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

?

Nice09_CA 6

• 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

Nice09_CA 17

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

Nice09_CA 18

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

Nice09_CA 19

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

Nice09_CA 22

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…