31/03/2009 the photanol process

The ‘Photanol’ Process:Cyanobacteria for simple solar fuel

Kornel Golebski, Andreas Angermayr, Ginny Anemaet

Joost Teixeira de Mattos & Klaas J. Hellingwerf

Swammerdam Institute for Life Sciences &

Netherlands Institute for Systems Biology

University of Amsterdam

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A little bit of history

A Round-Table Discussion held during the 10th FEBS Meeting in Paris (July 25, 1975) considered the different approaches by which Biological Systems might be used to convert ambient solar energy into more useful energy forms.

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The problem:

“Man does not have much choice. Either we trust the physicist to make us a sun without blowing us up, or we let the bioenergeticists use our present one. Otherwise, we won’t last more than a hundred years or so. This is an exciting challenge for the bioenergetics of tomorrow.”

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The proposed solution:

PSII PSIe-

H2O

O2

H+

macrosco

picm

emb

rane

hydrogenase

H2

Brh

funding

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What is needed..

Antenna

ReactionCenter Catalytic

Site 2

CatalyticSite 1

e-

e-

R (H+, CO2)

P (H2, MeOH)

R (H2O, HA) P (O2, A, H+)

EET

e-Antenna

ReactionCenter Catalytic

Site 2

CatalyticSite 1

e-

e-

R (H+, CO2)

P (H2, MeOH)

R (H2O, HA) P (O2, A, H+)

EET

e-

For any large-scale process, only H2O is a realistic electron donor

fuels

• Use the auto-regenerative capacity of living organisms• A solution for solar fuel with as few conversions as possible (0.334 = 0.01!)

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Some current biofuel technologies (1)

From: Esper, Badura and Rögner (2006) Trends in Plant Science 11: 543-549.

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Some current biofuel technologies (2)

2 Grow algae in ponds

biofuel+

Waste

Harvest cells

extraction

&

modification

Transport to separator

1 Grow crops on land

Harvest organic matter

fermentation

Transport to bioreactor& fractionate

Mostly ethanol Biodiesel (e.g. fatty acid methyl esters)

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The photanol approach

• First generation: Starch from corn or sugar cane fermented into ethanol by yeasts or palm oil trans-esterified to biodiesel.• Second generation:More recalcitrant bio-polymers fermented to alcohol(s) or biodiesel produced by marine algae.• Third generation: “Photanol”

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CO2 + H2O Cells + O2

(plants, bacteria)

E

(animals, fungi, bacteria)

fossil fuels

CO2

Earth’ surface

Unity of life & the broken circle

energy

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1 Light-dependent life (plants, bacteria)

H2O reducing power + ATP + O2

Reducing power + CO2 + ATP

The 2 modes of life

Cells

((Chloro)Phototrophy)

Organic C

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Chloro-Phototrophy; optimized during billions of generations

The light reactionsof photosynthesis:

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Glyceraldehyde-3-P

CO2

H2O

NADPH + ATP

O2

Light reaction

Dark reaction

Chloro-Phototrophy; optimized during billions of generations

1/3 GAP

Dark reaction

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Light reaction

PS II

PS I

NADP

NADPH

H2O O2

ATP

h

Dark reaction

Cells

GAP

CO2

ADP

Phototrophy

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2 Organic matter-dependent life

Organic C + O2 ATP + CO2 + H2O

(animals, fungi, bacteria)

(Chemotrophy)

a) respiration

Organic C + ATP

Organic C + O2

Cells

Organic C Cells + FERMENTATION PRODUCTS

b) fermentation (fungi, bacteria)

The 2 modes of life

b): occurs when O2 is lacking or organic C is abundant; well-known as “overflow metabolism” in E. coli, LAB and yeast

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(Ethanol, propanol, butanol, propanediol, glycerol, acetone, lactate, acetate, ..........)

Organic matter

Fermentation products

Pyruvate

Glyceraldehyde-3-P (GAP)

F-1,6-BP

Chemotrophy: optimized for billions of generations

NAD(P)H, ATP

NAD(P)H, (ATP)

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Light reaction

PS II

PS I

NADP

NADPH

H2O O2

ATP

h

Dark reaction

CO2

GAP

Cells

ADP

Fermentation

Fermentationproducts

Photofermentation

CO2 + H2O fuel + O2!!

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Fermentation pathways

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Bermejo et al. 1998 (Acetone production in E. coli (Clostridium acetobutylicum pathway) )

Deng and Coleman. 1999 (EtOH production in Synechococcus sp. (pdc and adh from Zymomonas mobilis) )

Takahama et al. 2003 (Ethylene in Synechococcus sp. (efe from P. syringiae) )

Fu. 2008 (EtOH production in Synechocystis sp. PCC 6803)

Pirkov et al. 2008 (Ethylene production in S. cerevisiae (efe from P. syringiae) )

Shen and Liao. 2008 (1-Butanol and 1-Propanol in E. coli)

Tang et al. 2009 (Propanediol in E. coli (genes from Clostridium butyricum))

Some successful pathway insertions

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Unicellular prokaryote Genome sequenced Auto- and heterotrophic Effective photosynthesis Model organism for photosynthesis Defined (simple) growth media Naturally transformable Grows to high densities

Circadian rhythm doubling time ~8h 6 to 10 genomes per cell Low maintenance energy req.

EM photograph, scale bar 200nm

Synechocystis sp. PCC 6803

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Host: phototrophicSynechocystis PCC6803

EtOH genes

PCR

recombination

expression

GAP

Constructing a photofermentative strainDonor: chemotrophicbacterial species

HOM1 HOM2

HOM1 HOM2

wt genome

plasmid

pAAA170 9 1 bp

p s b up (H O M 1 )

p s b dn (H O M 2 )

f1 (+ ) o r iA m p R

e fe

P la c

T7 pro m o te r

T3 pro m o te r

P A m pR

R B S

K a nR

Eco R I (2530)

Sa c I (4 8 93)

X ba I (4 8 65)

X hoI (6 6 9)

Sa l I (14 59 )

Bam H I (4 0 4 9)

Pst I (254 0 )

Pst I (3129 )

Sma I (254 4 )

Sma I (4 0 45)

K pnI (6 58 )

K pnI (4 158 )

K pnI (4 59 0)

goi

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(in)complete segregation

Tested clonesM C- C+

2kbp

5kbp

Example of incomplete segregation

M P N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Colony PCR of pAAA2 transformants. M is marker. P is positive control. N is negative control. Transformants grown on 4ug/ml kanamycin. No correct insertion in transformants 4, 5, 7, 8, 10; not fully segregated transformants 1, 2, 3, 6, 9; full segregation in 11, 12, 13, 14, 15, 16, 17

Cloning in the psbA2 locus of Synechocystis

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Ethanol synthesis by geneticengineering in Cyanobacteria

From: Ming-De Deng and John R. Coleman (1999) Applied & Environm. Microbiol. 65: 523-528 FIG. 4. Cell growth and

ethanol synthesis in Synechococcus sp. strain PCC 7942 transformed with pCB4-LRpa. Cells were grown at 30°C in the presence of light in a 500-ml liquid batch culture aerated by forcing air through a Pasteur pipette. Samples were taken at intervals in order to monitor cell growth (OD730) and ethanol accumulation in the culture medium. The PDC and ADH activities in cell lysates on day 5 were 320 and 170 nmol · min 1 · mg of total protein 1, respectively.

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CO2 + H2O fuel + O2!!

‘Photofermentation’: the best of both worlds

cellscells

• Cells are auto-regenerative catalysts of the process• The fuel molecules can stably coexist with oxygen• Production is not limited by the storage capacity of the cells• It is possible to form the product in volatile form• Process can be run in a closed large-scale photobioreactor

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CO2H2

Formyl-MFR

Methenyl-H4MPT

Formyl-H4MPT

Methyl-H4MPT

Methyl-S-CoM

HS-CoBCH4

H2H2F420

Fdred

Biological incompatibility: methanogenesis

Enzymes involved are extremely oxygen-sensitive and have several very uncommon cofactors

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Regulation of fuel formation: The GAP branchpoint

A~CO2B

GAPA

DE

Fluxgrowth = [Eg].vmax. [PGA]

Km + [PGA]

Fluxproduct = [Ep].vmax. [PGA]

Km + [PGA]

cassette

EgEp

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A~CO2B

DE

A

E

cassette

Promoter

product

NH4

+

GAP

The Photanol Process: Genetic Process control

Ammonia availability is often used as a control parameter to regulate biomass formation(cells: “C4H7O2N”)

CO2

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N-excess-oxoglutarate + NH4

+ glutamate

proteinsNtcA+

NtcA-aOG

PSigE SigE X E

Gene cassette-

Pgap1 gap1

N-depletion

Gene cassette+

Pgap1 gap1

-oxoglutarate + NH4+ glutamate

proteinsNtcA+

NtcA-aOG

PSigE SigE E~

Nitrogen sensing in Synechocystis

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6 CO25 R1,5bP

12 3-P-Glycerate 12 1,3-bPG

Growth 5 FbPHexose-P

N-dependent fuel cassette expression

time

NH4

+

growth

N-excess2OG + N Glu protein

N starvationNtca

2 GAP

Pth

l

crt

4h

bd

etf

bd

h

ald

Butanol

P

10 GAP +

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N-excess

6 CO25 R1,5bP

12 3-P-Glycerate 12 1,3-bPG

Growth 5 FbPHexose-P

2OG + N GluN starvation

Ntca + 2OG

2 GAP

Ntca~2OG

+

+P

thl

crt

4h

bd

etf

bd

h

ald

Butanol

time

growth

e

protein

10 GAP +

product

NH4

+

growth

N-dependent fuel cassette expression

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‘Back-of-the-envelope’ calculation

• 1 acre = 4 . 103 m2

• 1 year has 107 seconds of sunlight (3600 . 12 . 235)• Sunlight intensity (PAR): 600 μE.m-2.s-1

24 . 106 Einstein/acre/year

• Complete conversion of light energy to ethanol:

• 12 photons per ethanol: 2 CO2 + 3 H2O C2H6O + 3 O2

Maximal productivity:

2 . 106 moles ethanol/year/acre

~ 100 ton ethanol/year/acre

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Large-scale culturing

Tubular system Raceway pond Flat panel system

• Extensive expertise is being generated with respect to the scale-up of culturing systems; systems can be used in ‘open’ and ‘closed’ form (e.g. Wijffels c.s.)• All systems have in common that the fuel-producing cells are exposed to oscillating light regimes, with typical frequencies ranging from minutes (depending on mixing regime) to 24 hrs.

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Some regulatory mechanisms in the photosynthesis of Synechocystis

a] State transitions of phycobilisomes b] Non-photochemical, IsiA and/or OCP-mediated quenching c] zeaxanthin cycle d] Regulation of expression ratio of PSI/PSII/Antennae e] Circadian regulation of gene (photosystem) expressionf] NDH (and FNR) mediated cyclic electron transfer around PSI g] Cyclic electron transfer around PSII h] PSI trimerization, PSII dimerization, IsiA and iron limitation i] Variation of antenna size (j] Chromatic adaptation)

a Systems Biology-based optimization is necessary

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Circadian regulation of gene expression

Dong G and Golden SS (2008) How a cyanobacterium tells time. Curr Opin Microbiol. 11: 541-546.

7 sigma factors of three different classes

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Cyanobacteria do it during the day

• Two interesting physiologies may occur at night:

1] oxidative catabolism (‘glycogen’ CO2)

2] anaerobic fermentation

(‘glycogen’ organic acids)

• Feasibility of supportive LED illumination during the night?

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Summary of the Photanol Process

xCO2 + yH2O

CxH2yOz + (x+0.5y-0.5z)O2

ATP, NADPH

cells

Clean fuel production

CO2 consuming

Cheap technology

Not competing with food stocks

Principle generally applicable: ethanol, butanol, etc

Yield per year per surface: up to 20x higher than plant crops

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Dreams