Claude Aflalo,
Microalgal Biotechnology Laboratory, French Associates Institute for Agriculture and
Biotechnology of Drylands Institutes for Desert Research,Ben Gurion University, Israel
Villefranche 2009
The need, application and results of microalgal biomass analysis to study
Carbon flux and its control under growth and stress conditions for biofuel production
In cooperation with
MBL: S. Boussiba, Z. HaCohen, I. Khozin, E. Kleiman, S. Didiand A. Freberg, visiting student (UMB, Norway)
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1930 1950 1970 19900
10
20
30
40
50
60 Tbl/y
History
Use
DiscoveryProduction
2010 2030 2050
Forecast
Demand + 2% growth
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.
Quo vadis fossil fuels?
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Quo vadis Terra?
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.
[CH2]n + 1.5n O2
n CO2 + n H2O + energy
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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
CO2 CO2
Carbo-hydrate
Lipid
Protein
Optimal growth Stress
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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.
SensingMetabolic message = relative excess of
light
Secondary metabolites
production and accumulation
initiated
Division resumes; secondary
metabolites dilution in
daughter cells
Encystment; secondary
metabolites , cell wall and lipid accumulate
+Nutrients,acclimatation
Response: accommodation
mechanisms induced; division stops
-Nutrients,commitment
Check point
Vegetative growth; primary
metabolism Stress
?
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• 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
Microalgae have evolved to fulfill their needs, not ours…
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Catabolism splits organic molecules into inorganic compounds. It is mostly oxidative and generates available energy (ATP) and reducing equivalents (NADH).
An idealized view of central aerobic metabolism
Anabolism involves the reductive production of building blocks to sustain growth, at the expense of ATP and NADPH.
PolymersProteins Polysugars Lipids
blocksBuildingAmino acids Sugars Fatty acids
Intermediates
PEP/Pyr
AcCoA
ketone bodies
compoundsInorganicNH3 2CO2H O
TCA
CATABO
LIS
M
ADP + Pi
ADP + Pi
ATP
e-
O2
NAD+
NADH
+
ADP + PiADP + PiADP + Pi
ATPATPATP
ADP + PiADP + Pi
ATP ATPANABO
LIS
M
ADP + Pi
ATP
ATPNADP
NADPH
Photosynthesis in plants transduces light energy to generate ATP and NADPH used to fix atmospheric CO2 into sugar.ADP + Pi
ATP
NADPH
NADP+O2
H2O
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Metabolic versatility of the pentose phosphate pathway
Biosynthetic demands for cofactors and intermediary metabolites of central metabolism for the accumulation of 1 g starch, protein or lipid (Schwender et al. 2004).
The costs of macromolecules biosynthesis
Overproduction of lipid seems to be the strategy of choice to relieve oxidative stress (reduce excess ATP and NADPH.
In phototrophic organisms (e.g., algae and plants), the energy of light is transduced into chemical and reductive energy to support growth (macromolecules) and/or counter various stresses.
Starch
CO2
G6P
GAP
Pyr
PEP
AcCoAOAA
Mal
R5P
DHAP
MaCoA
C18:1C20:2C22:3
GlyP
TAG
GAP
Pyr
PEP
G6P
OAA
Mal
DHAP
C18:1
AcCoA
OAA
Cit
MaCoA
Cit
AApoolAA
pool
AApool
CO2
CH
Lip
Prot
So what’s in that box?
H2O, NADP+ ADP, Pi
O2, NADPH ATP
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Overview of lipid metabolismBeopoulos et al, 2008
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Total lipid and total carbohydrate determination
I. Harsh acid hydrolysis yields >95% monomers
II. Color reactions linear from 5-150 ug sugar or fatty acid
C+H
OMe
H
C
R
R H
H
O
P OH
O
OHO+
CH
OMe
H
C
R
R H
H
O
P OH
O
OHO
P OH
O
OHO
C+H O
OMe+
RH
C+R H
H
phosphovanillin
Provides a single aliquot, balanced and ready for direct colorimetric analysis of both compounds.
O
O
O
H2SO4H2SO4
OO
CO
O
OO
OO
O
O
O
OO
OR1 C
O R
R H
HR
H
C+R H
H+
H2SO4
OHR1
OH C
O
+H2SO4
+
anthrone
[colored adduct]
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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
N-deprived Medium
Batch culture at constant incident light intensity (decreasingly effective upon growth)
2% CO2 0.5% CO2
C1 C2
2% CO2 0.5% CO2
D1 D2
Growth Stress
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General growth parameters Parietochloris incisa
Col C1 -Full, 2% CO2
0
30
60
90
120
150
180
210
240
0 10 20 30 40
Age - day
Pigm
ent -
mg/
L
0
1
2
3
4
5
6
7
8
DW
- g/
LChlCarDW
Col C2 -Full, 0.5% CO2
0
30
60
90
120
150
180
210
240
0 10 20 30 40Age - day
Pigm
ent -
mg/
L
0
1
2
3
4
5
6
7
8
DW
- g/
L
Full-2%: After N is depleted (arrow), the pigment content diminishes and biomass growth gradually stops.
Full-0.5%: Biomass growth is slower but sustained after N is depleted.
Col D2 -Stress, 0.5% CO2
0
30
60
90
120
150
180
210
240
0 10 20 30 40Age - day
Pigm
ent -
mg/
L
0
1
2
3
4
5
6
7
8
DW
- g/
L
Col D1 -Stress, 2% CO2
0
30
60
90
120
150
180
210
240
0 10 20 30 40Age - day
Pigm
ent -
mg/
L
0
1
2
3
4
5
6
7
8
DW
- g/
L
Stress-2%: Biomass growth rapidly stops. The dilute culture ‘senses’ a relatively high light intensity.
Stress-0.5%: Same general behavior, indicating CO2 is saturating under these conditions.
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Pigments and CO2Chl/DW
0
1
2
3
4
5
6
7
8
0 10 20 30 40Age - day
Chl
/DW
- %
C1C2D1D2
Car/DW
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0 10 20 30 40Age - day
Car
/DW
- %
pH
6
6.5
7
7.5
8
8.5
9
0 10 20 30 40Age - day
Dar
k pH
Car/Chl
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0 10 20 30 40Age - day
Car
/Chl
Pigments content, and especially their ratio represent a good index for the depth of the stress perceived by the culture.
The pH value in non-flushed culture aliquots equilibrated in the dark may represent a sensitive indicator of the steady-state CO2 concentration under the real culture conditions.
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Lipid and carbohydrate accumulationCarbohydrate Volumetric
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0 10 20 30 40Age - day
Car
bohy
drat
e - g
/L
TFA Volumetric
0.0
0.5
1.0
1.5
2.0
2.5
0 10 20 30 40Age - day
TFA
- g/
L C1C2D1D2
Lipids Volumetric
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0 10 20 30 40
Age - dayLi
pids
- m
g/m
l
TFA Content
0
10
20
30
40
50
0 10 20 30 40Age - day
TFA
- %
DW
Carbohydrate Content
0
10
20
30
40
50
0 10 20 30 40Age - day
Car
bohy
drat
e - %
DW
Lipids Content
0
10
20
30
40
50
60
0 10 20 30 40Age
Lipi
ds -
%D
W
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Probing elongation (processing gas chromatograms)
+N 2%
0
10
20
30
40
50
60
70
0 10 20 30 40Age - day
% T
FA
18
20
16
22
+N 0.5%
0
10
20
30
40
50
60
70
0 10 20 30 40Age - day
% T
FA
18
20
16
22
+N 2%
0
5
10
15
20
25
30
35
0 10 20 30 40Age - day
% D
W
TFA
18
20
16
22
+N 0.5%
0
5
10
15
20
25
30
0 10 20 30 40Age - day
% D
W
TFA
18
20
16
22
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Probing desaturation
-N 0.5%
05
101520253035404550
0 10 20 30 40Age - day
% T
FA
0
1
2
3
4
5
-N 2%
0
5
10
15
20
25
30
35
40
45
0 10 20 30 40Age - day
% D
W
TFA
0
1
2
3
-N 0.5%
0
5
10
15
20
25
30
35
40
45
0 10 20 30 40Age - day
% D
W
TFA
0
1
2
3
-N 2%
05
101520253035404550
0 10 20 30 40Age - day
% T
FA
0
1
2
3
4
5
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Summary of kinetic lipid biosynthesis
-N 2%
0
10
20
30
40
50
60
70
0 10 20 30 40Age - day
% T
FA
18
20
16
22
-N 0.5%
0
10
20
30
40
50
60
70
0 10 20 30 40Age - day
% T
FA
18
20
16
22
-N 2%
0
5
10
15
20
25
30
35
40
45
0 10 20 30 40Age - day
% D
W
TFA
18
20
16
22
-N 0.5%
0
5
10
15
20
25
30
35
40
45
50
0 10 20 30 40Age - day
% D
W
TFA
18
20
16
22
-N 0.5%
05
101520253035404550
0 10 20 30 40Age - day
% T
FA0
1
2
3
4
5
-N 2%
0
5
10
15
20
25
30
35
40
45
0 10 20 30 40Age - day
% D
W
TFA
0
1
2
3
-N 0.5%
0
5
10
15
20
25
30
35
40
45
0 10 20 30 40Age - day
% D
W
TFA
0
1
2
3
-N 2%
05
101520253035404550
0 10 20 30 40Age - day
% T
FA
0
1
2
3
4
5
+N 2%
0
5
10
15
20
25
30
35
40
0 10 20 30 40Age - day
% T
FA
0
1
2
3
4
5
+N 0.5%
0
5
10
15
20
25
30
35
40
45
0 10 20 30 40Age - day
% T
FA
0
1
2
3
4
5
+N 2%
0
5
10
15
20
25
30
0 10 20 30 40Age - day
% D
W
TFA
0
1
2
3
+N 0.5%
0
5
10
15
20
25
30
0 10 20 30 40Age - day
% D
W
TFA
0
1
2
3
+N 2%
0
10
20
30
40
50
60
70
0 10 20 30 40Age - day
% T
FA
18
20
16
22
+N 0.5%
0
10
20
30
40
50
60
70
0 10 20 30 40Age - day
% T
FA
18
20
16
22
+N 2%
0
5
10
15
20
25
30
35
0 10 20 30 40Age - day
% D
W
TFA
18
20
16
22
+N 0.5%
0
5
10
15
20
25
30
0 10 20 30 40Age - day%
DW
TFA
18
20
16
22
DesaturationElongation
Full 2.0%
Full 0.5%
-N 0.5%
-N 2.0%
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Using meaningful indices: Lip:CH and Car/Chl
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.
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.
P. incisa, ratio vs. ‘stress index’
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0.0 0.2 0.4 0.6 0.8 1.0Car/Chl
Lip:
CH
P. incisa, ratio vs. time
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 5 10 15 20 25 30Age - day
Lip:
CH
C1C2D1D2
How general are these features ?
Stress: 0 mild harsh
Nice09_CA 21
Similar effect in a marine alga
Nannochloropsis sp.3
0
10
20
30
40
50
60
70
0.35 0.45 0.55 0.65 0.75
Tcar/Chl
Lip
or C
H -
%D
W0
0.5
1
1.5
2
2.5
3
3.5
L/C
CHsLipsL/Cs
Nannochloropsis was grown under a day/night cycle either in•full medium at low light intensity•N-depleted medium at high light intensityThe cultures were analyzed in terms of DW, pigments, as well as total carbohydrate and lipid content.
012345
0 2 4 6 8Age - day
DW
- g/
L
Nannochloropsis sp.3
0
10
20
30
40
50
60
70
0 2 4 6 8
Age - day
Lip
or C
H -
%D
W
0
0.5
1
1.5
2
2.5
3
3.5
L/C
CHLipL/C
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0
1
2
0 7 14Age -day
DW
- g/
LChlorella emersonii
0
10
20
30
40
50
60
70
0.0 0.5 1.0 1.5
Tcar/Chl
Lip
or C
H -
%D
W
0
0.2
0.4
0.6
0.8
1
1.2
Lip/
CH
CHLipL/C
Haematococcus pluvialis
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12 14
Tcar/Chl
Lip
or C
H -
%D
W0
0.2
0.4
0.6
0.8
1
1.2
Lip/
CH
CHLipL/C
The preferential accumulation of lipids upon stress appears to be also conserved in species of stable or variable sweet water
ponds.
Batch day/night cultures under variable light intensities
Different extents of stress were reached along batch growth allowing for N depletion.
Both Chlorella and Haematococcus accumulated biomass during the course of
the experiment. LI
Nice09_CA 23
?
Thank you…