Progress Toward Efficient and Sustainable Hydrogen Production

Transcription

1 GCEP Research Symposium 2013; Stanford University Progress Toward Efficient and Sustainable Hydrogen Production To Nature and Beyond Jim Swartz, Department of Chemical Engineering Department of Bioengineering [FeFe] Hydrogenase

2 Sustainable Energy Future? : Planet Earth Solar Energy H 2 O Photosynthetic Organisms Our Overall Objective Directly or Indirectly Energy: H O 2 2 Economy H 2 Power Plants, Fertilizer, Cement, Automobiles Current H 2 Market $135 Billion Made from CH 4 1.3% of CO 2 Emissions

3 Large Potential for Photosynthetic H 2 Production Current U.S. Consumption Of Liquid Fuels plus Nat. Gas 60 x Btu / yr Assume: Solar Incidence of 7 kwh/m 2 -day and 7.5 % Energy Efficiency (Overall) Need 35,000 Square Miles = 5 % of Current Cropland (But Do Not Need to Convert Current Crop Land)

4 But in (Bio)Chemical Engineering: In order to Dream BIG, We have to think SMALL 4 nm [FeFe] Hydrogenase (CpI) (from Clostridium pasteurainum) 2 H e - H 2 Up to 20,000 Reactions per Second!!

5 Can We Engineer An Organism to Make Hydrogen Directly From Sunlight and Water? h Ferredoxin H 2 PSI H + e e H + Hydrogenase H 2 O O 2 thylakoid lumen FNR Calvin cycle Sugars stroma H + ATP O 2 CO 2

6 Even Better, Can we Hard-Wire the Hydrogenase to the Photosystem? O 2 O 2 O 2 PSII Cytb 6 f PSI PQ PQH 2 e - e - PC FNR 2H + H 2 Fd CpI H 2 O 2H + + ½O 2

7 In Addition, Could We Go From This CH 4 + N 2 NH 3 + CO 2 To A More Sustainable Process? Biomass Enzymatic depolymerization NH 3 Fertilizer Sugars Enzymatic electron transfer to hydrogenase H 2 + CO 2 Chemicals/Fuel Need: High Conversion Efficiency and High Productivity

8 But Can We Make this Complex Enzyme? How is it Made Naturally? The Active Site of Fe-Fe Hydrogenases is Complicated: Stabilized by Cysteines, Carbon Monoxide, and Cyanide CO CN CO CN CO 1 Peters JW, Lanzilotta WN, Lemon BJ, Seefeldt LC Science 282(5395): An NREL Group* Discovered Three Required Maturases HydE, HydF, and HydG *Posewitz et al., JBC: 279:25711 (2004)

9 We Developed an in vitro Hydrogenase Maturation Procedure Using Individually Expressed Maturases & Optimized Substrates Used ΔiscR Host Strain Added GTP Added Pyridoxal-P i Optimized Concentrations E. coli Extract was Required Kuchenreuther et al.; PloS ONE 6(5) e

10 FTIR Spectroscopy of Apo-Hydrogenase Matured with Isotopically Labeled Tyrosine Confirmed that All the CO and CN Was Derived from Tyrosine These results show that the CO comes from the -CO 2 group on tyrosine and the CN comes from the alpha carbon and amino nitrogen Kuchenreuther et al (Swartz, Cramer, George).; PloS ONE 6(5) e

11 Further Work with the Dave Britt Lab at UC Davis Explained the First Steps in Building the All-Important Catalytic Center Tyrosine, SAM Reductant Tyrosine, SAM Reductant Accepted for Publication in Science

12 This work also enabled Improvement of Hydrogenase and Maturase Production in E.coli SDS-PAGE Gel of Lysate Supernatant (unpurified) Yields are at least 30 times higher than any previously reported. But, Not All of the Hydrogenase is Active Kuchenreuther, et al. PLoS ONE 2010

13 We Developed an in vitro Hydrogenase Maturation Procedure Using Individually Expressed Maturases & Optimized Substrates Used ΔiscR Host Strain Added GTP Added Pyridoxal-P i Optimized Concentrations E. coli Extract was Required Kuchenreuther et al.; PloS ONE 6(5) e

14 % Ac ve (log scale) The ISC Proteins Were NOT The Magic Factor(s) Liliana De La Paz Hydrogenase Apoenzyme (inactive) Hydrogenase Holoenzyme (active) isc operon Isc proteins were produced and confirmed to be active. They were then evaluated. 100 Chemical Substrates Fe +2 S -2 SAM GTP Cysteine Tyrosine Dithionite PLP Maturases Cell Extract ISC? % Lysate (Posi ve Control) No Lysate (Nega ve Control) isc proteins, No lysate No more than 1% activation was observed with ISC proteins alone.

15 Liliana is Now on a Magic Factor Safari Specific Ac vity (μmolh2 cons/min/mg CpI) Anion Exchange Chromatography Results Cell Extract Active Fractions Anion Exchange Hydrophobic Interaction Size Exclusion Measure hydrogenase activity over time using methyl viologen reduction assay Load B11 B10 B9 B8 Frac on B7 B6 t=4hrs t=12hrs Reducing SDS-PAGE 10% Bis-Tris Gel CysK Load FT W4 W5 B12 B11 B10 B9 B8 B7 B6 CsyK identified as a candidate protein Involved in cysteine metabolism Desulfurase activity observed in E. coli However, She Produced CysK -- NOT the Magic Factor

16 Back to Business: How Do We Efficiently Convert Sugars into Hydrogen? To Minimize Cost, We Will Use Unpurified Cell Extracts First Mobilize the 24 Electrons Available from the Glucose Using The Pentose Phosphate Pathway (in Red) 6 H 2 O Glucose Glucose-6-P 6 CO 2 (C 6 H 12 O 6 ) Oxidative Phosphorylation ATP H 2 O ADP ½O 2 P i 1 NADPH + 1 H + 12 NADPH + 12 H + 1 NADP + 11 NADPH + 11 H + FNR Pentose P i Pathway 22 Fd ox 11H 2 22 Fd red 22 H + H 2 ase Biomass Enzymatic depolymerization NH 3 Fertilizer Sugars Enzymatic electron transfer to hydrogenase H 2 + CO 2 Chemicals/Fuel 11 NADP +

17 Back to Business: How Do We Efficiently Convert Sugars into Hydrogen? To Minimize Cost, We Will Use Unpurified Cell Extracts Then Harvest Most of the Electrons to Produce Hydrogen (in Green) 6 H 2 O Glucose Glucose-6-P 6 CO 2 (C 6 H 12 O 6 ) Oxidative Phosphorylation ATP H 2 O ADP ½O 2 1 NADPH + 1 H + FNR = Ferredoxin NADPH Reductase Fd red = Reduced Ferredoxin Fd ox = Oxidized Ferredoxin H 2 ase = Hydrogenase P i 12 NADPH + 12 H + 1 NADP + 11 NADPH + 11 H + FNR 11 NADP + Pentose P i Pathway 22 Fd ox 11H 2 22 Fd red 22 H + H 2 ase

18 Back to Business: How Do We Efficiently Convert Sugars into Hydrogen? To Minimize Cost, We Will Use Unpurified Cell Extracts But We Need to Phosphorylate the Glucose 6 H 2 O Glucose Glucose-6-P 6 CO 2 (C 6 H 12 O 6 ) ATP Oxidative Phosphorylation H 2 O ADP ½O 2 1 NADPH + 1 H + Using Oxidative Phosphorylation; We should only need 1 NADPH P i 12 NADPH + 12 H + 1 NADP + 11 NADPH + 11 H + FNR 11 NADP + Pentose P i Pathway 22 Fd ox 11H 2 22 Fd red 22 H + Thus, the Conversion Yield Should Exceed 90% Fermentation Yields are Less Than 30% H 2 ase

19 Proof of Principle: Hydrogen Production from Glucose Cell Lysate plus Purified FNR, Fd, & H 2 ase Produced H 2 The FNR/Fd/H 2 ase pathway is limiting Phil Smith (now at BMS) Franklin Lu

20 For Commercial Conversion of Glucose (and Xylose) to H 2 Need HIGH Productivity for Distributed Facilities Published enzyme turnover numbers and expected expression levels suggest possible Fuel Value Productivity of 500 kj/liter-hr* About 10 times higher than current ethanol plants Cell Growth & Enzyme Expression *Assumes FNR Turnover Rate of 10/sec Homogenizer Glucose Glucose Glucose Air Air H 2 CO 2 N 2 Air

21 Volumetric Productivity (mmol H 2 L -1 hr -1 ) We Investigated the natural diversity of Ferredoxin NADPH Reductases (FNRs) Zhang et al (2007) Franklin Lu EcFNR SynFd Stirring, 37C EcFNR CpFd AnFNR CpFd *Karplus et al (2004) Structural aspects of plant ferredoxin: NADP+ oxidoreductases Photosyn Res 81: RootFNR CpFd Relationships between known FNR sequences* EcFNR: Ecoli FNR AnFNR: Anabaena FNR, SynFd: Synechocystis ferrodoxin, CpFd: Clostridial ferrodoxin

22 Improving the FNRs by Mutation and Screening Ferredoxin Sylvie Liong FNR We have observed Sufficient FNR Activity in Several Formats We are now Modifying the Cell Extracts for More Efficient Conversion

23 Now, Back to Direct Photosynthetic Hydrogen e - e - e - e - hv e - Thylakoid Membranes e -

24 Can we Hard-Wire the Hydrogenase to the Photosystem? O 2 O 2 O 2 FNR 2H + H 2 Fd CpI PSII Cytb 6 f PSI PQ PQH 2 e - e - PC H 2 O 2H + + ½O 2

25 Purified PSI Complex is Active for H 2 Production Yacoby, et al N-terminus PSI e - psaf Stacey Shiigi

26 Using the Ferredoxin from Clostridium pasteurainum (CpFd) Could Provide a Direct Connection 2H + H 2 2H + H 2 CpI CpI CpI CpI SynFd SynFd SynFd CpFd PSI PSI PSI PSI But Will It Couple with PSI??

27 Hydrogen Production from the Clostridial Ferredoxin: Potential for a hard-wired Connection hv CpI Stacey Shiigi

28 We Needed a Device for High Throughput Screen for Hydrogen Production Capability and Oxygen Tolerance For Calibration, We Inject Hydrogen Gas Below the 96- well plate. Inject Oxygen to test for Oxygen Tolerance A The 96-well Plate Sits on the Frame and is Covered By a Gasket (the hardest part) B A Glass Plate Coated with Paladium and Tungsten Oxide is then Clamped on Top Jamin Koo and Tim Schnabel C

29 The Screening Device is Working -- We can also Regenerate the Sensor Plates A Digital Camera Takes Images Over Time and Image Analysis Software Interprets the Rate of Color Formation Color Color formation rates: 10 mm DTH + 10 um CpFd + CpI [CpI] 0.08 nm 0.16 nm 0.32 nm 0.00 nm Linear (0.08 nm) Linear (0.16 nm) Linear (0.32 nm) Linear (0.00 nm) Jamin Koo and Tim Schnabel Time (s)

30 Conclusions 1. The in vitro H 2 ase maturation system is a valuable tool. 2. Cell-free metabolic engineering offers efficient and highly productive conversion of cellulosic biomass to hydrogen. 3. New Tools and Knowledge suggest feasibility for photosynthetic hydrogen production ---- BUT, we still need a productive, oxygen-tolerant hydrogenase. Thanks To our generous funding sources: GCEP, DOE, NSF To all the students, postdocs, and collaborators