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

2

.

Auxenochlorella protothecoides

5

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

4

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)

5

http://www.goodwholefood.com/epa-and-dha-krill-oil/

http://www.bioenergyconsult.com/algal-biomass/

A. Protothecoides antioxidants potentials

6

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

8

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

9

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

11

*

*

*

*

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

13

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

15

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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Acknowldeges

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