Source:
http://www.commerce.gov/sites/default/files/images/2011/june/cyantoc
h_aerial_shot_of_algae_farms_at_keahole_point.jpg
Algae Farm in Hawaii
Why Microalgae?
Microalgae, specifically autotrophic, are
considered the most viable option for
many reasons:
1. Mass production abilities
2. High neutral lipid content
3. Not a viable food source
4. Minimum land use
5. Carbon sink
3
Mass Production Abilities
Some strains of microalgae grows very
quickly compared to other terrestrial
crops
Chlorella vulgaris is the fastest growing
strain of autotrophic microalgae

4
High Neutral Lipid Content
Neutral Lipids are the only part of
microalgae commonly used in biofuel
production
Polar lipids tend to stick to themselves,
forming a ring, which is hard to break
during conversion
Neochloris oleoabundans has a very
high neutral lipid content (around 37%)
5
Minimal Land Use
Microaglae has the potential to produce
10X the biodiesel per unit of land
compared to other crops

6
http://electrictreehouse.c
om/wp-
content/uploads/2011/01/
algae-valcent-biofuel-
1.jpg
Carbon Sink
Autotrophic strains consume CO
2,
a major
contributor to global warming, during
photosynthesis
Autotrophic microalgae can also use
sodium bicarbonate as a carbon source
Heterotrophic strains use glucose, an
expense alternative

7
Project Goal and Objectives
Goal: To use autotrophic growth of microalgae
in high dissolved inorganic carbon mediums to
maximize growth and lipid productivity for use in
biofuels.
Objectives:
1. To study the growth and lipid accumulation
rates of Neochloris oleoabundans
2. To extract lipids from the microalgae and
determine lipid productivity
3. To create a growth model to predict and find
conditions of maximum productivity

8

Light Model for Chlorella vulgaris
created with data from previous
batch
9
dW
W
dt
=
max
n
n n
k
I
I I

=
+
2 2 2 0.5
0 0 0
0 0
( ) exp{ [( ) cos ( ( ) sin ) ]}
r
in
I
I W W r r r r r d dr
r
t
o u u u
t
= +
} }
10
Model fitted to data
o Model takes into
account shading
effect and algal
growth under different
average light
intensities
o Accuracy of model
depends on
parameters
determined under
specific growth and
average light intensity
o Model can be used to
design any
photobioreactor by
adding
hydrodynamics
Project Tasks
Literature Research
Gather background information from other studies involving
microalgae growth, especially in regards to lipids
Learning how to operate lab equipment
Autoclaving culture mediums
Cell Density: UV Spectrophotometer
Lipid analysis: FL Spectrophotometer
Dissolved inorganic carbon: Titration method
Nitrogen: Ammonia probe, Nitrate agent
Phosphorous: UV spectrophotometer
Lipid extraction technique: Gravimetric method
Lipid productivity: Gas Chromatography Mass Spectrum
11

Project Tasks (cont.)
Conducting growth kinetic study of Neochloris
oleoabundans
Autotrophic growth using sodium bicarbonate
(NaHCO
3
)
Compiling information, come to conclusions
Analyze data, including lipid productivity by volume
Prepare Deliverables:
1. Technical Paper
2. Poster
3. PowerPoint Presentation
4. Project Summary for NSF

12

Schedule and Progress Made
Tasks Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
Lab Traning


Literature Research



Growth Kinteics Study
Stage 1


Growth Kinteics Study
Stage 2



Lipid Extraction



Data Analysis



Technical Paper




Technical Poster


Presentation




13
The Two Step Method
Lipid production and growth are not
simultaneous, so two step method needed
Step 1 = Microalgae Growth
Step 2 = Lipid Production

14
http://www.nrel.gov/news/features/images/20101102_al
gae1_pix18070_large.jpg
http://aquaticbiofuel.files.
wordpress.com/2009/11/al
gae.jpg
Step 1: Microalgae Growth
15
To maximize growth,
different culture
conditions are
applied to growing
microalgae
Varied DIC
concentrations

Dissolved Inorganic Carbon
from NaHCO
3

16
DIC =
CO
2
(aq) + HCO
3
-

Sodium bicarbonate:
Keeps high DIC in
open system
Acceptable pH level
When CO
2
dissolves
in water, H
+
ion
produced and
lowers pH
CO
2
versus NaHCO
3

17
pCO
2

DIC
(mg/L)
pH
NaHCO
3

(mg/L) at
pCO
2
=0.037%
DIC
(mg/L)
pH
0.037%
0.2 5.6 1.3 0.2 7.7
25% 102 4.2 718 102 8.3
50% 202 4.1 1430 200 8.3
75% 300 4.0 2130 300 8.3
100% 396 3.9 2800 396 8.3
NaHCO
3
can keep a high DIC level at an acceptable pH window.
Table used with permission of Dr. Lee
Controlled pH daily using CO
2

18
Step 1 Results
19
Experimental Setup
Growth of Neochloris
oleoabundans increases as DIC
concentration increases
20
Fluorescence Spectroscopy
showed little change in lipid
content
21
Step 2: Lipid Production
After growth is at a maximum, different
nutrient conditions are applied to
maximize lipid accumulation
Specifically, nitrogen deficiencies will be
applied and light intensities varied

22
Why Nitrogen Deficiencies?
Microalgae needs nitrogen to grow
By cutting off a nitrogen source, one can
stunt growth, causing lipids to
accumulate

23
http://www.ars.usda.gov/sp2userfiles/ad_hoc/19000000S
afetyHealthandEnvironmentalTraining/graphics/Nitrogen
Atom.jpg
Set Up of Phase Two
24
Measured light
intensity (255,90,30
mol/m
2
/s)
Secured
fluorescent lights
(light temperature=
6500 K )

Finished Set Up
25
Stage 2 Preliminary Results

26
27
Biomass of Neochloris oleoabundans
decreases in Stage 2
Preliminary Fluorescence
Spectrophotometry shows increased
lipid content with increased light
intensity
28
Light Intensity seems to have
little effect on TOTAL lipids
29
0
10
20
30
40
30 90 255
%

W
e
i
g
h
t

o
f

B
i
o
m
a
s
s

Light Intensity (mol/m
2
*s)
Total Lipids
Total Lipids
Lipid Separation: The Final Step
Neutral lipids from Stage 1
(varying DIC)
Polar lipids Neutral
lipids
30
Lipid Extraction
Adding solvents
to silica gel
column
Column
separation
Rotary
evaporator
31
Light Intensity in Stage 2 had
negligible effect on lipid
production
32
0
5
10
15
20
25
30
35
40
30 90 255
%

W
e
i
g
h
t

o
f

B
i
o
m
a
s
s

Light Intensity (mol/m2*s)
Total
Lipids
Neutral
Lipids
0
10
20
30
40
50
60
30 90 255
N
e
u
t
r
a
l

L
i
p
i
d
s

(
%

o
f

t
o
t
a
l

l
i
p
i
d
s
)

Light Intensity (mol/m2*s)
Neutral
Lipids
Conclusion
Increased light intensity and DIC
concentration increases biomass
production
No conclusion can be made about lipid
production based on light intensity in
Stage 2
Further testing is necessary to create a
scalable model for Neochloris
oleoabundans
33
References
Alonzo F., and Mayzaud P. (1999) Marine Chemistry, 67, 289-301.
Bligh E., and Dyer W. (1959) Canadian Journal of Biochemistry and Physiology, 37, 911-917.
Brennan, L., Owende, P. (2013).Advanced Biofuels and Bioproducts. Springer Science + Business Media.
Chen W., Sommerfeld M., and Hu, Q. (2011) Bioresource Technology, 102, 135-141.
Chien, L., Gomes, J., Lin, Y., Liou, J., Su, C., Syru, R. (2011). Factors affecting lipid accumulation by Nannochloropsis oculata in a two-stage cultivation process, J Appl Phycol, 23,
903908.
Cooksey K., Guckert J., Williams S., and Callis P (1987). Journal of Microbiological Methods, 6, 333-345.
Danquah, M.K., Halim, R., and Webley, P.A. (2012). Extraction of oil from microalgae for biodiesel production: A review, Biotechnology Advances, 30, 709-732.
Fischer R., and Peters D. (1968) "Basic theory and practice of quantitative chemical analysis.," W. B. Saunders Company., Philadelphia, London, Toronto.
Go, S., Jeong, G., Kim, S., and Lee, S. (2012). Factors affecting the growth and the oil accumulation of marine microalgae, Tetraselmis suecica, Bioprocess Biosyst Eng, 35, 145-150.
Gordon, R., and Seckbach, J. (2012). The Science of Algal Fuels: Phycology, Geology, Biophotonics, Genomics and Nanotechnology. Springer.
Lam, M.K., and Lee, K.T. (2012). Microalgae biofuels: A critical review of issues, problems and the way forward, Biotechnology Advances, 30, 673-690.
Solovchenko, A.E., (2012). Physiological Role of Neutral Lipid Accumulation in Eukaryotic Microalgae under Stresses, Russian Journal of Plant Physiology, 59(2), 167-176.
Wang, G., Wang, T. (2012). Characterization of Lipid Components in Two Microalgae for Biofuel Application, J Am Oil Chem Soc, 89, 135143.
Widjaja, A., Chien, C., Ju, Y. (2009) Study of increasing lipid production from fresh water microalgae Chlorella vulgaris, Journal of the Taiwan Institute of Chemical Engineers, 40,
13-20.
Zhou, Y., Zhang, Z. (1989) "The toxicity methods in aquatic living," Agriculture Press.

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Questions?
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/servlet/JiveServlet/showIm
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question-mark.jpg