Cyanobacteria for solar fuel

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Cyanobacteria for solar fuel

• Klaas J. Hellingwerf
• Swammerdam Institute for Life Sciences
• Netherlands Institute for Systems Biology
• University of Amsterdam

MJ Teixeira de Mattos Photanol KNAW Symposium,
Jan., 2008
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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 wont 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
membrane
H2
PSI
or
hydrogenase
H2
macroscopic membrane
O2
e-
hydrogenase
PSI
PSII
PSII
e-
O2
H
H2O
bilayer
H2O
H
Brh
Brh
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Result after 10 years1
In de 1970er jaren heeft men getracht systemen
te ontwikkelen met chloroplasten voor
het genereren van een elektronenstroom uit
zonlicht en hydrogenasen voor productie van H2
uit deze elektronen en H. Deze systemen bleken
niet stabiel. Na de oliecrisis van 1973 is
in Nederland op initiatief van Prof. E.C. Slater
in 1975 een project gestart met het doel om
te onderzoeken of de fotolyse (met zonlicht) van
water in H2 en O2 te realiseren is
met biochemische systemen (fotosysteem-II en
hydrogenasen), of met chemische
afgeleiden daarvan. In de jaren 1975-85 heeft
ZWO/SON het onderzoek gefinancierd via een
speciaal programma het Hydrogenase Project.
Doelstelling was 1. bepaling van structuur en
werkingsmechanisme van de H2-producerende
biokatalysator hydrogenase (door biochemici,
biofysici en microbiologen) 2. ontwikkeling van
chemische alternatieven, want biokatalysatoren
zijn niet altijd even stabiel. In 1985 werd het
programma gestaakt mede als gevolg van de daling
van de olieprijs. Twee Nederlandse
onderzoeksgroepen (Hagen, TUD en Albracht, UvA)
werken nog steeds aan hydrogenasen. 1 REPORT OF
THE WORKSHOP BIOLOGISCHE H2 PRODUCTIE, donderdag
4 oktober 2001, Novem, Utrecht
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What is needed..

• Photovoltaics for electricity
• A solar solution for fuel (with as few
  conversions as possible (0.334 0.01!)

For any large-scale process, only H2O is a
realistic electron donor
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Some current biofuel technologies
1 Grow crops on land
2 Grow Algae in ponds
Harvest organic matter
Harvest cells
Transport to bioreactor fractionate
Transport to separator
extraction modification
fermentation
biofuel
Waste
Biodiesel (fatty acid methyl ester) or H2
Mostly ethanol
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First-, second- and third-generation technologies

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

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The 2 modes of life
1 Light-dependent life (plants, bacteria)
((Chloro)Phototrophy)
H2O
reducing power ATP O2
Organic C
Reducing power CO2 ATP
Cells
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The 2 modes of life
2 Organic matter-dependent life
(Chemotrophy)
a) respiration
(animals, fungi, bacteria)
Organic C O2
Organic C O2
ATP CO2 H2O
(redox reaction!)
Cells
Organic C ATP
b) fermentation
(fungi, bacteria)
Cells FERMENTATION
PRODUCTS
Organic C
(when organic C is abundant or O2 is lacking)
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The circle of life is driven by the sun
(plants, bacteria)
CO2 H2O
Cells O2
(animals, fungi, bacteria)
Earth surface
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The broken circle
(plants, bacteria)
CO2 H2O
Cells O2
CO2
(animals, fungi, bacteria)
Earth surface
fossil fuels
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Chloro-Phototrophy optimized during billions of
generations
CO2
Dark reaction
1/3 GAP
Glyceraldehyde-3-P
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Phototrophy
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Chemotrophy optimized for billions of generations
Organic matter
F-1,6-BP
Glyceraldehyde-3-P (GAP)
Pyruvate
Fermentation products
(Ethanol, propanol, butanol, propanediol,
glycerol, acetone, lactate, acetate, ..........)
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Photofermentation
Fermentation
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Biological incompatibility methanogenesis
Fdred
CO2
H2
Formyl-MFR
Formyl-H4MPT
Methenyl-H4MPT
H2
H2F420
Enzymes involved are extremely oxygen-sensitive
and have several very uncommon cofactors
Methyl-H4MPT
Methyl-S-CoM
HS-CoB
CH4
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Constructing a Photofermentative strain
Host phototrophic Synechocystis PCC6803
Donor chemotrophic bacterial species
GAP
EtOH genes
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The Photanol Process Product cassettes
CO2
Ethanol S. cerevisiae 1 pyr decarboxylase 2
alcohol DH I
Butanol L. brevis 1 thiolase 2 OHbutyrylCoA DH 3
crotonase 4 butyryl-CoA DH 5 Butyraldehyde DH 6
Butanol DH
The octane rating of n-butanol is similar to that
of gasoline but lower than that of ethanol and
methanol.
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Solventogenesis

• History Chaim Weizmann in early 1900 in Britain
  because of lack of natural rubber few years
  later acetone
• For many years primarily studied in Clostridium
  acetobutylicum
• However, the process of solventogenesis is also
  observed in aerotolerant organisms (like
  Saccharomyces cerevisiae and Lactococcus lactis
• Recent example Propane-diol fermentation in
  Escherichia coli (Monsanto)
• Example of recent innovation mixed solvent
  production in E.coli (Nature 451 86-90 (2008))

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Regulation of fuel formation The GAP branchpoint
ACO2
B
GAP
A
D
E
cassette
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The Photanol Process Genetic Process control
Ammonia availability is often used as a control
parameter to regulate biomass formation
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N sensing in Synechocystis
N-excess
glutamate

proteins
NtcA
NtcA-aOG
-
sE
X
PSigE
SigE
Gene cassette
Pgap1
gap1
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N sensing in Synechocystis
N-depletion
glutamate

proteins
NtcA
NtcA-aOG
sE
PSigE
SigE


Pgap1
Gene cassette
gap1
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N-dependent fuel cassette expression
N-excess
N starvation
Glu
protein
2OG N
Ntca
12 3-P-Glycerate
12 1,3-bPG
6 CO2
2 GAP
5 R1,5bP
10 GAP
P
P
5 FbP
Growth
Hexose-P
thl
crt
etf
4hbd
ald
bdh
Butanol
time
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N-dependent fuel cassette expression
N-excess
N starvation
Glu
protein
2OG N
Ntca 2OG
Ntca2OG
12 3-P-Glycerate
12 1,3-bPG
se
6 CO2

10 GAP
2 GAP
5 R1,5bP
P

5 FbP
Growth
Hexose-P
thl
etf
ald
bdh
Butanol
growth
growth
time
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Summary of the Photanol Process
cells
Clean fuel production CO2 consuming Cheap
technology Not competing with food stocks Yield
per year per surface up to 20x higher than plant
crops Principle generally applicable ethanol,
butanol, etc
xCO2 yH2O
CxH2yOz (x0.5y-0.5z)O2
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1st and 2nd vs 3rd generation biofuels

• Plants have lower productivity per unit surface
  than algae
• 2nd generation Lipid synthesis occurs during
  growth-restriction in nitrogen-deficient media
  (cells C4H7O2N)
• 3rd generation Cells are merely catalysts
  reaction CO2 h? H2O ? fuel O2

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Why Synechocystis?

• Prokaryote simple metabolism and lowest
  maintenance energy requirements of all living
  organisms
• Naturally transformable, which implies facile
  genetic alterations
• Has been subjected to all known genomics
  techniques
• A systems biology description is underway (e.g.
  The Plant Cell 13 793806 (2001) micro-arrays
  Phytochem. 68 23022312 (2007) metabolic flux
  analysis)

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Large-scale culturing
Tubular system Raceway pond Flat
panel system

• Extensive expertise is available in the scale-up
  of culturing systems systems can be used in
  open and closed form
• Many problems in down-stream processing are
  remaining
• 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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Current- Synthetic Biology
1 Energy conservation in the photosynthesis of
cyanobacteria in the form of conversion of light
energy into biomass at a constant (sun) light
energy of approximately 100 to 200
?Einstein.m-2.s-1 proceeds close to the
(biological) theoretical maximum (maximal biomass
yield gt 100 tonnes/ha/yr). 2 Very large free
energy losses may occur at high light
intensities, and transiently, because of delay of
adaptation of the cell to altering extra-cellular
conditions. 3 Multiple synthetic improvements
in the energy-conversion performance of
cyanobacteria can be perceived, like (i)
Addition of an IR-absorbing PS3 as an extra
proton pump (ii) engineering of extra
proton-translocating loops in the
electron-transfer chain (iii) cutting of
high-energy photons to achieve gt 1
charge-separations per photon etc.
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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 photosystem expression f
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