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Claude Aflalo , Microalgal Biotechnology Laboratory, French Associates Institute for Agriculture and Biotechnology of D

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

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Claude Aflalo , Microalgal Biotechnology Laboratory, French Associates Institute for Agriculture and Biotechnology of D

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  1. 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

  2. Forecast 60 Tbl/y History 50 2010 2030 2050 40 30 20 10 0 1930 1950 1970 1990 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. Demand + 2% growth Use Production Discovery Nice09_CA

  3. 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. Nice09_CA

  4. 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 Carbo-hydrate Lipid Nice09_CA

  5. ? 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. Sensing Metabolic message = relative excess of light Vegetative growth; primary metabolism Stress Response: accommodation mechanisms induced; division stops Division resumes; secondary metabolites dilution in daughter cells +Nutrients, acclimatation Check point Secondary metabolites production and accumulation initiated Encystment; secondary metabolites, cell wall and lipid accumulate -Nutrients, commitment Nice09_CA

  6. 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 Nice09_CA

  7. Polymers Polysugars Lipids Proteins ADP + Pi ADP + Pi ADP + Pi ATP ATP ATP ATP ATP ATP Building Sugars Fatty acids Amino acids blocks ADP + Pi NADPH ADP + Pi O2 ADP + Pi NADP+ H2O ATP ADP + Pi ATP + NADP ANABOLISM PEP/Pyr CATABOLISM NADPH ADP + Pi Intermediates AcCoA ketone bodies + TCA NAD - e NADH ADP + Pi O 2 ATP Inorganic H O NH CO 2 2 3 compounds An idealized view of central aerobic metabolism • Catabolism splits organic molecules into inorganic compounds. It is mostly oxidative and generates available energy (ATP) and reducing equivalents (NADH). • Anabolism involves the reductive production of building blocks to sustain growth, at the expense of ATP and NADPH. • Photosynthesisin plants transduces light energy to generate ATP and NADPHused to fix atmospheric CO2 into sugar. Nice09_CA

  8. Metabolic versatility of the pentose phosphate pathway The costs of macromolecules biosynthesis • Biosynthetic demands for cofactors and intermediary metabolites of central metabolism for the accumulation of 1 gstarch, 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. Nice09_CA

  9. AA pool AA pool Starch Prot G6P G6P CO2 CH R5P Lip GAP DHAP GAP DHAP AA pool PEP PEP OAA Pyr Pyr Cit Cit OAA OAA AcCoA AcCoA MaCoA Mal Mal MaCoA C18:1 GlyP C18:1 C20:2 C22:3 So what’s in that box? 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. CO2 H2O, NADP+ ADP, Pi O2, NADPH ATP TAG

  10. Overview of lipid metabolism Beopoulos et al, 2008 Nice09_CA

  11. O H R H O C O + R1 H H H H SO SO SO SO 2 2 2 2 4 4 4 4 C O R H + R1 O H R R H H H H + + C C R R H H O O O O M e H H O O P P O O H H + H O P O H O O + O O M e + C O M e O H R H R H H H + C C H O C H O C H + R H [colored adduct] R anthrone phosphovanillin O O O O O O O C O O O O O O O O O Total lipid and total carbohydrate determination I. Harsh acid hydrolysis yields >95% monomers Provides a single aliquot, balanced and ready for direct colorimetric analysis of both compounds. II. Color reactions linear from 5-150 ugsugarorfatty acid Nice09_CA

  12. 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) Nice09_CA

  13. Full Medium N-deprived Medium 2% CO2 0.5% CO2 2% CO2 0.5% CO2 C1 C2 D1 D2 Stress Growth General experimental design Healthy Culture Batch culture at constant incident light intensity (decreasingly effective upon growth) Nice09_CA

  14. Col C2 -Full, 0.5% CO2 Col C1 -Full, 2% CO2 240 8 240 8 210 7 210 7 180 6 180 6 150 5 150 5 Chl Pigment - mg/L Pigment - mg/L DW - g/L DW - g/L 120 4 120 4 Car DW 90 3 90 3 60 2 60 2 30 1 30 1 0 0 0 0 0 10 20 30 40 0 10 20 30 40 Age - day Age - day Col D2 -Stress, 0.5% CO2 Col D1 -Stress, 2% CO2 240 8 240 8 210 7 210 7 180 6 180 6 150 5 150 5 Pigment - mg/L DW - g/L DW - g/L Pigment - mg/L 120 4 120 4 90 3 90 3 60 2 60 2 30 1 30 1 0 0 0 0 0 10 20 30 40 0 10 20 30 40 Age - day Age - day General growth parameters Parietochloris incisa Full-0.5%:Biomass growth is slower but sustained after N is depleted. Full-2%: After N is depleted (arrow), the pigment content diminishes and biomass growth gradually stops. Stress-0.5%: Same general behavior, indicating CO2 is saturating under these conditions. Stress-2%: Biomass growth rapidly stops. The dilute culture ‘senses’ a relatively high light intensity. Nice09_CA

  15. Chl/DW Car/DW 8 1.4 7 1.2 6 1.0 C1 5 Chl/DW - % C2 0.8 Car/DW - % 4 D1 0.6 D2 3 0.4 2 1 0.2 0 0.0 0 10 20 30 40 0 10 20 30 40 Age - day Age - day Car/Chl pH 1.4 9 1.2 8.5 1.0 8 Dark pH Car/Chl 0.8 7.5 0.6 7 0.4 6.5 0.2 0.0 6 0 10 20 30 40 0 10 20 30 40 Age - day Age - day Pigments and CO2 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. Nice09_CA

  16. TFA Volumetric Carbohydrate Volumetric Lipids Volumetric 2.5 3.5 3.5 3.0 3.0 2.0 C1 2.5 C2 2.5 1.5 D1 TFA - g/L 2.0 2.0 Carbohydrate - g/L D2 Lipids - mg/ml 1.5 1.5 1.0 1.0 1.0 0.5 0.5 0.5 0.0 0.0 0 10 20 30 40 0.0 0 10 20 30 40 0 10 20 30 40 Age - day Age - day Age - day Carbohydrate Content TFA Content Lipids Content 50 50 60 40 50 40 40 30 TFA - % DW 30 Lipids - %DW Carbohydrate - %DW 30 20 20 20 10 10 10 0 0 0 0 10 20 30 40 0 10 20 30 40 Age Age - day 0 10 20 30 40 Age - day Lipid and carbohydrate accumulation Nice09_CA

  17. +N 2% +N 2% 70 35 60 30 TFA 18 50 25 18 20 40 20 20 % TFA 16 % DW 16 30 15 22 22 20 10 10 5 0 0 0 10 20 30 40 0 10 20 30 40 Age - day Age - day +N 0.5% +N 0.5% 70 30 60 25 TFA 50 18 20 18 20 40 20 % TFA 16 % DW 15 16 30 22 22 10 20 5 10 0 0 0 10 20 30 40 0 10 20 30 40 Age - day Age - day Probing elongation (processing gas chromatograms) Nice09_CA

  18. Probing desaturation Nice09_CA

  19. Summary of kinetic lipid biosynthesis Elongation Desaturation Full 2.0% Full 0.5% -N 2.0% -N 0.5% Nice09_CA

  20. P. incisa , ratio vs. time P. incisa , ratio vs. ‘stress index’ 1.4 1.4 1.2 1.2 1 1 0.8 0.8 Lip:CH Lip:CH 0.6 0.6 C1 C2 0.4 0.4 D1 0.2 0.2 D2 Stress: 0 mild harsh 0 0 0.0 0.2 0.4 0.6 0.8 1.0 0 5 10 15 20 25 30 Age - day Car/Chl 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. How general are these features ? Nice09_CA

  21. Similar effect in a marine alga • 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. Nice09_CA

  22. LI Chlorella emersonii Haematococcus pluvialis 70 1.2 70 1.2 60 60 1 1 50 50 0.8 0.8 40 40 Lip or CH - %DW Lip or CH - %DW Lip/CH Lip/CH 0.6 0.6 30 30 0.4 0.4 CH CH 20 20 Lip Lip 0.2 0.2 L/C L/C 10 10 0 0 0 0 0 2 4 6 8 10 12 14 0.0 0.5 1.0 1.5 Tcar/Chl Tcar/Chl 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. The preferential accumulation of lipids upon stress appears to be also conserved in species of stable or variable sweet water ponds. Nice09_CA

  23. ? Thank you… Nice09_CA

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