G. C. Dismukes. Renewable Bio-solar Hydrogen Production from Robust Oxygenic Phototrophs

Transcription

1 Renewable Bio-solar Hydrogen Production from Robust Oxygenic Phototrophs G. C. Dismukes Princeton University Department of Chemistry Princeton Materials Institute Princeton Environmental Institute Pacific Rim Summit on Industrial Biotechnology & Bioenergy Honolulu January 11-13, 2006

2 PRINCETON PENNSTATE HAWAII Renewable Bio-solar Hydrogen Production from Robust Oxygenic Phototrophs -BioSolarH 2 Team G. Charles Dismukes PU photosynthesis/engineering Edward I. Stiefel PU enzymology/inorganic Donald A. Bryant PSU genetics/mol biology cyanos Matthew Posewitz CSM genetics/mol biology algae Eric Hegg UU enzymology/inorganic Robert Bidigare UH microbial physiology/ecology

3 Renewable Bio-solar H 2 Production from Robust Oxygenic Phototrophs mutants field screening genome Achievements June 2005 Identified novel H 2 photoproducers Developed high throughput screen for H 2 Built ultra-sensitive H 2 detection instruments Engineered mutants in a green alga Payoff Clean H 2 from non-fossil source H 2 O Oxygenic Photosynthesis H 2 genes GMOs HoxE HoxF ORF? HoxU HoxY HoxH Bam HI (1484) Nco I (1099) Nco I (3375) Nco I (6934) Cla I (2981) Eco RI (4977) Cla I (7047) O 2 diaphorase moiety genomic sequence around hox genes in S. platensis Ni-Fe hydrogenase moiety 7098 bp Objectives Screening for the right organisms, photosystems & hydrogenases Down-regulation of competing biochemical pathways Random & specific mutagenesis for higher H 2 production Genetically combining host phototrophs with optimal hydrogenases

4 Solar Bio-H 2 Goals: Economic viability 4 ~10% efficiency at ambient light levels (eg., antenna deletion mutants) Requires strict temporal or sptial separation of H 2 and O 2 Achieve current cost estimates for large scale bioreactors 4. Independent predictions by NREL and Thiess Engineering Solar Bio-H 2 Current Status Chlamydomonas reinhardtii: two stage light + dark + S deprivation Strain IncidentPhoton Conv. Eff. H 2 rate ml (liter culture) -1 hr -1 WT 1 ~ 0.1% 1.5 LHC insertion mutant 2 (3-4 x expected) minimal H 2 observed stm6 mutant 3 ~1.5% (5-13 x) 1. NREL team UCB team, Melis 3. Univ. Bielfeld & Univ Queensland: Kruse & Hankamer, JBC (2005)

5 Colonies on agar plate NREL WO 3 H 2 gas sensor Metabolomics & Proteomics Instrumentation Development Fast repetition rate fluorimeter PSII Quantum Efficiency Dissolved H 2 & O 2 LED + Clark cells Triple Quadrupole-TOF mass spectrometer & electrospray ion

6 Ultrasensitive cells for dissolved H 2 & O 2 with multi-wavelength light-emitting diode illuminator Clark type Cells for dissolved O 2 & H Molar H 2 or O 2 5 μl volume Sensitivity 50 femtomoles 0.1 sec response time Wavelength selective illumination via LEDs: blue, orange, red, infrared, white

7 -BioSolarH 2 Photo-H 2 and O 2 in WT Chlamydomonas reinhardtii CC124 hydrogen & oxygen concentration, rel.u Pulses light/dark 10s/90s on 200 pulses Hydrogen Oxygen off Conclusions Major Photo-H 2 Minimal dark-h 2 Co-production of H 2 and O 2 H 2 yield is O 2 limited incubation time, hours Cells from Petri dish resuspended in TAP liquid medium. Anaerobic pre-incubation time 12 min Pulse illumination via white LED at 130 μe m -2 s -1 G. Ananyev

8 Light pulses produce two kinetic phases of photo-h 2 in C. reinhardtii WT Photo-H light pulse duration, s 300 post light H 2 Peak 1 Light H 2 Photo-H off hydrogen evolution (peak) 0 Light + Dark H 2 Total Area Photo-H 2 + Post light H integrated hydrogen evolution (area) on time, min light pulse duration, s Conclusion: NADP +? Two pools of photo-electron acceptors in PSI capable of light-induced H 2 production secondary e - acceptor pool?? Integrated H 2 yield is linear with pulse duration competing pathways for e - /H + consumption are either fixed or not present G. Ananyev Fd? e - acceptor pool hν PSI H 2 -ase slow H 2

9 -BioSolarH 2 Chlamydomonas reinhardtii cc124 Alternative e - donor e - acceptor H 2 -ase pool H 2 H 2 concentration, rel.u. Anaerobic -O 2 pre-incubation hr 0.2 hr 4 hr 0.2 hr -Δhyd E/F light on, 10 s pulses time, min hν hν H 2 O PSII PQ PSI 670 nm PSI + PSII 730 nm PSI only 670 nm PSI + PSII Conclusions: Photo-H 2 & dark-h 2 requires H 2 ase genes hyd E/F Maximum photo-h2 production requires illumination of both PSI and PSII The major source of reductant required for photo-h2 comes from water via PSII turnover The minor PSI only pathway presumably involves PQ/other donor G. Ananyev & M. Posewitz

10 Arthrospira (spirulina) maxima Habitat: volcanic soda lakes Alkalophile, ph = M carbonate No N 2 fixation system Fastest PSII turnover 3-5x larger PQ pool vs algae genes hox-efuyh encode NAD-dependent bidirectional H2ase No hup genes reported (uptake H 2 ase)

11 Princeton: Fast Repetition Rate Laser Fluorometer 50 ns pulses 0.5 W; 660 nm laser 20 µs duration pulse train at full light saturation Programmable pulse flexibility Fast sampling, 20 mhz detection Chlorophyll Fo, Fv, Fm, cross section of PSII Q A- oxidation kinetics STF single turnover laser flash train 660 nm Variables to be measured Fv/Fm = PSII quantum efficiency PQ pool size 10 ms Dark or other light 20 μs Ananyev & Dismukes, Photosyn Res. (2005)

12 PSII Variable Fluorescence at 100Hz flash repetition rate; Arthrospira p. whole cells PSII- WOC PQ pool PSI NADP + Rubisco [CO2] Kautsky curve demonstrates transition from single turnover events in PSII to multiple electron transfer in whole electron transfer chain Arthrospira sp. have a larger PQ pool capacity 3-5X vs. green algae! P e Fv/Fm B 60 μs single turnover flash cluster 10 ms dark 0.46 dark preincubation period flash number 20 sec

13 Turnover Frequency of PSII is highest by 5x for Arthrospira maxima 8 B 7 Quality factor, 1/(α+β) Spirulina Chlorella X5 dark time between STF, ms 0.56 Kok α & β Kok parameters: photochemical misses (α) & double hit/misses (β) Fv/Fm Experiment PSII quantum efficiency ~54% 0.50 Fitting Personal use only, no 0publication * Ananyev & Dismukes, Photosyn Res. rights (2005) allowed, G. C. Dismukes STF number

14 hydrogen concentration, rel.u Induction of dark H 2 (Left) & PSII quantum efficiency (Right) during photoautotrophic growth at 1.7 μm Ni 2+ of A. maxima 5 4 Days of photoautotrophic growth prior to anaerobisis Transient reductant pool Fv/Fm PQ pool oxidized reduced time, hours STF number Ni 2+ addition to standard cyanobacteria growth media (Zarrouk) is essential for H 2 ase 3-4 days lag to fermentative H 2, yields transient reductant pool ( 2 or 3 peaks) Second lag phase 1-2 days later, oscillations in H 2 Culture survives for days in batch culturing no additives Microscopy shows that most active dark-h 2 producing cells have no gas vacuoles

15 PSI + PSII Illumination by 640 nm red light produces additional H 2 hydrogen, mv Pulses light/dark 1s/99s h Conclusions Dark (fermentative) and a light-induced pathways to H 2 are present These pathways share a common intermediate that is precursor to H h dark time, hours

16 PSI Illumination by IR 735 nm stops fermentative H 2 & stores H 2 precursor 1 s light/ 99 s dark 5 s light/95 s dark hydrogen, mv Light H 2 On 735 nm cycle of dark H 2 Dark H time, hours Dark H 2 reductant is stored under IR illumination Off H2 recovers hydrogen, mv nm delay 32 min h 0-12 h time, hours 2 x greater yield IR Light suppresses dark H 2 Fermentative H 2 restored in dark after IR light stopped at 2x higher rate

17 A. maxima 400 hydrogen, mv nm PSI illumination Suppresses fermentative H 2 Slow photo-h nm time, min PSI+PSII illumination Adds to fermentative H 2 Fast photo-h 2

18 Model for Dark and Light H 2 production by Arthrospira m. glycogen glucose pyruvate NAD-dependent NiFe-H 2 ase NAD + NDH Complex I H 2 H 2 O O 2 PSII PQH 2 PQ PSI Fd FNR NADP +

19 Sustained H 2 production rates: representative reports of cyanobacteria using water as electron donor via hydrogenase probably via nitrogenase In light Our current results [nitrate in media] Removal of nitrate from media In Dark 30 C 23 Est. rate of H 2 photoproduction in green algae C. reinhardtii under S deprivation (2005) NREL, UQ/UB Arthrospira maxima Arthrospira platensis Synechocystis sp 6803 mutant M55 Cyanothece PCC 7822 Oscillatoria sp Miami BG7

20 -BioSolarH 2 Conclusions New tools for measuring H 2, O 2 and redox status provide reliable quantitative data on concentrations and fluxes greatly simplify and clarify the fundamental mechanisms of H 2 production in microalgae and cyanobacteria Cyanobacteria & microalgae have different pathways to H 2 Ideal temporal separation of H 2 and O 2 production in cyanobacteria solves gas separation problem

21 -BioSolarH 2 Princeton Edward Stiefel, co-i Dr. Gennady Ananyev Dr. Derrick Kolling Dr. Tuan Nguyen Damian Carrieri,GS2 Kelsey McNeely,GS1 Nick Benette, GS1 Tyler Brown, UG06 Co-Investigators Matthew Posewitz, CSM Donald A. Bryant, PSU Robert Bidigare, UH Eric Hegg, UU