The need, application and results of microalgal
biomass analysis to study Carbon flux and its
control under growth and stress conditions for
biofuel production
Claude Aflalo,
Microalgal Biotechnology Laboratory,
French Associates Institute for Agriculture and
Biotechnology of Drylands
Institutes for Desert Research,
Ben Gurion University, Israel
In cooperation with
MBL: S. Boussiba, Z. HaCohen, I. Khozin,
E. Kleiman, S. Didi
and A. Freberg, visiting student (UMB, Norway)
Villefranche 2009
Quo vadis fossil fuels?
Production and discovery of new sources of fossil fuel are decreasing.
The demand in energy is increasing. New, permanent condition
=> Imperative need for alternative sources.
60 Tbl/y
History
Forecast
50
Demand +
2% growth
40
30
Use
20
Production
10
Discovery
0
1930
1950
1970
1990
2010
2030
2050
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Quo vadis Terra?
[CH2]n + 1.5n O2

n CO2 + n H2O + energy
The (ab)use of fossil fuel, needed for
development, has an increasingly
negative effect on the environment.
The choice and management of
alternative energy sources ought to
consider global Carbon, Oxygen and
energy balance to minimize the impact.
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Carbon flux in phototrophic organisms
External sources: CO2, light (energy, reductive equivalents)
Biosynthetic output: protein, carbohydrate, lipids
 Growth: materials for new biomass (cells)
 Stress: no growth, storage
Optimal growth
Stress
Protein
CO2
CO2
Carbohydrate
Lipid
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The physiology behind stress management
H. pluvialis (as a model example) has evolved to fit in restricted
aqueous habitats, and to respond efficiently (overproduction of
astaxanthin) to the drastic changes expected to occur thereby.
Vegetative growth;
primary metabolism
Stress
Sensing
Metabolic message = relative excess of light
Response: accommodation
mechanisms induced;
division stops
Secondary metabolites
production and
accumulation initiated
+Nutrients,
Check acclimatation
point
Division resumes;
secondary
metabolites dilution
in daughter cells
?
-Nutrients,
commitment
Encystment;
secondary
metabolites , cell wall
and lipid accumulate
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Microalgae have evolved to fulfill their needs, not ours…
• Given favorable condition, they will grow at maximal rate.
• Under any stress, complex processes are initialized, whereby
 cell division stops, and biosynthesis is reduced;
 the relative excess of photosynthetic electron transfer rate,
results in oxidative stress;
 appropriate cellular responses are being induced, leading to
 accumulation of storage compounds to be used for maintenance
(energy, reducing power) and building blocks to be available
when favorable conditions are restored.
These properties should be well-defined and properly applied
for efficient biotechnological exploitation of the
photosynthetic organisms
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An idealized view of central aerobic metabolism
Polysugars
Proteins
ADP + Pi
ADP + Pi
ATP
ADP + Pi
ATP
Sugars
Amino acids
ATP
Building
blocks
ADP + Pi
Fatty acids
ADP + Pi
ADP + Pi
ANABOLISM
CATABOLISM
ATP
ATP
NADP+
NADPH
ADP + Pi
Intermediates
ATP
AcCoA
Catabolism splits organic
molecules into inorganic
compounds. It is mostly
oxidative and generates
available energy (ATP) and
reducing equivalents (NADH).
ATP
PEP/Pyr
Anabolism involves the
reductive production of
building blocks to sustain
growth, at the expense of ATP
and NADPH.
NADPH
ketone bodies
O2
NAD+
TCA
e-
NADH
Polymers
Lipids
H2O
NADP+
ATP
ADP + Pi
O2
ADP + Pi
Photosynthesis in plants
transduces light energy to
generate ATP and NADPH used to
fix atmospheric CO2 into sugar.
ATP
NH3
H2O
CO2
Inorganic
compounds
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The costs of macromolecules biosynthesis
Biosynthetic demands for
cofactors and intermediary
metabolites of central
metabolism for the
accumulation of 1 g starch,
protein or lipid (Schwender
et al. 2004).
Overproduction of lipid
seems to be the strategy of
choice to relieve oxidative
stress (reduce excess ATP
and NADPH.
Metabolic versatility of the pentose phosphate pathway
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So what’s in that box?
Prot
CO2
CH
Lip
In phototrophic organisms (e.g., algae and plants), CO2
the energy of light is transduced into chemical
and reductive energy to support growth
Starch
(macromolecules) and/or counter
various stresses.
G6P
G6P
AA
pool
R5P
DHA
P
AA
pool
GAP
DHAP GAP
PEP
PEP
Pyr
Pyr
OAA
Cit
OAA
Cit
AcCoA
Mal
OAA
TAG
MaCoA
Mal
C18:1
C18:1
AA
pool
AcCoA
MaCoA
GlyP
C20:2
C22:3
H2O, NADP+
ADP, Pi
O2, NADPH
ATP
Overview of lipid metabolism
Beopoulos et al, 2008
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Total lipid and total carbohydrate determination
I. Harsh acid hydrolysis yields >95% monomers
R1
O
R
R1 OH +
H
O
O
O
H2SO4
O
H2SO4
C
O
O
O
O
R
R
H
C+
H
Provides a single aliquot,
balanced and ready for direct
colorimetric analysis of both
compounds.
O
O
O
O
H
+
O C
H2SO4
O
H2SO4
H
R
O
O
O
II. Color reactions linear from 5-150 ug sugar or fatty acid
O
O
+
[colored adduct]
HO P OH
O
OMe
+
C+
H O
anthrone
H
O
HO P OH
O
OMe
R
H
C+
R
H
H O P OH
O+
OMe
H R H
C+
H O C
R
H
H R H
H
C
O C
R
phosphovanillin
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11
Experimental/Analytical tools
• Control of CO2 input (pH monitoring)
• Determination of total fixed Carbon into macromolecules
(carbohydrate, lipid, and protein)
• Design meaningful chemometric indices to detect and quantitate
preferential Carbon flow into accumulated lipids
• Elemental analysis (CNHS)
• Composition of accumulated lipids (GC FAME)
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General experimental design
Healthy Culture
Full
Medium
2% CO2
0.5% CO2
C1
C2
Growth
N-deprived
Medium
2% CO2
0.5% CO2
D1
D2
Stress
Batch culture at constant incident light intensity
(decreasingly effective upon growth)
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General growth parameters Parietochloris incisa
Col C2 -Full, 0.5% CO2
8
8
210
7
180
6
150
5
120
4
90
3
60
2
210
7
180
6
150
90
Chl 5
Car 4
DW 3
60
2
30
1
30
1
0
0
0
0
120
10
20
30
40
0
10
40
Col D2 -Stress, 0.5% CO2
Col D1 -Stress, 2% CO2
8
210
7
210
7
180
6
180
6
150
5
150
5
120
4
120
4
90
3
90
3
60
2
60
2
30
1
30
1
0
0
0
0
10
20
Age - day
30
40
Pigment - mg/L
240
240
DW - g/L
Pigment - mg/L
Biomass growth
rapidly stops.
The dilute
culture ‘senses’
a relatively high
light intensity.
30
Biomass
growth is
slower but
sustained after
N is depleted.
Age - day
Age - day
Stress-2%:
20
Full-0.5%:
8
Stress-0.5%:
DW - g/L
0
Pigment - mg/L
Pigment - mg/L
After N is
depleted
(arrow), the
pigment content
diminishes and
biomass growth
gradually stops.
240
DW - g/L
Full-2%:
240
DW - g/L
Col C1 -Full, 2% CO2
Same general
behavior,
indicating CO2
is saturating
under these
conditions.
0
0
10
20
30
40
Age - day
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Pigments and CO2
Chl/DW
Car/DW
8
1.2
6
Car/DW - %
Chl/DW - %
7
C1
C2
D1
D2
5
4
3
1.0
0.8
0.6
2
0.4
1
0.2
0
0.0
0
10
20
30
40
0
Age - day
10
20
30
40
Age - day
Car/Chl
The pH value in nonflushed culture aliquots
equilibrated in the dark
may represent a
sensitive indicator of
the steady-state CO2
concentration under the
real culture conditions.
pH
1.4
9
1.2
Dark pH
8.5
1.0
Car/Chl
Pigments content,
and especially their
ratio represent a good
index for the depth of
the stress perceived
by the culture.
1.4
0.8
8
7.5
0.6
7
0.4
0.2
6.5
0.0
6
0
10
20
Age - day
30
40
0
10
20
30
40
Age - day
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Lipid and carbohydrate accumulation
TFA Volumetric
Lipids Volumetric
2.5
1.5
1.0
0.5
Carbohydrate - g/L
C1
C2
D1
D2
Lipids - mg/ml
3.0
2.0
TFA - g/L
Carbohydrate Volumetric
3.5
3.5
2.5
2.0
1.5
1.0
0.5
0.0
0.0
0
10
20
30
0
40
10
20
30
TFA Content
1.0
0.5
0
10
20
10
40
30
20
40
40
30
20
10
0
0
30
40
50
10
0
30
Carbohydrate Content
Carbohydrate - %DW
30
20
Age - day
50
Lipids - %DW
TFA - % DW
40
20
1.5
0.0
40
60
Age - day
2.0
Lipids Content
50
10
2.5
Age - day
Age - day
0
3.0
0
10
20
Age
30
40
0
10
20
30
40
Age - day
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Probing elongation (processing gas chromatograms)
+N 2%
70
+N 2%
35
60
30
TFA
18
% TFA
20
40
16
30
22
25
% DW
50
18
20
20
15
16
22
20
10
10
5
0
0
0
10
20
Age - day
30
40
0
+N 0.5%
70
10
30
40
+N 0.5%
30
60
Age -20day
25
50
TFA
18
40
16
30
22
20
% DW
% TFA
20
18
20
15
16
22
10
20
5
10
0
0
0
10
20
Age - day
30
40
0
10
20
Age - day
30
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Probing desaturation
-N 2%
50
40
0
35
1
30
2
25
3
20
4
15
5
40
% DW
% TFA
45
10
35
TFA
30
0
25
1
20
2
15
3
10
5
5
0
0
10
20
30
0
40
0
Age - day
-N 0.5%
10
30
25
20
15
10
5
3
4
% DW
1
2
30
40
40
0
40
35
20
Age - day
-N 0.5%
45
50
45
% TFA
-N 2%
45
35
TFA
30
0
25
1
20
2
15
3
10
5
5
0
0
0
10
20
Age - day
30
40
0
10
20
Age - day
30
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Summary of kinetic lipid biosynthesis
Elongation
70
Desaturation
+N 2%
35
60
0
TFA
30
1
16
30
22
25
18
25
2
20
3
15
4
10
5
20
20
15
16
20
10
10
5
22
Full
2.0%
% TFA
% TFA
20
40
% DW
50
0
10
20
Age - day
30
0
+N 0.5%
70
10
Age 20
- day
30
3
20
Age - day
30
40
0
+N 0.5%
45
40
16
30
22
20
% DW
% TFA
20
18
20
15
16
22
10
20
10
5
0
0
Full
0.5%
35
0
30
1
2
25
3
20
4
15
30
40
5
10
30
40
16
30
22
20
35
TFA
30
18
25
20
20
16
15
22
-N
2.0%
1
2
25
3
20
4
15
5
5
-N 0.5%
70
10
20
Age - day
30
60
40
40
18
35
20
30
16
30
22
% DW
% TFA
50
TFA
18
20
25
16
20
22
15
20
10
10
5
0
0
0
10
20
Age - day
30
40
-N
0.5%
20
30
10
20
Age - day
TFA
30
0
25
1
20
2
15
3
0
40
0
-N 0.5%
30
40
10
1
30
25
20
2
15
10
5
3
4
30
40
40
0
40
35
20
Age - day
-N 0.5%
45
50
45
35
TFA
30
0
25
1
20
2
15
3
10
5
5
0
0
-N 2%
35
Age - day
45
40
5
10
-N 0.5%
50
30
10
0
40
20
40
0
0
10
45
30
5
40
0
-N 2%
0
% TFA
Age - day
30
3
Age - day
35
0
20
40
40
10
0
30
45
10
10
10
20
50
% TFA
20
40
0
10
Age - day
40
18
2
15
0
0
-N 2%
% DW
% TFA
20
Age - day
45
50
1
5
0
-N 2%
60
TFA
10
0
70
+N 0.5%
10
% DW
20
Age - day
40
0
% DW
10
30
20
5
0
20
Age - day
25
% DW
TFA
18
% TFA
25
50
10
30
40
60
2
0
10
40
+N 0.5%
30
1
15
5
0
40
20
10
5
0
0
TFA
25
0
0
+N 2%
30
35
30
18
+N 2%
40
% DW
+N 2%
0
0
10
20
Age - day
30
40
0
10
20
Age - day
30
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Using meaningful indices: Lip:CH and Car/Chl
P. incisa , ratio vs. ‘stress index’
1.4
1.4
1.2
1.2
1
1
Lip:CH
Lip:CH
P. incisa , ratio vs. time
0.8
0.6
C1
C2
D1
D2
0.4
0.2
5
10
15
20
25
0.6
0.4
0.2
Stress: 0 mild
0
0
0.8
30
Age - day
Upon stress induction, the lipid content
increases (at the expense of protein, but
not carbohydrate), resulting in an
increase of the Lip:CH ratio up to a
limit. The latter may reflect a constraint
in resources management imposed by
cellular physiology.
0
0.0
0.2
harsh
0.4
0.6
0.8
1.0
Car/Chl
The lack of full correlation between the
metabolic ratio Lip:CH and the pigments
ratio Car/Chl is indicative of subtle
variation in the manifestation of ‘stress’,
often leading to hysteretic behavior.
How general are these features ?
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0
8
Nannochloropsis sp.3
70
CH
Lip
L/C
60
Lip or CH - %DW
2
4
6
Age - day
3.5
70
3
60
50
2.5
40
2
30
1.5
20
1
10
0.5
0
0
0
2
4
Age - day
6
8
Nannochloropsis sp.3
CHs
Lips
L/Cs
3.5
3
50
2.5
40
2
30
1.5
20
1
10
0.5
0
0.35
0.45
0.55
0.65
L/C
2
1
0
Nannochloropsis was grown under a day/night cycle either
in
•full medium at low light intensity
•N-depleted medium at high light intensity
The cultures were analyzed in terms of DW, pigments, as
well as total carbohydrate and lipid content.
Lip or CH - %DW
5
4
3
L/C
DW - g/L
Similar effect in a marine alga
0
0.75
Tcar/Chl
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Batch day/night cultures under variable light intensities
Different extents of stress were reached along batch growth allowing for N depletion.
DW - g/L
2
Both Chlorella and Haematococcus
accumulated biomass during the course of
the experiment.
1
0
0
7
Age -day
14
LI
Haematococcus pluvialis
60
1
70
60
50
0.8
40
0.6
30
0.4
CH
Lip
L/C
20
10
0.2
0
0
0.0
0.5
1.0
Tcar/Chl
1.5
1.2
1
50
0.8
40
0.6
30
Lip/CH
1.2
Lip or CH - %DW
70
Lip/CH
Lip or CH - %DW
Chlorella emersonii
0.4
CH
Lip
L/C
20
10
0.2
0
0
0
2
4
6
8
10
12
14
Tcar/Chl
The preferential accumulation of lipids upon stress appears to be
also conserved in species of stable or variable sweet water ponds.
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?
Thank you…
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