auxenochlorella protothecoides as a source of lipids and antioxidants
TRANSCRIPT
Auxenochlorella Protothecoides as a sourceof lipids and antioxidants
MSc Mary C Ibarra Vidal, Hector De la Hoz Siegler
University of Calgary
Bioprocess Laboratory, Chemical Engineering
Calgary, Alberta, Canada
October 25th, 2016 Melbourne, VIC, Australia 1
https://www.pinterest.com/pin/502644008384226497/
Microalgae Importance
Microalgae few µM to a few hundreds
Lipids (biofuels)
High Value Products:
—Carotenoids (strain dependent)
—Antioxidants
—Pigments
—Food Supplements
—Others
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.
Auxenochlorella protothecoides
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High lipid content under
heterotrophic conditions
Important source lutein/zeaxanthin
Recently designated as GRAS by FDA
https://commons.wikimedia.org/wiki/File%3AAlpha-D-Glucopyranose.svg
A. Protothecoides heterotrophic culture
Low cost carbon source
Recycled from Biodiesel
Metabolized for A. protothecoides
Enhanced lipid production on A. Protothecoides
High lipid productivity
80% of Total Medium cost
Low lipid yield
Cells Bleaching affecting antioxidants
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Glucose Glycerol
Potential Applications for A. protothecoides lipids
Biofuels
Important source ofOmega-3 fatty acids (EPA and DHA)
Other Fatty Acids(palmitic, oleic, linoleic, stearic and arachidonic)
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http://www.goodwholefood.com/epa-and-dha-krill-oil/
http://www.bioenergyconsult.com/algal-biomass/
A. Protothecoides antioxidants potentials
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LUTEIN
Potent antioxidant
Retina damage
prevention and
protection
Infant Milk
Zeaxanthin
Cantaxanthinhttp://pharmsine.en.ecplaza.net/canthaxanthin-10-feed-grade--21536-785491.html
https://faculty.washington.edu/chudler/armd.html
Experimental Methods
• 100% Glycerol
• 75:25 Glycerol-Glucose
• 50: 50 Glycerol-Glucose
• 25:75 Glycerol-Glucose
• 100% Glucose
10: 1 Carbon-Nitrogen ratio
• 5 Glycerol-Glucose Combinations40:1
• 5 Glycerol-Glucose Combinations80:1
• 5 Glycerol-Glucose Combinations120:1
20 Culture Conditions with 4 replicates
(Total 30 g/L carbon source)
Culturing Time9 Days
Experimental Measurements
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Flask Culture
Centrifugation
HPLC Glycerol-Glucose Measurements
Substrate Concentration
Dry weigthtMicrotubes
Biomass concentration
Absorbance at 540 nm
Extraction
TEAC Test Lipid Content
supernatant biomass
End of the cultureDaily BasisDaily Basis
Substrate concentration and Biomass profiles
Lower glycerol concentrationin combination with Glucose
Equal glucose-glycerol initial Concentration
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0
5
10
15
20
25
30
0
2
4
6
8
10
12
14
16
18
0 1 2 3 4 5 6 7 8 9
Gly
cero
l-G
luco
se C
on
cen
tra
tion
(g
/L)
Dry
Wei
gh
t (g
/L)
Time (Days)
40:1 Dry weight 50:50 gly-gluGlycerolGlucose
0
5
10
15
20
25
30
0
2
4
6
8
10
12
14
16
18
0 1 2 3 4 5 6 7 8 9G
lyce
rol-
Glu
cose
Co
nce
ntr
ati
on
(g
/L)
Dry
Wei
gh
t (g
/L)
Time (Days)
40:1 Dry Weight 25:75 Gly-Glu
Glycerol
Glucose
NON- DIAUXIC GROWTH
Cells Bleaching
Glucose-Glycerol Ratio 100% Glycerol 25:75 50:50 25:75 100% Glucose
80
:1
12
0:1
10
12
0:1
8
0:1
Car
bo
n-N
itro
gen
Rat
io
55.6
129
113
149
50.6
64.8
86.5
139
0
20
40
60
80
100
120
140
160
180
10:1 40:1 80:1 120:1
Lip
id y
ield
(m
g l
ipid
s/g
su
bst
rate
)
Culture carbon-nitrogen ratio
100% Glycerol 75:25 Gly-Glu 50:50 Gly-Glu 25:75 Gly-Glu 100% Glucose
Lipid Substrate Yield
Lipids Yield increases with nitrogen limitation
Glycerol presented the best lipid yield
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*
*
*
*
C C
C
C
C **
Antioxidants to Substrate Yield
3.95
5.64
6.65
5.114.68
3.44
1.72
2.45
1.88
0
1
2
3
4
5
6
7
10:1 40:1 80:1 120:1
An
tiox
ida
nts
y
ield
(µm
ol
Tro
lox
eq
uiv
ale
nt/
gr
bio
ma
ss)
Culture carbon-nitrogen ratio
100% Glycerol 75:25 Gly-Glu 50:50 Gly-Glu 25:75 Gly-Glu 100% Glucose
12
*
C CC C
*
*
* *
*
*
* *
*
*
C
Conclusions
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Glycerol presence enhance Biomass yield beyond the theoretical limit
0.7640.785
1.04
0.809
0.544
0.689
0.615
0.684
0.5390.560
0.606
10:01 40:1 80:1 120:1
Bio
mass
Y
ield
(G
Bio
mass
/G S
ub
trate
)
CULTURE CARBON-NITROGEN RATIO
100% Glycerol 75:25 Gly-Gluc 50:50 Gly-Glu 25:75 Gly-Glu 100% Glucose
Autotrophic MetabolismCalvin Cycle
O2
CO2
Conclusions
In presence of Glycerol
Heterotrophic Metabolism14
STOP
Increase of Dark Calvin cycle activity
Conclusions
40:1 Carbon-Nitrogen ratio and 100% Glycerol
Best condition Lipids and Antioxidants
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Heterotrophic cultivation with Glycerol addition
Modulates chloroplast bleaching
Enhance the production of lipids
Increases lipids substrate yield
Enhance the production of antioxidants
Future Directions
Evaluate the effect of glycerol addition on the lipid profile.
Evaluate the effect of glycerol addition on the carotenoids profile
Compare heterotrophic culture with Mixotrophic.
Compare semi-continuous to continuous operation utilizing glycerol.
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