Hydrogen from water : Engineering photosynthetic metabolism for a

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Hydrogen from water :
Engineering photosynthetic metabolism for a
light-powered biohydrogen production
Matthias Rögner
Lehrstuhl für Biochemie der Pflanzen
Ruhr-Universität Bochum
LEOPOLDINA
Recommendation
for Bioenergy
07'2012
Recommendations concerning hydrogen production from water
• Considering the almost unlimited availability of water and sunlight,
the production of hydrogen via photolytic cleavage
of water could be an ideal energy source – renewable,
environmentally friendly, and sustainable......
• Molecular and synthetic biological techniques will help in
constructing genetically modified microorganisms with oxygenic
photosynthesis that have more stable and more efficient H2evolving systems......
Harnessing cyanobacteria
for energy.....
H2
Vision : regenerative energy (C-free)
Natural catalysts:
Photosystem 2 (PS2)
Hydrogenase (H2ase)
Photosynthesis
Tree
H2O + CO2
Spinach
e
_
sugar / biomass + O2
Cyanobacterium
Photosynthesis : Efficiency & Kinetics
Efficiency ≤ 99 %
≤ 38 %
≤ 27 %
≤ 1 % (Biomass!)
"Light reactions" : Light capture in fs-range
"Dark reactions" :
PS-electron transport with TOF = 50 - 200 s-1
TOF = 0.3 s-1 (Rubisco)
Conclusions:
•  Direct coupling to primary events of PS for high efficiency !
•  Tremendous over-capacity of PS light reactions should be
used – large energy loss due to dark reactions!
Do such cells exist
in nature ?
Photosynthesis: Direct coupling
to H2-production in green algae
Chlamydomonas reinhardtii
Advantage : Very high activity of FeFe-type H2ase (TOF ≤ 10,000 s-1)
Problems :
- Anaerobic conditions (due to O2-sensitivity of H2ase)
reduce PS-capacity to about 5 %
- Genetic manipulation of C. reinhardtii difficult
Cyanobacteria –
ideal design organisms
...in hot springs
•  Require fresh- or sea
water, inorganic nutrients,
air & (sun-)light
•  > 10.000 species known,
> 100.000 unknown
...on sand of deserts
•  Adapted to extreme
conditions
•  Easy mass culture &
genetic transformation
...on ice
•  Short generation time
Coupling PS with H2ase : Cyanobacteria
Ni-Fe H2ase
•  low activity, inefficient
(1% of FeFe-H2-ase)
•  too many steps
Synechocystis 6803
Coupling PS with H2ase : Green algae
FeFe-H2ase
•  most simple structure
•  ≤100-fold higher activity
than NiFe H2ase
•  direct coupling via Fd
Conclusion:
Cyanobacteria are ideal
design-organisms, but
with "wrong" H2-ase
Strategy
"Chassis"
"Engine"
McLaren-Mercedes
Eukaryote :
Chlamydomonas
reinhardtii
Prokaryote :
Synechocystis
PCC 6803
("Engine")
("Chassis")
"Design cell"
Strategy in detail (1)
WT cell
①
Optimization of
(existing) PS-ET
for H2 production
②
Coupling ET with engineered external H2ase
Cell engineering: Prerequisite for economical
bio-H2 production (acc. to LCA)
•  Present H2-production per L cell culture
(∼ 2 ml H2 L-1 h-1) has to be increased
by a factor ≥ 100
•  Continuous H2-production under aerobic
conditions (mass fermentation)
H.-J. Wagner
RUB
1) Optimisation of PS-electron transport
for H2-production
Phycobilisomes (PBS)
PBS antenna size reduction
•  ≤ 6-fold increased linear ET
due to increased PS2 / PS1
•  Saving metabolic energy
(PBS ≤ 60% of cell-protein)
•  ≤ 3-fold higher cell densities
•  ≤ 4-fold higher light intensities
with ≤ 4-fold higher ET
M. Broekmans G. Bernát
WT
Olive
PAL
Increase linear ET by uncoupling
ATP-synthesis (εΔC-mutant)
1)  Cell growth unimpaired !
2)  ΔpH < 20%, 2.5 x slower
3)  max. ET-rate ≈ doubled
(collab. T. Hisabori, Tokyo Institute of Technology)
Imashimizu, Bernát, Isato, Broekmans,
Konno, Sunamura, Rögner & Hisabori
(2011) JBC 286, 26595-26602
Engineering PS1-Fd-FNR-H2ase interaction
FNR
Biomass
(< 25 %)
T. Hase
G. Kurisu
(Osaka Univ.)
P. Liauw
Fd
H2ase
2) Coupling ET with engineered
"external" H2ase
2) Coupling ET with engineered
"external" H2ase
Prerequisites :
+
FeFe-H2ase expressed
in cyanobacteria :
> 500-fold H2-evolution
+
NiFe-H2ase MBH (R. eutropha) :
Structural reasons for O2-tolerance
(Fritsch et al. 2011 Nature)
(Ducat et al. 2011 PNAS 108)
FeFe-H2ase (Chlamy) : Design for O2-tolerance
Anaerobic work station in tent
Directed evolution combined with high throughput screening...
T. Happe
Combination of various mutants for design cell
OL-mutant
Δε-mutant
Design cell
FNR-mutants
Summary & outlook (design cell)
Mutants
antenna red.
Present gain
(factor LET)
Future
expectation
Conclusion:
>6 (PS2
)
uncoupling
CO2-fix.
O2-tolerance
4-5
10-100
>2
Factor >100 for H2-production is possible,
if all mutants are pooled in one design cell !
Strategy in detail (2)
Semiartificial system
Model organism
(cyanobacterium)
Design
proteins
Cellular system
Biobattery : Simple model system to
H2ase
maximize ET H2O
Electrodes....
O2
T. Kothe
H2
Design of Photobioreaktor
Development of a low budget Photobioreactor
LED
5 L Flat panel reactor (coop. KSD)
•  30% better surface/vol. ratio than tubular
•  Transparent polymer; chem. sterilisation
•  Invest. < 10 % of commercial reactors !
Continuous cultivation system
for optimization of culture conditions
batch
Control of
pH
T
CO2
O2
turbidity
light
media supply
(online control
by LabVIEW)
online
cont. culture
Illumination
pH
NaO
H
Turbidity
Media
Aeration Biomass
HCl
•  Steady state characterization & optimization of design cells
•  Long-term cultivation > 9 months successful
J.-H. Kwon
Monitoring cellular metabolism
by steady state proteome analysis
WT
Olive
Log2 (Olive / WT)
PAL
Log2 (PAL / WT)
Identification of >2,500 proteins (>70 % of all proteins)
to find bottlenecks = prerequisite for improvement
PBR-scaling up
20 x 5 L
(Coop. KSD, Hattingen)
100 L
Impact factors over life cycle (100 L PBR)
• 
Operation >99 % (19 % process materials, 80 % process energy)
• 
Production & disposal <1%
T-control 9 %
Cumulated
energy demand
(CED) –
operational phase
Sterilisation
30 %
CO2 14 %
Illumination
30 %
Conclusion:
Optimization potential in energy reduction for sterilization & illumination
H.-J. Wagner
Outlook
Future ?
Architecture with algae
(IBA 2013 Hamburg-Wilhelmsburg)
(BIQ & SSC, Hamburg)
Future : Bio-H2 as buffer-energy
in energy-mix?
Wind
PV
(collab. K. Hakamada / J. Miyake, Osaka Univ.)
Bio-H2
H2
H 2O
Blue-green biotechnology !
Acknowledgements RUB
Adrian Badura
Gábor Bernát
Martin Broekmans
Marta Kopczak
Tim Kothe
Jong-Hee Kwon
Pasqual Liauw
Marc Nowaczyk
Sascha Rexroth
Julia Sander
Nadine Waschewski
Katrin Wiegand
T. Happe (Photobiotechnology)
H.-J. Wagner (LEE)
W. Schuhmann (Analyt. Chem.)
Ext. coop. & funding
HU Berlin
KSD Innovations com.
B. Friedrich
O. Lenz
Osaka University
T. Hase
G. Kurisu
MPI MH
W. Lubitz
W. Gärtner
Univ. Köln
A. Berkessel
DFG
BMBF
Tokyo Institute Technology
T. Hisabori
M. Imashimizu
EU
Gründung eines Interessenverbundes.....
Solar Biofuels Ruhr
Fraunhofer
UMSICHT
Ruhr-Universität
Bochum
MPI für Bioanorganische
Chemie
Fa. KSD

Hydrogen from water : Engineering photosynthetic metabolism for a

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