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The ‘Photanol’ Process: Cyanobacteria for simple solar fuel

The ‘Photanol’ Process: Cyanobacteria for simple solar fuel. Kornel Golebski, Andreas Angermayr, Ginny Anemaet Joost Teixeira de Mattos & Klaas J. Hellingwerf Swammerdam Institute for Life Sciences & Netherlands Institute for Systems Biology University of Amsterdam .

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The ‘Photanol’ Process: Cyanobacteria for simple solar fuel

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  1. The ‘Photanol’ Process:Cyanobacteria for simple solar fuel Kornel Golebski, Andreas Angermayr, Ginny Anemaet Joost Teixeira de Mattos & Klaas J. Hellingwerf Swammerdam Institute for Life Sciences & Netherlands Institute for Systems Biology University of Amsterdam the photanol process

  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. the photanol process

  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.” the photanol process

  4. H2 macroscopic membrane O2 hydrogenase PSI PSII e- H2O H+ Brh The proposed solution: funding the photanol process

  5. What is needed.. • Use the auto-regenerative capacity of living organisms • A solution for solar fuel with as few conversions as possible (0.334 = 0.01!) fuels For any large-scale process, only H2O is a realistic electron donor the photanol process

  6. Some current biofuel technologies (1) From: Esper, Badura and Rögner (2006) Trends in Plant Science 11: 543-549. the photanol process

  7. Some current biofuel technologies (2) 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 (e.g. fatty acid methyl esters) Mostly ethanol the photanol process

  8. The photanol approach • First generation: Starch from corn or sugar cane fermented into ethanol by yeasts or palm oil trans-esterified to biodiesel. • Second generation: More recalcitrant bio-polymers fermented to alcohol(s) or biodiesel produced by marine algae. • Third generation: “Photanol” the photanol process

  9. Unity of life & the broken circle (plants, bacteria) E CO2 + H2O Cells + O2 CO2 (animals, fungi, bacteria)  energy Earth’ surface fossil fuels the photanol process

  10. 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 the photanol process

  11. Chloro-Phototrophy; optimized during billions of generations The light reactions of photosynthesis: the photanol process

  12. Chloro-Phototrophy; optimized during billions of generations H2O Light reaction O2 NADPH + ATP CO2 Dark reaction Dark reaction 1/3GAP Glyceraldehyde-3-P the photanol process

  13. Phototrophy Light reaction Dark reaction Cells NADP hn NADPH GAP ADP ATP PS II H2O O2 CO2 PS I the photanol process

  14. 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 Cells Organic C + ATP (fungi, bacteria) b) fermentation Cells + FERMENTATION PRODUCTS Organic C b): occurs when O2 is lacking or organic C is abundant; well-known as “overflow metabolism” in E. coli, LAB and yeast the photanol process

  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, ..........) NAD(P)H, ATP NAD(P)H, (ATP) the photanol process

  16. Light reaction Dark reaction CO2 NADP hn NADPH GAP Fermentation products ADP ATP PS II Cells H2O O2 PS I Photofermentation Fermentation CO2 + H2O  fuel + O2!! the photanol process

  17. Fermentation pathways the photanol process

  18. Some successful pathway insertions • Bermejo et al. 1998 (Acetone production in E. coli (Clostridium acetobutylicum pathway) ) • Deng and Coleman. 1999 (EtOH production in Synechococcus sp. (pdc and adh from Zymomonas mobilis) ) • Takahama et al. 2003 (Ethylene in Synechococcus sp. (efe from P. syringiae) ) • Fu. 2008 (EtOH production in Synechocystis sp. PCC 6803) • Pirkov et al. 2008 (Ethylene production in S. cerevisiae (efe from P. syringiae) ) • Shen and Liao. 2008 (1-Butanol and 1-Propanol in E. coli) • Tang et al. 2009 (Propanediol in E. coli (genes from Clostridium butyricum)) the photanol process

  19. Unicellular prokaryote Genome sequenced Auto- and heterotrophic Effective photosynthesis Model organism for photosynthesis Defined (simple) growth media Naturally transformable Grows to high densities Circadian rhythm doubling time ~8h 6 to 10 genomes per cell Low maintenance energy req. Synechocystis sp. PCC 6803 EM photograph, scale bar 200nm the photanol process

  20. Constructing a photofermentative strain goi Host: phototrophic Synechocystis PCC6803 Donor: chemotrophic bacterial species EtOH genes GAP PCR recombination HOM1 HOM2 wt genome expression plasmid the photanol process HOM1 HOM2

  21. M P N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Example of incomplete segregation Tested clones M C- C+ 5kbp 2kbp (in)complete segregation Cloning in the psbA2 locus of Synechocystis Colony PCR of pAAA2 transformants. M is marker. P is positive control. N is negative control. Transformants grown on 4ug/ml kanamycin. No correct insertion in transformants 4, 5, 7, 8, 10; not fully segregated transformants 1, 2, 3, 6, 9; full segregation in 11, 12, 13, 14, 15, 16, 17 the photanol process

  22. Ethanol synthesis by geneticengineering in Cyanobacteria From: Ming-De Deng and John R. Coleman (1999) Applied & Environm. Microbiol. 65: 523-528 FIG. 4.   Cell growth and ethanol synthesis in Synechococcus sp. strain PCC 7942 transformed with pCB4-LRpa. Cells were grown at 30°C in the presence of light in a 500-ml liquid batch culture aerated by forcing air through a Pasteur pipette. Samples were taken at intervals in order to monitor cell growth (OD730) and ethanol accumulation in the culture medium. The PDC and ADH activities in cell lysates on day 5 were 320 and 170 nmol · min 1 · mg of total protein 1, respectively. the photanol process

  23. ‘Photofermentation’: the best of both worlds cells CO2 + H2O fuel + O2!! • Cells are auto-regenerative catalysts of the process • The fuel molecules can stably coexist with oxygen • Production is not limited by the storage capacity of the cells • It is possible to form the product in volatile form • Process can be run in a closed large-scale photobioreactor the photanol process

  24. 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 the photanol process

  25. Regulation of fuel formation: The GAP branchpoint Fluxgrowth = [Eg].vmax. [PGA] Km + [PGA] Fluxproduct = [Ep].vmax. [PGA] Km + [PGA] A~CO2 B GAP A Eg Ep D E cassette the photanol process

  26. The Photanol Process: Genetic Process control A~CO2 B GAP A + Promoter NH4 D E E cassette product CO2 Ammonia availability is often used as a control parameter to regulate biomass formation (cells: “C4H7O2N”) the photanol process

  27. Nitrogen sensing in Synechocystis + + a-oxoglutarate + NH4 a-oxoglutarate + NH4 N-depletion glutamate + proteins NtcA NtcA-aOG sE PSigE SigE + ~ Pgap1 Gene cassette gap1 N-excess glutamate + proteins NtcA NtcA-aOG X sE - PSigE SigE Pgap1 Gene cassette gap1 the photanol process

  28. + 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 the photanol process time

  29. + 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 the photanol process time

  30. ‘Back-of-the-envelope’ calculation • 1 acre = 4 . 103 m2 • 1 year has 107 seconds of sunlight (3600 . 12 . 235) • Sunlight intensity (PAR): 600 μE.m-2.s-1  24 . 106 Einstein/acre/year • Complete conversion of light energy to ethanol: • 12 photons per ethanol: 2 CO2 + 3 H2O  C2H6O + 3 O2 • Maximal productivity: 2 . 106 moles ethanol/year/acre ~ 100 ton ethanol/year/acre the photanol process

  31. Large-scale culturing Tubular system Raceway pond Flat panel system • Extensive expertise is being generated with respect to the scale-up of culturing systems; systems can be used in ‘open’ and ‘closed’ form (e.g. Wijffels c.s.) • 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. the photanol process

  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 gene (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 the photanol process

  33. Circadian regulation of gene expression Dong G and Golden SS(2008) How a cyanobacterium tells time.Curr Opin Microbiol. 11: 541-546. 7 sigma factors of three different classes the photanol process

  34. Cyanobacteria do it during the day • Two interesting physiologies may occur at night: 1] oxidative catabolism (‘glycogen’  CO2) 2] anaerobic fermentation (‘glycogen’  organic acids) • Feasibility of supportive LED illumination during the night? the photanol process

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

  36. Dreams the photanol process

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