olaf kruse algae biotech group - lorentz center bubbler photoautotrophic ... considering the light...
TRANSCRIPT
Hydrogen production withHydrogen production withChlamydomonasChlamydomonas
Olaf Kruse Algae Biotech Group
University of Bielefeld University of Queensland University of Karlsruhe University of Münster
Olaf Kruse Ben Hankamer Clemens Posten Michael Hippler
Our approachppEffizient use of Sun light energy(2.73 YJ pro Jahr worldwide, ~5500x of the global energy consumption)
Use of molcular biology foroptimisation of algae biomass production and its use
( p g gy p )
for biofuels production and valuable products in a biorefinery concept
G l t ti l d t f l f biGeneral potential advantages of algae for bioenergy:a) Biomass contains no Ligno-cellulose, but high amounts of lipid, protein and starch)
b) Production can occur on non-arable land
c) Low water requirement) q
d) Up to 30 times higher biomass yields per ha land mass
e) Easy to genetically engineer C. reinhardtii
Projects:Projects:Projects:Projects:
Creating a mixed fermentative/photosynthetic Creating a mixed fermentative/photosynthetic g p yg p yHH22 production systemproduction systemOptimizing light captureOptimizing light captureOptimizing light captureOptimizing light captureIncreasing starch degradation processesIncreasing starch degradation processesIncrease cultivation efficiencyIncrease cultivation efficiencyU f lt tU f lt tUse of salt waterUse of salt waterUsing microalgae for Using microalgae for biomethanebiomethane productionproductiong gg g ppIdentifying valuable side productsIdentifying valuable side productsE bli hiE bli hi bi fibi fiEstablishing a Establishing a biorefinerybiorefinery systemsystem
Work in progressDevelopment of salt-tolerance
as % salt water
(seawater = 3.5% salinity)
Evan Stevens unpublished
INCOMPLETE FACTORIAL DESIGN SCREENINGS• to optimise factors involved in media and cultivation
MIXOTROPHIC CULTIVATION• Achieved 1 0g/L/day biomass dry weight (batch average)• Achieved 1.0g/L/day biomass dry weight (batch average)
• Achieved 2.4g/L/day biomass dry weight (on best day)
• Maximum total biomass = 4.5g/LConditions:C reinhardtii cc124 in TAP media with 5x normal acetate
l
Conditions:C.reinhardtii cc124 in TAP media with 5x normal acetate volume microinjected over cultivation period and pH stabilised
Ph t t t hipressure regulator
0.2μm filter
autoclavableair bubbler
Photoautotrophic CultivationNew research is focused
th ti i ti fflow regulators
air bubbler upon the optimisation of cultivation using CO2 as the only carbon source(A) prototype gas manifold
A B C D
(B) lab scale bioreactor
(C) gas injection
(D) active photoautotrophic culture
1516
89
101112131415
g/L] Zugabe
von 10x
2345678
BTM
[g von 10x
TAP-Medium
Biomass increases until
01
0 100 200 300 400 500 600 700 800 900 1000Zeit [Stunden]
For plate reactor AR / AG ≈ 1 / dRdR ≈ LP ~ cX
Biomass increases untildistal side is nearly dark
Hydrogen PathwayHydrogen Pathwayy g yy g y
Light energy can drive metabolite and biofuel production
Hydrogenase
FerredoxinFerredoxin2H2O O2↑ + 4H+ + 4e-
4H+ + 4e- 2H2↑
Photosystem II
2
N t ti yNet reaction2H2O 2H2↑ + O2↑
Focus: Economic solar-powered H2 production from H2O using engineered green algal cells
S lS l
2 g g g g
Solar EnergySolar
Energy
Salt Solar Energy 2 H + O
Water Desalination
++2 H2O2 H2OAlgaeAlgae 2 H2O
gy
Fuel Cell
Marine Algae2 H2 + O2
Fresh2 H2O
++2 H2 O22 H2 O2
Fuel Cell2
Solar-driven H2 production discovered in algae in 1939 (H. Gaffron)
Hydrogenase is ~100x more efficient than known bacterial enzymes, but oxygen-sensitive
Major breakthrough: Two-phase process (A. Melis, 2000)>>> Separation of O2 producing & O2 sensible processes
Considering the light intensity used, corresponding to ~1,4% Energy Conversion Efficiency
If efficiency of 7 10% at high solar levels can be achieved thenIf efficiency of 7-10% at high solar levels can be achieved, then economically viable
The High-Hydrogen Producing Mutant Stm6 …
… the first step to increase H2 productionp 2 p
Which pathways are involved in the H2 production?
H2 production in Chlamydomonas is O2 sensitive. It allows the alga to survive under anaerobic conditions.
Olaf Kruse, Jens Rupprecht, Jan H. Mussgnug, G. Charles Dismukes and Ben Hankamer, PPS 2005
g
PSII is down PSII is down
Stm6 - Properties
Cyclic electron transfer isCyclic electron transfer isregulatedregulated
Cyclic electron transfer is blockedCyclic electron transfer is blocked
Less oxygen productionLess oxygen production No competition with linear e- transferNo competition with linear e- transfer
PhotosynthesisPhotosynthesis
Olaf Kruse, Jens Rupprecht, Klaus-Peter Bader, Peer Martin Schenk, Giovanni Finazzi, and Ben Hankamer, JBC 2005
300Hydrogen production Chlamydomonas reinhardtii strains
200Glc4cc503cc406[m
l/l]
100
cc406cc137cc125NPQ4
-Pro
duct
ion
[
0
H2-
0 24 48 72 96 120 144 168time [h]
The next generation high H2 production g g 2 pstrain Stm6glc4
Stm6glc4
Hup1
Anja Döbbe, Julia Beckmann, Jens Rupprecht, Armin Hallmann, Ben Hankamer, Olaf Kruse, J.Biotech. 2007
Cloning of a glucose transporter for externalsubstrat supply of H2 production
1mM glucose supplement
Stm6glc4 + 1mM glucose
supplement causes a 50% increase
In H2 production
Stm6glc4
Anja Döbbe, Julia Beckmann, Jens Rupprecht, Armin Hallmann, Ben Hankamer, Olaf Kruse, J.Biotech. 2007
Increasing light capture efficiencyIncreasing light capture efficiency
Th j bl f li ht tt tiThe major problems of light attenuation
Light limitationLight limitationor starvationIncident light time
dependent
Light saturationor inhibition Low concentrations,
mutual shadingmutual shading,turbulences
Improving the light to biomass efficiency
MEMelisBERKELEY
Normal system: Up to 95% of energy wasted as heat and fluorescenceEngineered cell lines: Energy wastage through fluorescence largely eliminated
(Mussgnug et al 2007 Plant Biotech J)
LHCII proteins
• most abundant integral membrane proteins on earth
• nine individual isoforms in C. reinhardtii (LHCBM1-6, 8, 9, 11)
• nucleus encoded
Di i f i f h i f d i f h i hi h• Distinct functions for each isoform despite of their highhomology?
Overcoming energy loss by optimizing the LHC antenna
110,0
RNAi of LHCII mRNA
80,0
90,0
100,0
S (%
Stm
3)
50,0
60,0
70,0
ance
rel.
18S
20,0
30,0
40,0
RN
A a
bund
a
0,0
10,0
,
1-1
1-2
1-3
1-4
1-5
1-6
1-7
1-8
1-9
cb4
cb5
m1
m2
m3
m4
m5
m6
m8
m11 8S
mR
Lhc
Lhc
Lhc
Lhc
Lhc
Lhc
Lhc
Lhc
Lhc
Lhc
Lhc
Lhcb
Lhcb
Lhcb
Lhcb
Lhcb
Lhcb
Lhcb
Lhcb
m 1
Mussgnug et al 2007 Plant Biotech J
EACH LR3 CELL ABSORBS AND WASTES LESS LIGHT
Mussgnug et al., 2007
BETTER LIGHT PENETRATION
LOW FLUOR. LOSSES
t lhl l 60S
Mussgnug et al., 2005 NAB1, an LHC translation controllercytosol
P700*
Fd
chloroplasthν
NAB1
60S 40S
PQPSII
PSI
Cyt b6fPC
P680*
P700e-NAB1
repression oftranslation
2 H2O O2
P680
LHCBM protein?
t anslation
2 2
80S
PQH2/PQ
lhcbm mRNA
translation
LHCII (lhcbm)-transcription
PQH2/PQ
PQH2/PQ
LHCII mRNA-Pool
Isoform-selectivefine-tuning on the levelof translation by NAB1
nucleus
Q 2 Q of translation by NAB1
Overexpressiong a mutated (permanently active) version of theLHC translation repressor NAB1 to reduce antenna size
Mussgnug et al., 2005 Plant CellWobbe et al., 2009 under revision
Systems Biology on HSystems Biology on H productionproductionSystems Biology on HSystems Biology on H22 productionproduction
CoordinatedCoordinated transcriptomicstranscriptomics, , proteomicsproteomics andandmetabolomicsmetabolomicsCreationCreation ofof an AGILENT an AGILENT fullfull genomegenome chipchipandand istist useuse forfor differentialdifferential transcriptomicstranscriptomicsandand ist ist useuse forfor differential differential transcriptomicstranscriptomicsIsotope Isotope labelinglabeling andand GCGC--MS, MS, GCxGCGCxGC--TOFMS TOFMS andand NMR NMR studiesstudiesIsotopeIsotope labellinglabelling andand MALDIMALDIIsotope Isotope labellinglabelling andand MALDIMALDI
TranscriptomicsProteomicsWild type ProteomicsMetabolomics
Wild-typeCC406
• Transcriptomics• Proteomics• Proteomics• Metabolomics
MutantGlc4
T0 T1 T2 T3 T4 T5+s -s
RNA RNA RNA RNA RNA RNA
cDNA
L b l d
cDNA
L b l dLabeled cDNA
Labeled cDNA
Hybridisation on Microarray chipsy p
Fluorescence detection
QuantifyRed/Gree
Expression profiles
2 00
1,00
2,00
Glc4 T1
Glc4 T2
0,25
0,50Glc4 T3
Glc4 T4
CC406 T1
0,13
CC406 T1
CC406 T2
CC406 T3
0,03
0,06CC406 T4
CC406 T5
0,02CC406 T6
512
128
256
512 Glc4 T1
Glc4 T2
Gl 4 T3
32
64
128 Glc4 T3
Glc4 T4
CC406
8
16
32 T1CC406 T2CC406 T3
2
4
8 T3CC406 T4CC406 T5CC406
1
2
LHCSR1 LHCSR3 LHCBM9
CC406 T6
600
QY change during S deprivation in Glc4 and cc406
400
500
glc4 7
300
QY
x 10
00
glc4_7
100
200cc406_1
020 40 60 80 100 120 140
Time after S deprivation (h)
Some (e.g. Asp, Glu,Cys) amino acids were most abundant before hydrogen production
intermediates of cytric acid cycle were prevalent before hydrogen production
intermediates of glycolysis were most abundant during hydrogen production
GCxGC-TOFMS was established and confirmed many results of GC/MS
fatty acids increase during hydrogen production
Cut bands of interest ProteomicsMichael Hippler (Münster)
Tryptic digest
Analysis with LC-MS/MS
Identification ofpeptides with SEQUEST,
OMSSA and GPFOMSSA and GPF
Selection of Arg t i i tidSpectra count analysis containing peptides
Quantification with
Spectra count analysis
Quantification with LC-MS/MS
Arg 13C6/Arg 12C6
Using SILAC for comparative proteomics
AN/AR
g p pin Chlamydomonas reinhardtii
AN/AR
PsaD (3; 19)PsaB (1; 6)PsaA (1; 6)
ATPB (2; 17)ATPA (1; 8)
LhcbM6 (1; 2)LhcbM3 (1; 9)LhcbM1 (1; 4)
D2 (2; 6)CP43 (2; 17)CP47 (2; 12)
D1 (3; 20)PsaF (1; 5)
PsaD (3; 19)
158275 GGR (1; 5)168660 2Fe2S ferredoxin (1; 3)
166082 Chl27A (2; 8)172115 ChlL1 (2; 7)170911 HCP4 (1; 3)170988 HCP2 (1; 1)154962 PFL (4; 24)
170524 ADH1 (1; 5)LhcbM6 (1; 2)
Prot
ein
191668 ICL (1 3)154121 ICDH alpha subunit (1; 7)
157092 NADP/FAD dep. Oxidoreductase (1;4)170957 Putative AAT1 (1; 9)
YCF3 (1; 5)156523 PSII Luminal protein (3; 13)
160578 VIPP1 (1; 7)152426 Ca sensing (4; 17)
158275 GGR (1; 5)
167714 Transketolase-like (2; 10)170326 Putative TAL2 (3; 11)
172283 Fumerate reductase / succinate DH (1; 4)158171 CIS2 (1; 9)194915 CIS1 (2; 8)
168551 MAS (4; 26)191668 ICL (1; 3)
0 1 2 3 4 5 6
Relative Ratio
Labscale 20L bioreactor tests for biogas production from algae
Scheme of 20l-labscale reactors. 1. Thermostat for heating water; 2. sample port; 3. Substrate for fermentation; 4. Substrate injection; 5. stirrer; 6. Gas collection bag 7. Magnetic ventil; 8.gas counter; 9. gas
l i 10 C t R1 R10 R tanalysis; 10. Computer R1-R10: Reactors
454-Fast-Sequencing
1. Methanoculleus marisnigri2. Clostridium thermocellum3 Thermosinus carboxydivorans3. Thermosinus carboxydivorans
Genome Sequencer FLX