ARPA-E Adams-Kelly 10-21-09...Chalcopyrite, t = 0 days with M. sedula Chalcopyrite, t = 21 days with...

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H 2 -driven CO 2 Fixation by Extremely Thermoacidophilic Archaea Michael W.W. Adams, University of Georgia Robert M. Kelly, North Carolina State University ARPA-E Workshop on Direct Solar Fuels October 21, 2009 LIGHT DARK H 2 O H 2 Electron Source Primary Reduced Product Biofuel CH/O CO 2

Transcript of ARPA-E Adams-Kelly 10-21-09...Chalcopyrite, t = 0 days with M. sedula Chalcopyrite, t = 21 days with...

Page 1: ARPA-E Adams-Kelly 10-21-09...Chalcopyrite, t = 0 days with M. sedula Chalcopyrite, t = 21 days with M. sedula CuFeS 2 solids 10g/L pH 2 Heavy metal tolerance Iron oxidation Sulfur

H2-driven CO2 Fixation by Extremely Thermoacidophilic Archaea

Michael W.W. Adams, University of Georgia

Robert M. Kelly, North Carolina State University

ARPA-E Workshop on Direct Solar FuelsOctober 21, 2009

LIGHT DARK

H2O H2

ElectronSource

PrimaryReducedProduct

Biofuel

CH/O

CO2

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ElectronSource

PrimaryReducedProduct

Biofuel

SOLAR BIOFUEL PRODUCTION

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OxidizedElectronSourceO2

SecondaryReducedProductSUGARS

OxidizedSubstrate

CO2

ElectronSourceH2O

PrimaryReducedProductNADPH

SUGARS

Green Plants

CO2

VISIBLE LIGHT

Biofuel(of choice*)

BiomassConversion

MicrobialConversion

PS CC

PS = photosynthesis (I+II)CF = CO2 fixation (Calvin cycle)

ElectronSource

PrimaryReducedProduct

Biofuel

SOLAR BIOFUEL PRODUCTION

*CxHy / CaHbOc

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OxidizedElectronSourceO2

SecondaryReducedProductSUGARS

OxidizedSubstrate

CO2

ElectronSourceH2O

PrimaryReducedProductNADPH

Algae

CO2

VISIBLE LIGHT

Biofuel

H2

PS CC

PS = photosynthesis (I+II)CF = CO2 fixation (Calvin cycle)

ElectronSource

PrimaryReducedProduct

Biofuel

SOLAR BIOFUEL PRODUCTION

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OxidizedElectronSourceO2

SecondaryReducedProductSUGARS

OxidizedSubstrate

CO2

ElectronSourceH2O

PrimaryReducedProductNADPH

CO2

VISIBLE LIGHT

MetabolicEngineering Biofuel

(of choice)

Algae

Simplified (Biomimetic)Scheme?

PS CC

PS = photosynthesis (I+II)CF = CO2 fixation (Calvin cycle)

ElectronSource

PrimaryReducedProduct

Biofuel

SOLAR BIOFUEL PRODUCTION

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OxidizedElectronSourceO2

OxidizedSubstrate

CO2

ElectronSourceH2O

PrimaryReducedProduct

H2

CO2

VISIBLE LIGHT

Biofuel(of choice)

PS = photosynthesis (I+II)CF = CO2 fixation (Calvin cycle)

CFModule

PSModule

ElectronSource

PrimaryReducedProduct

Biofuel

SOLAR BIOFUEL PRODUCTION

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OxidizedElectronSourceO2

OxidizedSubstrate

CO2

ElectronSourceH2O

PrimaryReducedProduct

H2

CO2

VISIBLE LIGHT

Biofuel(of choice)

PS = photosynthesis (I+II)CF = CO2 fixation (Calvin cycle)

O2

CO2 + H2O

Fuel use withoxidized source(O2) regenerates

substrates (CO2, H2O)

IDEAL SOLARENERGY CONVERSION

SYSTEM

ElectronSource

PrimaryReducedProduct

Biofuel

SOLAR BIOFUEL PRODUCTION

CFModule

PSModule

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OxidizedElectronSourceO2

OxidizedSubstrate

CO2

ElectronSourceH2O

PrimaryReducedProduct

H2

CO2

VISIBLE LIGHT

Biofuel(of choice)

PS = photosynthesis (I+II)CF = CO2 fixation (Calvin cycle)

O2

CO2 + H2O

Fuel use withoxidized source(O2) regenerates

substrates (CO2, H2O)

IDEAL SOLARENERGY CONVERSION

SYSTEM

LIGHT DARK

H2O H2

ElectronSource

PrimaryReducedProduct

Biofuel

SOLAR BIOFUEL PRODUCTION

CH/O

CFModule

PSModule

CO2

PSM CFM

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OxidizedElectronSourceO2

OxidizedSubstrate

CO2

ElectronSourceH2O

PrimaryReducedProduct

H2

CO2

VISIBLE LIGHT

Biofuel(of choice)

PS = photosynthesis (I+II)CF = CO2 fixation 

Key Research

Areas

PS

LIGHT DARK

H2O H2

ElectronSource

PrimaryReducedProduct

Biofuel

SOLAR BIOFUEL PRODUCTION

CH/OPSM CFM

CFModule

PSModule

CO2

CO2 CHO/CH

2H+ + 2e- H2

H2

H2

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OxidizedElectronSourceO2

OxidizedSubstrate

CO2

ElectronSourceH2O

PrimaryReducedProduct

H2

CO2

VISIBLE LIGHT

Biofuel(of choice)

PS = photosynthesis (I+II)CF = CO2 fixation 

Key Research

Areas

PS

LIGHT DARK

H2O H2

ElectronSource

PrimaryReducedProduct

Biofuel

SOLAR BIOFUEL PRODUCTION

CH/OPSM CFM

CFModule

PSModule

CO2

CO2 CHO/CH

2H+ + 2e- H2

H2

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Key Biological ReactionsPSM:1. Water photolysis (visible light):

H2O O2 + 2H+ + 2e-

2. Hydrogen production2H+ + 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

LIGHT DARK

H2O H2

ElectronSource

PrimaryReducedProduct

Biofuel

SOLAR BIOFUEL PRODUCTION

CH/OPSM CFM

CO2

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Structure/Function Studies of NiFe-hydrogenaseStructure/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

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Hydrogen Production from Polyglucose(13 enzymes, Pi, NADP – catalytic)

(C6H10O5)n + 7 H2O 12 H2 + 6 CO2

PPP(Pentose

PhosphatePathway)

#13: NADPH-dependentH2-evolving

Pyrococcus Hydrogenase

Zhang et al. PLoS One 5, e456, 2008

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Calvin Cycle

Reverse Tricarboxylic Acid Cycle Reductive Acetyl-CoA Pathway

Adapted from http://lecturer.ukdw.ac.id/dhira/Metabolism/CarbonAssim.html

plants, cyanobacteria, purple and green bacteria

Chlorobium limicola

Desulfobacter

hydrogenophilus

Hydrogenobacter thermophilus

Acetobacterium woodii

Defulfobacterium autotrophicum

Microbial CO2Fixation Pathways

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

Ramos-Vera, W. H. et al. (2009) Berg, I. A. et al. (2007)

3-Hydroxypropionate Cycle

Chloroflexus aurantiacusAlber, B. et al. (2006)

Ignicoccus hospitalis Metallosphaera sedula

Microbial CO2Fixation Pathways

(Thermophiles)

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The Extremely Thermoacidophilic Archaeon Metallosphaera sedula

The Extremely Thermoacidophilic Archaeon Metallosphaera sedula

M. sedula genes closest sequence matches

S. solfataricus

S. tokodaii

S. acidocaldarius

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

Ferroplasma species

Thermoplasma species

Pyrobaculum species

Other / No top hit

SsoSac

Sto

other

Huber et al. 1989

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.

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

Topt = 73°C; pHopt = 2.0

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Comparative genomics: Extreme thermoacidophilesComparative genomics: Extreme thermoacidophiles

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Comparative genomics: Extreme thermoacidophilesComparative genomics: Extreme thermoacidophiles

Page 19: ARPA-E Adams-Kelly 10-21-09...Chalcopyrite, t = 0 days with M. sedula Chalcopyrite, t = 21 days with M. sedula CuFeS 2 solids 10g/L pH 2 Heavy metal tolerance Iron oxidation Sulfur

Growing E. coliGrowing E. coli

Oxoid

Page 20: ARPA-E Adams-Kelly 10-21-09...Chalcopyrite, t = 0 days with M. sedula Chalcopyrite, t = 21 days with M. sedula CuFeS 2 solids 10g/L pH 2 Heavy metal tolerance Iron oxidation Sulfur

Metal mobilization by M. sedulaChalcopyrite, t = 0 days with M. sedula Chalcopyrite, t = 21 days with M. sedula

CuFeS2 solids10g/LpH 2

Heavy metal tolerance

Iron oxidation

Sulfur oxidation

(Inorganic) Carbon fixation

Adhesion to solids

Cu2+

Auernik, KS, Y. Maezato, PH Blum, and RM Kelly. 2008. Appl. Environ. Microbiol. 74(3): 682-692Auernik, KS, CR Cooper, and RM Kelly. 2008. Curr. Opin. Biotechnol. 19(5):445-453

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Auernik, KS and RM Kelly. 2008. Appl. Environ. Microbiol. 74(24): 7723-7732

Metal and RISC oxidation components on Fe2+, S°

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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)

Page 23: ARPA-E Adams-Kelly 10-21-09...Chalcopyrite, t = 0 days with M. sedula Chalcopyrite, t = 21 days with M. sedula CuFeS 2 solids 10g/L pH 2 Heavy metal tolerance Iron oxidation Sulfur

Versatile growth physiology of M. sedula

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

Autotrophy (12 hrs)

Heterotrophy (5 hrs)Mixotrophy (< 4hrs)

Page 24: ARPA-E Adams-Kelly 10-21-09...Chalcopyrite, t = 0 days with M. sedula Chalcopyrite, t = 21 days with M. sedula CuFeS 2 solids 10g/L pH 2 Heavy metal tolerance Iron oxidation Sulfur

M. sedula Hydrogenases

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

Page 25: ARPA-E Adams-Kelly 10-21-09...Chalcopyrite, t = 0 days with M. sedula Chalcopyrite, t = 21 days with M. sedula CuFeS 2 solids 10g/L pH 2 Heavy metal tolerance Iron oxidation Sulfur

Auernik et al. (2008) Curr. Opin. Biotech. 19, 445-453

ATP

2 NADPH

NADPHATP

ATP

ATP

NAD

2 NADPH

M. sedula 3-HP/4-HB CO2 fixation pathway

Overall: 2CO2 + 4ATP + 5NADPH + CoA + NAD Acetyl CoA + 3ADP+AMP + 3Pi + PPi + 5NADP + NADH

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Auernik, KS, CR Cooper, and RM Kelly. 2008. Curr. Opin. Biotechnol. 19(5):445-453 Berg, I et al. 2007. Science. 318:1782-1786

H1H2 A1A2 M1M2

M. sedula 3-HP/4-HB CO2 fixation pathway

4-hydroxybutyryl-CoA dehydratase

Page 27: ARPA-E Adams-Kelly 10-21-09...Chalcopyrite, t = 0 days with M. sedula Chalcopyrite, t = 21 days with M. sedula CuFeS 2 solids 10g/L pH 2 Heavy metal tolerance Iron oxidation Sulfur

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

M. sedula 3-HP/4-HB CO2 fixation pathway

Page 28: ARPA-E Adams-Kelly 10-21-09...Chalcopyrite, t = 0 days with M. sedula Chalcopyrite, t = 21 days with M. sedula CuFeS 2 solids 10g/L pH 2 Heavy metal tolerance Iron oxidation Sulfur

3-HP/4-HB CO2 Fixation Pathway in Metallosphaera sedula

Page 29: ARPA-E Adams-Kelly 10-21-09...Chalcopyrite, t = 0 days with M. sedula Chalcopyrite, t = 21 days with M. sedula CuFeS 2 solids 10g/L pH 2 Heavy metal tolerance Iron oxidation Sulfur

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?

LIGHT DARK

H2O H2

ElectronSource

PrimaryReducedProduct

Biofuel

SOLAR BIOFUEL PRODUCTION

CH/OPSM CFM

CO2