Bio-Inspired Abiotic Catalysts

Nature's Renewable Energy Blueprint: Future Fuel from Photosynthesis & Biomimics

Direct Bio-Solar H2 Production

2 H2O O2 + 2 H2 (H+/e+)

2 H2O +

WAVE WIND PV BIOMASS SOLAR TOWER HYDROELECTRIC TIDAL POWER

•ALMOST ALL CLEAN ENERGY TECHNOLOGIES GENERATE ELECTRICITY•GLOBAL ENERGY USE: 33% ELECTRICITY: 67% FUELS (eg OIL, COAL, GAS)

FUTURE TECHNOLOGY ASSESSMENT SOLAR H2 FROM H2O

RENEWABLE ENERGY ANNUAL ENERGY x GLOBAL ENERGYSOURCE SIZE (TW-YR) RESOURCE DEMAND @ 46 TW-YR

SOLAR 178,000 3869xGEOTHERMAL 92,063 2001xWIND 194 420xOCEAN THERMAL 10WAVE 2HYDROELECTRIC 1.6TIDAL 0.1TOTAL 270,271 5875x

13TW-YR

IEA source

Can we develop a practical H2-producing system to meet our energy needs?

Microalgae:Chlamydomonas reiinhardti

Cyanobacteria:Arthrospira (Spirulina) sp.

Sunlight

CO2 H20

Nutrients

Cell Factories

Chemical energy

Native & GMOs that produce more energy

Microbial cell factories directly produce chemical energy w/o sacrificing the microbe

H2 alcohols, acids,hydrocarbons

PENNSTATE HAWAIIPRINCETON

Renewable Bio-solar Hydrogen Production from Robust Oxygenic Phototrophs

-BioSolarH2 TeamG. Charles Dismukes PU photosynthesis/chemistryEdward I. Stiefel PU enzymology/inorganicDonald A. Bryant PSU genetics/mol biology cyanosMatthew Posewitz CSM genetics/mol biology algaeEric Hegg UU enzymology/inorganicRobert Bidigare UH microbial physiology/ecology>2006John Peters MSU crystallography & directed evolnRobert Austin PU microchemostats & cellular evoln

Objectives•Screening for the right organisms, photosystems & hydrogenases•Down-regulation of competing biochemical pathways•Random & specific mutagenesis for higher H2 production•Genetically combining host phototrophs with optimal hydrogenases

Renewable Bio-solar H2 Production from Robust Oxygenic Phototrophs

screeningfield

genomic sequence around hox genes in S. platensis7098 bp

HoxE HoxF HoxU HoxY HoxHORF?

diaphorase moiety Ni-Fe hydrogenase moiety

Bam HI (1484)Eco RI (4977)Cla I (2981) Cla I (7047)Nco I (1099)

Nco I (3375) Nco I (6934)

H2Oxygenic PhotosynthesisH2O

O2

genome

mutants

genes

GMOs

Achievements June 2005•Identified novel H2 photoproducers•Developed high throughput screen for H2•Built ultra-sensitive H2 detection instruments•Engineered mutants in a green alga

PayoffClean H2 from non-fossil source

Solar Bio-H2 Goals: Economic feasibility

• ~10% solar-to-H2 efficiency at ambient light levels• Requires temporal or spatial separation of H2 and O2

• Achieve current cost estimates for large scale bioreactors

Two independent feasibility studies: • NREL , Amos et al.• Thiess Engineering (Australia) + U. Queensland

(Hankamer)

Losses in solar energy conversion efficiency to H2

light energy capture e- & H+ competing pathways

SECE(H2) = Λi (1-ΛNPQ)(1-ΩII) Φ1Φ2 (1-ΧC)(1- ΧA)(1- ΧN)(1-XO)

H2O PSII PQ PSI Fd H2ase H2

H+

Kruse et al. (2005) Photochem. Photobiol. Science; 4, 957 – 970.Prince, R. & Kheshgi, H. (2005) Crit Rev Microbiol. 31, 19-31.

Incident Sun light(100%)

PigmentsAbsorbed

LightStored Energy

RC H2 energy

(42% visible)(24% max)

(12% max)0% Solar

flux100

e- & H+

alternatepathways

PV

PEC

HydrogenasesCyanobacteria• NiFe enzymes• H2 ↓ Uptake (N2ase)• H2 Bidirectional enzyme

(↑↓)• NADH or NADPH

cognate reductant (-320 mv)

• Slower• Less O2 sensitive

Microalgae• FeFe enzyme• H2↑ evolution only• Ferredoxin cognate

reductant (-420 mV)• Faster• Very O2 sensitive

H2ase-dependent Solar Biological Hydrogen from Water

Microalgae

Direct H2, 2 quanta/e-, 1 stage, no gas separation

H2O H2 + ½ O2

(NREL, ORNL, UCB) Indirect H2 via starch, 3 quanta/e-, 2 temporally resolved stages

2 H2O + CO2 light O2 + [CH2O] + H2O PS

2 H2O + [CH2O] anaeorbic PSII light 2 H2 + CO2 + H2O Resp + low % PSII

CyanobacteriaIndirect H2 via glycogen, 2 quanta/e-, 2 temporally resolved stages

2 H2O + CO2light [CH2O] + O2 + H2O PS

[CH2O] + H2O anaeorbic + dark 2 H2 + CO2 Fermentation

H2O PSII - PSI Ferredoxin “CH2O”Calvin Cycle

ATPe-

e-

Fe-Hydrogenase

CO2

Self-AnaerobisisSulfur Deprivation

Ghirardi, Zhang, Lee, Flynn, Seibert, Greenbaum, Melis, Tr. Biotechnology, 2000. 18(12): p. 506-511.

e-

3 Quanta / e-, 2 stage light + anaeorbic, temporal gas separation

Eukaryotic Microalgae: Chlamydomonas R.

reductase

GlycolysisTricarboxylic

Acid Cycle

½O2+2H+ NADPHe-

e-

e-e-

H2

+ Respiration

Indirect Biophotolysis

Instrumentation Development

PS charge separation ratesFast repetition rate fluorimeter

Metabolomics & Proteomics

Triple Quadrupole-TOF mass spectrometer & electrospray ion

ETA Summer 2006

Dissolved H2 or O2LED + Clark cells

Screening Colonies on plates/wells

NREL WO3 H2 gas sensorETA Spring 2006

LED illuminator400 – 730 nm100 mW opticalpower

Sample (5 µL)

Membrane

Platinum-IridiumElectrode (+0.4 V)

LED 0 2 4 6

0

50

100

rate

of g

as e

volu

tion

incubation time, hours

O2

H2

Light on

Light off

Chlamydomonas r. CC124

Ag/ AgCl electrode

Dissolved H2 & O2 rates measured with 1000x improved sensitivity

5 µL volumeSensitivity = 2 nanomolar0.1 sec response time

LEDS: blue, orange, red, infrared, white

Conclusions•Major Photo-H2 •Minimal dark-H2•Co-production of H2 and O2

-BioSolarH2

H2O PSII PQ PSI

Alternative e-

donore- acceptor

pool H2H2-ase

hνhν

0 10 20 30 40

0

50

100

0.2 hr

670 nmPSI + PSII0.2 hr

4 hr

730 nmPSI only

0.2 hr

H2

conc

entr

atio

n, re

l.u.

time, min

light on, 10 s pulses

670 nmPSI + PSII

4 hr

Anaerobic -O2pre-incubation

-∆hyd E/FConclusions: • Photo-H2 & dark-H2 requires H2ase

genes hyd E/F• Maximum photo-H2 production

requires illumination of both PSI and PSII

• The major source of reductantrequired for photo-H2 comes from water via PSII turnover

• The minor PSI only pathway involves PQ/other donor

G. Ananyev & M. Posewitz

Chlamydomonas reinhardtii cc124

0 5 10 15 20

0

100

200

300

hydr

ogen

con

cent

ratio

n, re

l.u.

time, min

1 2 5 10 20 40 60 80

light pulseduration, s

Light produces two kinetic phases of photo-H2 in C. reinhardtii WT

0 20 40 60 80

0

100

200

300

light pulse duration, s

H2 e

volu

tion

(pea

k) Light H2

Light + Dark H2

0

50

100

integrated H2 evolution (area

)

Photo-H2post light H2

Peak Photo-H2

Total AreaPhoto-H2 +Post light H2

PSI

e- acceptorpool

secondarye- acceptor

pool

?

H2H2-ase

?

slow

hν

Conclusion: •Two pools of photo-electron acceptors in PSI capable of light-induced H2 production

•Integrated H2 yield is linear with pulse duration competing pathways for e-/H+ consumption

are either fixed or not present

on

off

NADP+?

Fd

G. Ananyev

CyanobacteriaArthrospira (spirulina) maxima

Habitat: volcanic soda lakesAlkalophile, pH = 9.5-11

0.2-1.2 M carbonateNo N2 fixation systemFastest PSII turnover e-/H+ flux-5x larger PQ pool vs algae

enes hox-EFUYH encodeNAD-dependent bidirectional H2ase No hup genes reported (uptake H2ase)

0 10 20 30 40 50

0.50

0.52

0.54

0.56

0.58

Fv/F

m

STF number

5 6 7 8 9 10

X5

Experiment

Fitting

1 10 100 10002

3

4

5

6

7

8

Q

ualit

y fa

ctor

, 1/(α

+β)

dark time between STF, ms

B

Chlorella

Spirulina

Water oxidation rate by Arthrospira maxima cells is highest by 5x

PSII quantum efficiency ~54%

Cyanos can split water 3-5x faster athigher photon flux vs. plants & algaeDue to 3-5x larger PQ pool size

* Ananyev & Dismukes, Photosyn Res. (2005)

Kok α & β

Cells with NiCl2 supplementation 3 days after inoculation in continuous illumination 130µE/m2sec light flux (above)12/05/05

0.01

0.1

1

10

0 100 200 300 400 500 600 700 800 900

Time (Hours)

Chl A

bsor

ptio

n

0.0uM Ni0.25uM Ni1.0uM NiuM Ni 4.0

06/16/06

A. [Ni2+] causes chlorosis during photoautotrophic growth (130µE/m2sec).

C. Ni2+ stimulates H2 production rate by 20 x in reactor purged of H2.

6/19/06. 25mL samples + 0.200g sodium dithionite25 days old, 12hr light/dark growth18(+/-2) mg chla / L

0

1

2

3

4

5

6

7

8

-5 5 15 25 35 45 55

time (hours)

0.0uM Ni (w/o glucose)0.0uM Ni (+ glucose)1.0uM Ni (w/o glucose)1.0uM Ni (+ glucose)3.0uM Ni (w/o glucose)3.0uM Ni (+ glucose)

Damian Carrieri

B. photoautotrophic growth rate is independent low levels of Ni2+,

at high Ni2+ growth lags but recovers.

D. and by 3 x in headspace of bulk cultures with H2 back pressure.

Dynamics of Dark Fermentative H2 Production RateArthrospira maxima

Logarithmic growth Stage 1

Stationary growth Stage 2

12 days

+ pyruvate

Phase-2

Phase-1

0 20 40 60 800

100

200

300

400

hy

drog

en, m

V

time, min

640 nm

735 nm

PSI illumination (735 nm)Suppresses fermentative H2Slow photo-H2

PSI+PSII illumination (640 nm)Adds to fermentative H2Fast photo-H2

A. maxima

Pathways for Dark and Light H2production by Arthrospira m.

PSII

H2O O2

PQH2

PQPSI

Fd FNR NADP+

NAD-dependentNiFe-H2ase

glycogen

glucose

pyruvate

NAD+

NDHComplex I

H2

ICM-thylakoid

Sustained H2 production from water by cyanobacteria

Synechocystis pcc 6803 mutant –ΔNDHB2

*Not sustainable

Arthrospiraplatensis1

Arthrospiramaxima

BatchpH 10. 5 & 35°C + Ni2+

[nitrate in media] Excess H2removed

In DarkIn light

photo-H2 in alga C. reinhardtiiunder Sulfur depletion

- nitrate

2. Cournac et al (2004)BioSolarH2 team (2006)

2006 NREL immobilized 0.35%

2004 NREL batch 0.15%

% E

ffici

ency

Sol

ar-to

-H2

1. Aoyama et al. (1997);

-BioSolarH2

Conclusions

•New tools for measuring H2, O2 and redox status provide reliable quantitative data on concentrations and fluxes

•greatly simplify and clarify the fundamental mechanisms of H2 production in microalgae and cyanobacteria

•Cyanobacteria & microalgae have different pathways to H2

•Ideal temporal separation of H2 and O2 production in cyanobacteria (fermentation) solves gas separation issue

•Strain selection and media optimization yield 20x improvement in cyanobacterial fermentative H2

Synthetic Biology Goals in Cyanobacteria

1. Replace native NiFe-H2ase with foreign FeFe-H2ase(s)• Couple to cognate Fd donor, eg. Eliminate loss to NAD(P)H

2. Over-express native NiFe-H2ase(s) (strong RUBISCO promotor)• Deliver e- potential: Knock-out competing ferm. pathways draining reductant

NADH (-lactate, -ethanol)• Deliver H+ potential: ATP hydrolysis or lower external pH (add CO2)

3. Directed evolution under forced fermentative growth, eg. cell division at suppressed photoautotrophic metabolism

4. Chimeric NiFe-H2ases (Ralstonia etc. gene shuffling)

5. Develop a genetic transformation system for Arthrospira species.

glycogen G6P

pyruvate

acetyl-CoA+ CO2

acetyl phosphate

CH3COOH

acetaldehyde

Manipulating Fermentative Pathways of Synechococcus 7002 & Synechocystis 6803

(whole genome predictions)

Fd(ox)

Fd(red)

NAD(P)H

NAD(P)

H+

H2

NAD(P)H

NAD(P)H

ATP

2 NAD(P)H + 3 ATP

CH3CH2OH

6803 only

NAD(P)

H+H+

NAD(P)+

2 NAD(P) + 3 ADP, Pi

ADP

7002 only

6803 & 7002

NAD(P)H

NAD(P)

lactate dehyd.

aldehyde dehyd.

alcohol dehyd.

pyruvate:Fdoxidoreductase

Fd: NAD(P) reductaseNiFe-H2ase

acetate kinase

phosphotransacetylase

glycolysis

acetyl-CoA

synthetase

OHOH

O

Kelsey McNeely

-BioSolarH2 Team 2005+PrincetonGennady AnanyevDerrick KollingDamian Carrieri GS2Kelsey McNeely GS1Nick Bennette GS1Tyler Brown ’06

Christine Louie 06, Adam Litterman 07Peter Curtin 08, Anita Ma 09

Edward I. Stiefel PUDonald A. Bryant PSUMatthew Posewitz CSMEric Hegg UURobert Bidigare UH

>2006John Peters MSURobert Austin PU

Future Challenges (DOE*)

•Synthesize durable catalysts for efficient water splitting and reversible O2 reduction without noble metals

•Functionally integrate these with solar photocells (semiconductors) for water oxidation, and with fuel cell cathodes for O2 reduction

*DOE/BES Publication:2003 http://www.sc.doe.gov/bes/reports/files/NHE_rpt.pdf

Part 2"Biomimetic Catalysts for Splitting Water"

Ferreira, Iverson, Maghlaoui, Barber, Iwata (2004) Science

Photosystem II - Water Oxidizing ComplexThermosynechococcus el.

Ferreira et al (2004) Science

The Catalytic Site of Water Oxidation in Oxygenic PhotosynthesisXRD Model by Imperial College –Barber and Iwata

4 hν

Hypothesized Mechanism of Photosynthetic O2 Evolution*

* Dasgupta, van Willigen, Dismukes (2004) Physical Chem: Chem. Physics

4 e- + 2 H+

Mn4(2III, 2IV)

O

Mn

Mn

O

Mn

O

O

Mn

OO

R2P

O

O

PR2O

OR2PO

O

PR2

O

OR2P O

OR2P

R = phenyl p-tolyl

2.8 MnII + 1.2 MnO4-

B)

A)

[(bpy)2Mn Mn(bpy)2]3+

bpy =N N

R2PO2-

R2PO2-

DMF

MeCN

Mn2(III, IV)

70-90 % yield

O

O

O OP

XX

Rüttinger, W., C. Campana and G.C. Dismukes, J. Am. Chem. Soc., 1997. 119(27): 6670-6671; Carrell, Cohen and Dismukes, J. Mol. Catal. A: Chem., 2002. 187: 3-15;

X = H, CH3, -CH2Br -OCH3

Synthesis of L6Mn4O4 cubane complex

Unusually long (weak) Mn-O bonds

• Mn-O distance: 1.88 ~ 1.98 Å• Mn-Mn distance: 2.90 ~ 2.95 Å

• O-O distance: 2.53 ~ 2.60 Å

Structure of (Ph2PO2)6Mn4O4 cubane complex

MnIII(µ-O)2MnIV complexesMn-O distance: 1.8 ÅMn-Mn distance: 2.6 ~ 2.7 Å

O-O distance in O2: 1.21 ÅO-O distance in H2O2: 1.50 Åvan der Waals radius

of O atom: 2.8 Å

Ruttinger, W., C. Campana, G.C. Dismukes, J.. Am. Chem. Soc. (1997) 119, 6670-71

Docking of phenol to XRD structure of cubane

800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100

2

2+

1+

1Mn4O2L

25

Mn4O4L26

Mn4O2L15

m/z

Mn4O4L16

[ “ ]+2+

Mn4O4L262

[ “ ]+1+

Mn4O4L161

m / z15 20 25 30 35 40

Inte

nsity

(a.u

.) H2O CO 16O218O2 L6Mn4(16O)4

L6Mn4(18O)4

Blank

O

P

O

O

O

CH3

CH3

Quadrupole MS

LDI-MS

Stronger electron-donating phosphinate ligands increase the QY of photodissociation In the gas phase

L2

Wu et al. (2005) Inorg Chem

Yagi et al (2001) Ang. Chem. Int.

Models: [Mn4O4]6+,7+ Chemistry to O2

O

Mn

Mn

O

Mn

O

O

Mn

OO

P

O

O

PR2O

OR2PO

O

PR2

O

OR2P O

OR2P

MnO

OMn

MnO

MnO

Ruettinger, Yagi, Wolf, Bernasek, Dismukes (2000) JACSWu, Selitto, Sheats, Yap, Dismukes (2004) Inorg. Chem.

2 H2O +

OO

P

NO2

NO2

ligands

OO

PO

O

P

MeO

OMe

O2 is released only if one phosphinate is dissociated

Dynamic- DFT Calculations of O2 release from L6Mn4O4 L- + L5Mn4O4

+

F. DeAngelis& R. Car

O2 release isexothermic ∆G = - 7.2 kcal/mol

Activation Barrier~ 27 kcal/mol

MnO

O

O

Mn

MnO

Mn or Ca

η2 −PeroxoE

nerg

y, k

cal/m

ol

[L5CaMn3O4]+

[L5Mn4O4]2+

[L5Mn4O4]+

DFT Calculations of the Transition State Energy on the path to O2 release

30

20

10

0

27,7

20.0

14.8

Reaction Coordinate

different Mn oxidation states

MnIII Ca2+ replacement

F. DeAngelis and R. Car

Conclusions:Lower kinetic barrier to O2 release

• The higher Mn oxidation state (III, 3IV) vs. (2III,2IV) gives a lower peroxo ∆E by 7.7 kcal/mol

• Replacing MnIII CaII lowers peroxo ∆E by 5.2 kcal/mol.

• Ca2+ serves as an “Electrostatic Ball Bearing” to lower the barrier to intramolecular O-O bond formation

Mn Mn

MnO

MnO

"tethered butterfly"

Ph2PO2- + O2

MnO

OMn

MnO

MnO

2 H2O

"oxidized butterfly"

O2

Electrodeor Solar Cell

e- + H+

Attach to Solar/ Fuel Cell Electrode

Electrode

The Holy Grail: Catalysts for Electro- and Photo- Sensitized Water Splitting*

MnO

OMn

MnO

MnO

X

Ying YuRuslan PryadunJohanna Scarino

Collaborators:

CSIRO + Monash U.Gerhard SwiegersLeone Spiccia

Spin Coating: 1µl of 10% Nafion solution is spotted on 3mm glassy carbon electrode and spun at 2000 rpm

ω

The Nafion coated electrode is soaked in 0.5mM solution of the oxidized cubane:

(bis(methoxyphenyl)phosphinic acid)6Mn4O4ClO4in acetonitrile.

Doping Nafion Coated Glassy Carbon Electrode with Cubane

F

F

F

FF

F

OO

S

F

CF3 F

FF

FF

F

O

O

OH

(CF2-CF2)x

MnO

OMn

MnO

MnO

MnO

OMn

MnO

MnO

MnO

OMn

MnO

MnO Mn

O

OMn

MnO

MnO

Nafion

Ion exchange of L6Mn4O4+ with counter cations in

hydrophilic sulfonate lined channels. channel "diameter" ~10-100 nm. Chou, et al. J. Phys. Chem. B 2005, 109, 3252.

ClO4-

MnO

OMn

MnO

MnO

=

+

Nafion

glassy carbon electrode

Johanna Scarino

4

3

2

1

0

curre

nt (u

A)

120100806040200time (min)

Oxidized Bismethoxy cubane doped nafion on GCE (solid), nafion GCE (dashed), in 0.1M NaSO4, held at 1V (just above cubane oxidation) vs Ag/AgCl, exposed to light at 5minutes

O

P

O

O

O

CH3

CH3

MnO

OMn

MnO

MnO

=

+

ClO4-

GCE Held at 1.0 V vs. Ag/AgCljust above cubane oxidation couple 1 ↔ 1+

Xenon source illumination

Collaborators: R. Brimblecome, G. Swiegers, L. Spiccia, J. Sheats, unpublished.

Breaking News: June-July 2006Sustained Photo-assisted Electrocatalytic Oxidation of Water

with Manganese-oxo Cubanes

Nafion coated Glassy Carbon Anode + 1+

Nafion coated Glassy Carbon Anode

Light

2 H2O O2 + 4 H+ + 4 e-

“Predictions are hard to make, especially about the future.”

Can we predict which technologies will solve the energy crisis?

Steven Chu delivers the 31st Donald Ross Hamilton Lecture at Princeton University March 30, 2006:"The energy problem: our current choices and future hopes"

When asked at the end of his public lecture which single new energy technology he would bet on to help solve the energy crisis he responded:

".....synthetic biology (genetic engineering) applied to algae looks to be promising as a means to produce fuels. Synthetic biomimeticapproaches should then rise to prominence that need to have durability and use less land.“

Steven Chu touring the facilities of the BioSolarH2 team at Princeton.

Dr. Chu is the director of the Lawrence Berkeley National Laboratory in California (3800 employees, $0.55B/yr budget) and the 1997 Nobel Prize winner in Physics.

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