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The Photanol ProcessCyanobacteria 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 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
funding
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What is needed..

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

fuels
For any large-scale process, only H2O is a
realistic electron donor
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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)
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 (e.g. fatty acid methyl esters)
Mostly ethanol
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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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Unity of life the broken circle
(plants, bacteria) E
CO2 H2O
Cells O2
CO2
(animals, fungi, bacteria)
? energy
Earth surface
fossil fuels
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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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Chloro-Phototrophy optimized during billions of
generations
The light reactions of photosynthesis
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Chloro-Phototrophy optimized during billions of
generations
CO2
Dark reaction
Dark reaction
1/3 GAP
Glyceraldehyde-3-P
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Phototrophy
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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
Cells
Organic C ATP
b) fermentation
(fungi, bacteria)
Cells FERMENTATION
PRODUCTS
Organic C
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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Chemotrophy optimized for billions of generations
NAD(P)H, ATP
NAD(P)H, (ATP)
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Photofermentation
Fermentation
CO2 H2O ? fuel O2!!
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Fermentation pathways
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Some successful pathway insertions

• 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))

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Synechocystis sp. PCC 6803

• 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
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Constructing a photofermentative strain
Host phototrophic Synechocystis PCC6803
Donor chemotrophic bacterial species
EtOH genes
GAP
PCR
recombination
expression
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(in)complete segregation
Cloning in the psbA2 locus of Synechocystis
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,
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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 30C 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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Photofermentation the best of both worlds
cells
CO2 H2O fuel O2!!

• 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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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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Regulation of fuel formation The GAP branchpoint
ACO2
B
GAP
A
Eg
Ep
D
E
cassette
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The Photanol Process Genetic Process control
CO2
Ammonia availability is often used as a control
parameter to regulate biomass formation (cells
C4H7O2N)
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Nitrogen sensing in Synechocystis
N-excess
glutamate

proteins
NtcA
NtcA-aOG
X
sE
-
PSigE
SigE
Gene cassette
Pgap1
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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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)
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
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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
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
xCO2 yH2O
CxH2yOz (x0.5y-0.5z)O2
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Dreams