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

Centre for Biotechnology at the University of Bielefeld

MICROALGAE: biomass for all biofuels

PHOTOSYNTHESIS

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

Systems Biotechnology for a Biorefinery concept ith i lwith microalgae

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Evan Stevens unpublished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

Chlamydomonas reinhardtii Stm6A hi h H d i i l- A high H2 producing microalga-

Stm6Stm6

WTWT

The Biochemistry Of Solar Powered H2 Production

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

Starch StoreStarch StoreStm6Stm6

WTWTWTWT

Provided by Uwe Kahmann

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

Stm6glc4T7gwith a more efficient sun light collection system

Julia Beckmann, Lutz Wobbe unpublished

T7 produces ~ 25% more H2 whenT7 produces 25% more H2 whenadjusted to equal cholrophyll content

WT

Stm6

Stm6glc4

Stm6glc4T7

What‘s next?

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

Harvesting points during H2 production

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)

80

100

60

40 C…Glc4

0

20

-20

0T1 T2 T3 T4 T5 T6

3 5

3

3,5

2

2,5

T0

T1

1,5

2 T1

T2

T3

T4

T5

0 5

1

T5

T6

0

0,5

Chl. a/b Glc4 2 Chl. a/b cc406 2

Compounds detected by GC/MS

120 detected compounds

48 identified metabolites by using references

584 peaks could be detected

• Nearly five times as many as with GC/MS (120 peaks)

Overlay of total ion current (TIC) chromatograms displayed as a contour plot

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

Bioenergy Consortium East-Westphalia

10.000L -plant

500L -plant

20L -plant

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

microalgae as a biomass source for biogas production

Combined H production and biogas fermentationCombined H2 production and biogas fermentation

454-Fast-Sequencing

1. Methanoculleus marisnigri2. Clostridium thermocellum3 Thermosinus carboxydivorans3. Thermosinus carboxydivorans

Genome Sequencer FLX

Th k !Thank you !