H2-Driven CO2 Fixation by Extremely Thermoacidophilic Archaea

H2-driven CO2 Fixation by Extremely Thermoacidophilic Archaea

Michael W.W. Adams, University of Georgia Robert M. Kelly, North Carolina State University

Primary CO2 Electron Biofuel Source Reduced LIGHT Product DARK

H2O H2 CH/O

ARPA-E Workshop on Direct Solar Fuels October 21, 2009 SOLAR BIOFUEL PRODUCTION

Primary Electron Biofuel Source Reduced Product SOLAR BIOFUEL PRODUCTION

Primary Electron Biofuel Source Reduced Product

Secondary Biomass Electron Green Plants Reduced Conversion Source Product SUGARS H O 2 SUGARS PS CC Microbial VISIBLE LIGHT Conversion Oxidized Oxidized Primary Substrate Biofuel Electron Reduced CO (of choice*) Source Product 2 O2 NADPH

CO PS = photosynthesis (I+II) 2

CF = CO2 fixation (Calvin cycle) *CxHy / CaHbOc SOLAR BIOFUEL PRODUCTION

Primary Electron Biofuel Source Reduced Product

Secondary Electron Algae Reduced Source Product H O 2 SUGARS PS CC VISIBLE LIGHT H Oxidized 2 Oxidized Primary Substrate Electron Reduced Biofuel CO Source Product 2 O2 NADPH

CO PS = photosynthesis (I+II) 2

CF = CO2 fixation (Calvin cycle) SOLAR BIOFUEL PRODUCTION

Primary Electron Biofuel Source Reduced Product

Secondary Metabolic Electron Algae Reduced Engineering Biofuel Source Product (of choice) H O 2 SUGARS PS CC VISIBLE LIGHT

Oxidized Oxidized Primary Substrate Electron Reduced CO Source Product 2 O2 NADPH

CO Simplified (Biomimetic) PS = photosynthesis (I+II) 2 Scheme? CF = CO2 fixation (Calvin cycle) SOLAR BIOFUEL PRODUCTION

Primary Electron Biofuel Source Reduced Product

Electron PS CF Source Module Module H2O Biofuel (of choice) VISIBLE LIGHT

Oxidized Oxidized Primary Substrate Electron Reduced CO Source Product 2 O2 H2

CO PS = photosynthesis (I+II) 2

CF = CO2 fixation (Calvin cycle) SOLAR BIOFUEL PRODUCTION

Primary Electron Biofuel Source Reduced Product

Electron PS CF Source Module Module H2O Biofuel (of choice) O2 VISIBLE LIGHT

Oxidized Fuel use with Oxidized Primary CO Substrate oxidized source 2 Electron Reduced + CO (O ) regenerates Source Product 2 2 H O O substrates 2 2 H 2 (CO2, H2O) IDEAL SOLAR CO2 PS = photosynthesis (I+II) ENERGY CONVERSION CF = CO2 fixation (Calvin cycle) SYSTEM SOLAR BIOFUEL PRODUCTION

Primary CO2 Electron Biofuel Source Reduced LIGHT Product DARK PSM CFM H2O H2 CH/O

Electron PS CF Source Module Module H2O Biofuel (of choice) O2 VISIBLE LIGHT

Primary CO2 Electron Biofuel Source Reduced LIGHT Product DARK PSM CFM H2O H2 CH/O

Electron PS CF Source Module Module H O Biofuel 2 H 2 (of choice) PS CO2 CHO/CH VISIBLE LIGHT H2 2H+ + 2e- H 2 Oxidized Oxidized Primary Substrate Electron Reduced Key Source CO2 Research Product Areas O2 H2

CO PS = photosynthesis (I+II) 2

CF = CO2 fixation SOLAR BIOFUEL PRODUCTION

Primary CO2 Electron Biofuel Source Reduced LIGHT Product DARK PSM CFM H2O H2 CH/O

Electron PS CF Source Module Module H2O Biofuel H2 CO CHO/CH (of choice) VISIBLE LIGHT PS 2 2H+ + 2e- H 2 Oxidized Oxidized Primary Substrate Electron Reduced Key Source CO2 Research Product Areas O2 H2

CO PS = photosynthesis (I+II) 2

CF = CO2 fixation SOLAR BIOFUEL PRODUCTION

Primary CO2 Electron Biofuel Source Reduced LIGHT Product DARK PSM CFM H2O H2 CH/O

Key Biological Reactions PSM: 1. Water photolysis (visible light): + - H2O O2 + 2H + 2e 2. Hydrogen production + - 2H + 2e H2 CFM: 1. Hydrogen activation + - H2 2H + 2e

2. CO2 to biofuel conversion (reduced carbon/CH/CHO) + - CO2 + 2H + 2e CxHy / CaHbOc

Much higher energy yield using H2 as reductant for CO2 fixation to biofuel compared to sugar to biofuel Structure/Function Studies of NiFe-hydrogenase

- Hydrogenase is not a ‘commodity enzyme’

- Very complex maturation process in vivo to assemble complex, O2-sensitive, NiFe-catalytic site (CO, CN ligands, etc.) - No genetically-tractable organism that produces hydrogenase available for detailed structure/function studies - Recent breakthrough – production of recombinant form of the NADPH- dependent Pyrococcus hydrogenase in E. coli

Fundamental structure/function studies to design: - ‘Minimal’ hydrogenases (NiFe-site plus peptides)

-H2 production and H2 activation catalysts

- Decreased O2-sensitivity - Electron carrier specificity - Pathways of electron transfer Hydrogen Production from Polyglucose (13 enzymes, Pi, NADP – catalytic)

PPP (Pentose Phosphate Pathway)

#13: NADPH-dependent

H2-evolving Pyrococcus Hydrogenase Zhang et al. PLoS One 5, e456, 2008

(C6H10O5)n + 7 H2O  12 H2 + 6 CO2 Calvin Cycle

plants, cyanobacteria, purple and green bacteria

Microbial CO2 Fixation Pathways

Reverse Tricarboxylic Acid Cycle Reductive Acetyl-CoA Pathway

Chlorobium limicola

Desulfobacter hydrogenophilus

Acetobacterium woodii Hydrogenobacter thermophilus Defulfobacterium autotrophicum

Adapted from http://lecturer.ukdw.ac.id/dhira/Metabolism/CarbonAssim.html 3-Hydroxypropionate Cycle

Microbial CO2 Chloroflexus aurantiacus Fixation Pathways Alber, B. et al. (2006) (Thermophiles)

Dicarboxylate/4-Hydroxybutyrate Cycle 3-Hydroxypropionate/4-Hydroxybutyrate Cycle

Ignicoccus hospitalis Metallosphaera sedula

Ramos-Vera, W. H. et al. (2009) Berg, I. A. et al. (2007) TheThe ExtremelyExtremely ThermoacidophilicThermoacidophilic ArchaeonArchaeon MetallosphaeraMetallosphaera sedulasedula

Genome: 2.2 Mb (46.2% G + C) 2304 ORFs Growth: Mixotrophic aerobe; metal mobilizer

Topt = 73°C; pHopt = 2.0

M. sedula genes closest sequence matches

S. solfataricus other S. tokodaii

S. acidocaldarius

Sso Other sulfolobus species Sac (islandicus, metallicus, shibatae) Acidianus species

Ferroplasma species

Sto Thermoplasma species Huber et al. 1989 Pyrobaculum species

Other / No top hit

Auernik, K. S., Y. Maezato, P. H. Blum, and R. M. Kelly. 2008. The genome sequence of the metal-mobilizing, extremely thermoacidophilic archaeon Metallosphaera sedula provides insights into bioleaching-associated metabolism. Appl. Environ. Microbiol. 74:682-92. ComparativeComparative genomics:genomics: ExtremeExtreme thermoacidophilesthermoacidophiles ComparativeComparative genomics:genomics: ExtremeExtreme thermoacidophilesthermoacidophiles GrowingGrowing E.E. colicoli

Oxoid Metal mobilization by M. sedula

Chalcopyrite, t = 0 days with M. sedula Chalcopyrite, t = 21 days with M. sedula

CuFeS2 solids 10g/L pH 2 Cu2+

Iron oxidation Sulfur oxidation (Inorganic) Carbon fixation Heavy metal tolerance Adhesion to solids

Auernik, KS, Y. Maezato, PH Blum, and RM Kelly. 2008. Appl. Environ. Microbiol. 74(3): 682-692 Auernik, KS, CR Cooper, and RM Kelly. 2008. Curr. Opin. Biotechnol. 19(5):445-453 Metal and RISC oxidation components on Fe2+, S°

Auernik, KS and RM Kelly. 2008. Appl. Environ. Microbiol. 74(24): 7723-7732 Metal and RISC oxidation components on CuFeS2

Auernik, KS, and RM Kelly. Molecular hydrogen impacts chalcopyrite bioleaching efficiency for the extremely thermoacidophilic archaeon Metallosphaera sedula. (submitted) Versatile growth physiology of M. sedula

Mixotrophy (< 4hrs) Heterotrophy (5 hrs)

Autotrophy (12 hrs)

Auernik, KS, and RM Kelly. Physiological versatility of the extremely thermoacidophilic archaeon Metallosphaera sedula supported by heterotrophy, autotrophy and mixotrophy transcriptomes (submitted) M. sedula Hydrogenases

ATP NAD

2 NADPH ATP

NADPH ATP ATP 2 NADPH

Overall:

2CO2 + 4ATP + 5NADPH + CoA + NAD Acetyl CoA + 3ADP+AMP + 3Pi + PPi + 5NADP + NADH Auernik et al. (2008) Curr. Opin. Biotech. 19, 445-453 M. sedula 3-HP/4-HB CO2 fixation pathway

4-hydroxybutyryl-CoA dehydratase

H1H2 A1A2 M1M2

Auernik, KS, CR Cooper, and RM Kelly. 2008. Curr. Opin. Biotechnol. 19(5):445-453 Berg, I et al. 2007. Science. 318:1782-1786 M. sedula 3-HP/4-HB CO2 fixation pathway

Primary CO2 Electron Biofuel Source Reduced LIGHT Product DARK PSM CFM H2O H2 CH/O

Fundamental Studies Needed:

1. Photosystems I and II

2. Hydrogenases

3. CO2 fixation pathways

Key issues:

1. Mechanisms of catalysis and design of biomimetic systems?

2. Electron transfer pathways between the three systems?