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Cyanobacteria for solar fuel

Cyanobacteria for solar fuel. Klaas J. Hellingwerf Swammerdam Institute for Life Sciences & Netherlands Institute for Systems Biology University of Amsterdam . MJ Teixeira de Mattos “Photanol” KNAW Symposium, Jan., 2008. A little bit of history.

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Cyanobacteria for solar fuel

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  1. Cyanobacteria for solar fuel Klaas J. Hellingwerf Swammerdam Institute for Life Sciences & Netherlands Institute for Systems Biology University of Amsterdam MJ Teixeira de Mattos “Photanol” KNAW Symposium, Jan., 2008 From Solar to Fuel with Bio

  2. A little bit of history A Round-Table Discussion held during the 10th FEBS Meeting in Paris (July 25, 1975) considered the different approaches by which Biological Systems might be used to convert ambient solar energy into more useful energy forms. From Solar to Fuel with Bio

  3. The problem: “Man does not have much choice. Either we trust the physicist to make us a sun without blowing us up, or we let the bioenergeticists use our present one. Otherwise, we won’t last more than a hundred years or so. This is an exciting challenge for the bioenergetics of tomorrow.” From Solar to Fuel with Bio

  4. The proposed solution: membrane H2 PSI or: hydrogenase H2 macroscopic membrane O2 e- hydrogenase PSII PSI PSII e- O2 + H+ H2O bilayer H2O H+ Brh Brh From Solar to Fuel with Bio

  5. Result after 10 years1 In de 1970’er jaren heeft men getracht systemen te ontwikkelen met chloroplasten voor het genereren van een elektronenstroom uit zonlicht en hydrogenasen voor productie van H2 uit deze elektronen en H+. Deze systemen bleken niet stabiel. Na de oliecrisis van 1973 is in Nederland op initiatief van Prof. E.C. Slater in 1975 een project gestart met het doel om te onderzoeken of de fotolyse (met zonlicht) van water in H2 en O2 te realiseren is met biochemische systemen (fotosysteem-II en hydrogenasen), of met chemische afgeleiden daarvan. In de jaren 1975-’85 heeft ZWO/SON het onderzoek gefinancierd via een speciaal programma: het ‘Hydrogenase Project’. Doelstelling was: 1. bepaling van structuur en werkingsmechanisme van de H2-producerende biokatalysator hydrogenase (door biochemici, biofysici en microbiologen); 2. ontwikkeling van chemische alternatieven, want biokatalysatoren zijn niet altijd even stabiel. In 1985 werd het programma gestaakt mede als gevolg van de daling van de olieprijs. Twee Nederlandse onderzoeksgroepen (Hagen, TUD en Albracht, UvA) werken nog steeds aan hydrogenasen. 1: REPORT OF THE “WORKSHOP BIOLOGISCHE H2 PRODUCTIE”, donderdag 4 oktober 2001, Novem, Utrecht From Solar to Fuel with Bio

  6. What is needed.. • Photovoltaics for electricity • A solar solution for fuel (with as few conversions as possible (0.334 = 0.01!) For any large-scale process, only H2O is a realistic electron donor From Solar to Fuel with Bio

  7. Some current biofuel technologies 1 Grow cropson land 2 Grow Algae in ponds Harvest organic matter Harvest cells Transport to bioreactor & fractionate Transport to separator extraction & modification fermentation biofuel + Waste Biodiesel (fatty acid methyl ester) or H2 Mostly ethanol From Solar to Fuel with Bio

  8. First-, second- and third-generation technologies: • First generation: Starch from corn or sugar cane fermented into ethanol by yeasts or palm oil trans-esterified to biodiesel. • Second generation: Bio-polymers fermented to alcohol(s) or biodiesel produced by (marine) algae. • Third generation: “Photanol” From Solar to Fuel with Bio

  9. The 2 modes of life 1 Light-dependent life (plants, bacteria) ((Chloro)Phototrophy) H2O reducing power + “ATP” + O2 Organic C Reducing power + CO2 + “ATP” Cells From Solar to Fuel with Bio

  10. The 2 modes of life 2 Organic matter-dependent life (Chemotrophy) a) respiration (animals, fungi, bacteria) Organic C + O2 Organic C + O2 “ATP” + CO2 + H2O (redox reaction!) Cells Organic C + “ATP” (fungi, bacteria) b) fermentation Cells + FERMENTATION PRODUCTS Organic C (when organic C is abundant or O2 is lacking) From Solar to Fuel with Bio

  11. The circle of life is driven by the sun (plants, bacteria) CO2 + H2O Cells + O2 (animals, fungi, bacteria) Earth’ surface From Solar to Fuel with Bio

  12. The broken circle (plants, bacteria) CO2 + H2O Cells + O2 CO2 (animals, fungi, bacteria) Earth’ surface fossil fuels From Solar to Fuel with Bio

  13. Chloro-Phototrophy; optimized during billions of generations H2O Light reaction O2 e- + ATP CO2 Dark reaction 1/3GAP Glyceraldehyde-3-P From Solar to Fuel with Bio

  14. Phototrophy Light reaction Dark reaction CO2 NADP hn NADPH GAP ADP ATP PS II Cells H2O O2 PS I From Solar to Fuel with Bio

  15. Chemotrophy: optimized for billions of generations Organic matter F-1,6-BP Glyceraldehyde-3-P (GAP) Pyruvate Fermentation products (Ethanol, propanol, butanol, propanediol, glycerol, acetone, lactate, acetate, ..........) From Solar to Fuel with Bio

  16. Phototrophy Fermentation Light reaction Dark reaction CO2 NADP hn NADPH GAP GAP GAP GAP GAP Fermentation products Fermentation products ADP Fermentation products ATP Fermentation products PS II Cells H2O O2 Fermentation products PS I Photofermentation Fermentation From Solar to Fuel with Bio

  17. Biological incompatibility: methanogenesis Fdred H2 CO2 Formyl-MFR Formyl-H4MPT Methenyl-H4MPT H2 H2F420 Enzymes involved are extremely oxygen-sensitive and have several very uncommon cofactors Methyl-H4MPT Methyl-S-CoM HS-CoB CH4 From Solar to Fuel with Bio

  18. Constructing a Photofermentative strain PCR recombination expression Host: phototrophic Synechocystis PCC6803 Donor: chemotrophic bacterial species GAP EtOH genes From Solar to Fuel with Bio

  19. The Photanol Process: Product cassettes ethanol + O2 propanol + O2 Solar energy + CO2 butanol + O2 propanediol + O2 acetone + O2 CO2 Ethanol: S. cerevisiae: 1 pyr decarboxylase 2 alcohol DH I Butanol: L. brevis 1 thiolase 2 OHbutyrylCoA DH 3 crotonase 4 butyryl-CoA DH 5 Butyraldehyde DH 6 Butanol DH The octane rating of n-butanol is similar to that of gasoline but lower than that of ethanol and methanol. From Solar to Fuel with Bio

  20. Solventogenesis: • History: Chaim Weizmann in early 1900 in Britain because of lack of natural rubber; few years later: acetone • For many years: primarily studied in Clostridium acetobutylicum • However, the process of ‘solventogenesis’ is also observed in ‘aerotolerant’ organisms (like Saccharomyces cerevisiae and Lactococcus lactis • Recent example: Propane-diol fermentation in Escherichia coli (Monsanto) • Example of recent innovation: mixed solvent production in E.coli (Nature 451: 86-90 (2008)) From Solar to Fuel with Bio

  21. Regulation of fuel formation: The GAP branchpoint Fluxgrowth = [Eg].vmax. [PGA] Km + [PGA Fluxproduct = [Ep].vmax. [PGA] Km + [PGA] A~CO2 B GAP A D E cassette From Solar to Fuel with Bio

  22. The Photanol Process: Genetic Process control A~CO2 B GAP A + Promoter NH4 D E E cassette product Ammonia availability is often used as a control parameter to regulate biomass formation From Solar to Fuel with Bio

  23. + a-oxoglutarate + NH4 N sensing in Synechocystis N-excess glutamate + proteins NtcA NtcA-aOG - sE X PSigE SigE Pgap1 Gene cassette gap1 From Solar to Fuel with Bio

  24. N sensing in Synechocystis + a-oxoglutarate + NH4 N-depletion glutamate + proteins NtcA NtcA-aOG sE PSigE SigE ~ + Pgap1 Gene cassette gap1 From Solar to Fuel with Bio

  25. + NH4 growth N-dependent fuel cassette expression N-excess N starvation Glu protein 2OG + N Ntca 12 3-P-Glycerate 12 1,3-bPG 6 CO2 2 GAP 5 R1,5bP 10 GAP + P P 5 FbP Growth Hexose-P thl crt etf 4hbd ald bdh Butanol From Solar to Fuel with Bio time

  26. + NH4 crt 4hbd product N-dependent fuel cassette expression N-excess N starvation Glu protein 2OG + N Ntca + 2OG Ntca~2OG 12 3-P-Glycerate 12 1,3-bPG se 6 CO2 + 10 GAP + 2 GAP 5 R1,5bP P + 5 FbP Growth Hexose-P thl etf ald bdh Butanol growth growth From Solar to Fuel with Bio time

  27. ATP, NADPH Summary of the Photanol Process cells Clean fuel production CO2 consuming Cheap technology Not competing with food stocks Yield per year per surface up to 20x higher than plant crops Principle generally applicable: ethanol, butanol, etc xCO2 + yH2O CxH2yOz + (x+0.5y-0.5z)O2 From Solar to Fuel with Bio

  28. 1st and 2nd vs 3rd generation biofuels: • Plants have lower productivity per unit surface than algae • 2nd generation: Lipid synthesis occurs during growth-restriction in nitrogen-deficient media (cells: “C4H7O2N”) • 3rd generation: Cells are merely catalysts; reaction: CO2 + hν+ H2O  fuel + O2 From Solar to Fuel with Bio

  29. Why Synechocystis? • Prokaryote: simple metabolism and lowest maintenance energy requirements of all living organisms • Naturally transformable, which implies facile genetic alterations • Has been subjected to all known genomics techniques • A systems biology description is underway (e.g.:The Plant Cell 13: 793–806 (2001) – micro-arrays; Phytochem. 68: 2302–2312 (2007) – metabolic flux analysis) From Solar to Fuel with Bio

  30. Large-scale culturing Tubular system Raceway pond Flat panel system • Extensive expertise is available in the scale-up of culturing systems; systems can be used in ‘open’ and ‘closed’ form • Many problems in down-stream processing are remaining • All systems have in common that the fuel-producing cells are exposed to oscillating light regimes, with typical frequencies ranging from minutes (depending on mixing regime) to 24 hrs. From Solar to Fuel with Bio

  31. Current- & Synthetic Biology 1] Energy conservation in the photosynthesis of cyanobacteria in the form of conversion of light energy into biomass at a constant (sun) light energy of approximately 100 to 200 Einstein.m-2.s-1 proceeds close to the (biological) theoretical maximum (maximal biomass yield: > 100 tonnes/ha/yr). 2] Very large free energy losses may occur at high light intensities, and transiently, because of delay of adaptation of the cell to altering extra-cellular conditions. 3] Multiple ‘synthetic’ improvements in the energy-conversion performance of cyanobacteria can be perceived, like: (i) Addition of an IR-absorbing PS3 as an extra proton pump; (ii) engineering of extra proton-translocating loops in the electron-transfer chain; (iii) ‘cutting’ of high-energy photons to achieve > 1 charge-separations per photon; etc. From Solar to Fuel with Bio

  32. Some regulatory mechanisms in the photosynthesis of Synechocystis a] State transitions of phycobilisomes b] Non-photochemical, IsiA and/or OCP-mediated quenching c] zeaxanthin cycle d] Regulation of expression ratio of PSI/PSII/Antennae e] Circadian regulation of photosystem expression f] NDH (and FNR) mediated cyclic electron transfer around PSI g] Cyclic electron transfer around PSII h] PSI trimerization, PSII dimerization, IsiA and iron limitation i] Variation of antenna size (j] Chromatic adaptation)  a Systems Biology-based optimization is necessary From Solar to Fuel with Bio

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