G. C. Dismukes - Princeton University

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Renewable Bio-solar Hydrogen Production from Robust Oxygenic Phototrophs

G. C. Dismukes

Princeton UniversityDepartment of Chemistry

Princeton Materials InstitutePrinceton Environmental Institute

Pacific Rim Summit on Industrial Biotechnology & Bioenergy

Honolulu January 11-13, 2006


PENNSTATE HAWAIIPRINCETON

Renewable Bio-solar Hydrogen Production from Robust Oxygenic Phototrophs

-BioSolarH2 TeamG. Charles Dismukes PU photosynthesis/engineeringEdward I. Stiefel PU enzymology/inorganicDonald A. Bryant PSU genetics/mol biology cyanosMatthew Posewitz CSM genetics/mol biology algaeEric Hegg UU enzymology/inorganicRobert Bidigare UH microbial physiology/ecology


Objectives•Screening for the right organisms, photosystems & hydrogenases•Down-regulation of competing biochemical pathways•Random & specific mutagenesis for higher H2 production•Genetically combining host phototrophs with optimal hydrogenases

Renewable Bio-solar H2 Production from Robust Oxygenic Phototrophs

screeningfield

genomic sequence around hox genes in S. platensis7098 bp

HoxE HoxF HoxU HoxY HoxHORF?

diaphorase moiety Ni-Fe hydrogenase moiety

Bam HI (1484)Eco RI (4977)Cla I (2981) Cla I (7047)Nco I (1099)

Nco I (3375) Nco I (6934)H2Oxygenic PhotosynthesisH2O

O2

genome

mutants

genes

GMOs

Achievements June 2005•Identified novel H2 photoproducers•Developed high throughput screen for H2•Built ultra-sensitive H2 detection instruments•Engineered mutants in a green alga

PayoffClean H2 from non-fossil source


Solar Bio-H2 Goals: Economic viability4

• ~10% efficiency at ambient light levels (eg., antenna deletion mutants)• Requires strict temporal or sptial separation of H2 and O2• Achieve current cost estimates for large scale bioreactors

4. Independent predictions by NREL and Thiess Engineering

Solar Bio-H2 Current Status

Chlamydomonas reinhardtii: two stage light + dark + S deprivation

Strain IncidentPhoton Conv. Eff. H2 rate ml (liter culture)-1 hr-1

• WT 1 ~ 0.1% 1.5 • LHC insertion mutant 2 (3-4 x expected) minimal H2 observed• stm6 mutant 3 ~1.5% (5 -13 x)

1. NREL team 20042. UCB team, Melis3. Univ. Bielfeld & Univ Queensland: Kruse & Hankamer, JBC (2005)


Instrumentation Development

Fast repetition rate fluorimeterPSII Quantum Efficiency

Metabolomics & Proteomics

Triple Quadrupole-TOF mass spectrometer & electrospray ion

Dissolved H2 & O2LED + Clark cells

Colonies on agar plate

NREL WO3 H2 gas sensor

Ultrasensitive cells for dissolved H2 & O2 with multi-wavelength light-emitting diode illuminator

Clark type Cells for dissolved O2 & H2

10-8 Molar H2 or O25 μL volumeSensitivity 50 femtomoles0.1 sec response time

Wavelength selective illumination via LEDs: blue, orange, red, infrared, white


-BioSolarH2

0 1 2 3 4 5 6 7

0

200

400

600

800

1000

hydr

ogen

& o

xyge

n co

ncen

tratio

n, re

l.u.

incubation time, hours

Oxygen

Hydrogen

on

off

Cells from Petri dish resuspended in TAP liquid medium.Anaerobic pre-incubation time 12 minPulse illumination via white LED at 130 μE m-2 s-1

Pulseslight/dark10s/90s

Photo-H2 and –O2 in WT Chlamydomonas reinhardtii CC124

G. Ananyev

Conclusions•Major Photo-H2•Minimal dark-H2•Co-production of H2 and O2•H2 yield is O2 limited

200 pulses


0 5 10 15 20

0

100

200

300

time, min

1 2 5 10 20 40 60 80

light pulseduration, s

Light pulses produce two kinetic phases of photo-H2 in C. reinhardtii WT

0 20 40 60 80

0

100

200

300

light pulse duration, s

hydr

ogen

evo

lutio

n (p

eak) Light H2

Light + Dark H2

0

50000

100000

integrated hydrogen evolution (area)

Photo-H2post light H2

Peak Photo-H2

Total AreaPhoto-H2 +Post light H2

PSI

e- acceptorpool

secondarye- acceptor

pool

?

H2H2-ase

?

slow

hν

Conclusion: •Two pools of photo-electron acceptors in PSI capable of light-induced H2 production

•Integrated H2 yield is linear with pulse duration competing pathways for e-/H+ consumption are either fixed or not present

on

off

NADP+?

Fd ?

G. Ananyev


-BioSolarH2

H2O PSII PQ PSI

Alternative e-

donore- acceptor

pool H2H2-ase

hνhν

0 10 20 30 40

0

50

100

0.2 hr

670 nmPSI + PSII0.2 hr

4 hr

730 nmPSI only

0.2 hr

H2

conc

entr

atio

n, re

l.u.

time, min

light on, 10 s pulses

670 nmPSI + PSII

4 hr

Anaerobic -O2pre-incubation

-Δhyd E/F Conclusions: • Photo-H2 & dark-H2 requires H2ase

genes hyd E/F• Maximum photo-H2 production

requires illumination of both PSI and PSII

• The major source of reductantrequired for photo-H2 comes from water via PSII turnover

• The minor PSI only pathway presumably involves PQ/other donor

G. Ananyev & M. Posewitz

Chlamydomonas reinhardtii cc124


Arthrospira (spirulina) maxima

Habitat: volcanic soda lakesAlkalophile, pH = 9.5-11

0.2-1.2 M carbonateNo N2 fixation systemFastest PSII turnover3-5x larger PQ pool vs algae

genes hox-EFUYH encodeNAD-dependent bidirectional H2ase No hup genes reported (uptake H2ase)


•≥50 ns pulses 0.5 W; 660 nm laser•≥20 µs duration pulse train at full light saturation•Programmable pulse flexibility•Fast sampling, 20 mHZ detection•Chlorophyll Fo, Fv, Fm, •cross section of PSII•QA

- oxidation kinetics

Princeton: Fast Repetition Rate Laser Fluorometer

10 msDark or

other light

≥ 20 μs

STF – single turnover laser flash train660 nm Variables to be measured

Fv/Fm = PSII quantum efficiencyPQ pool size

Ananyev & Dismukes, Photosyn Res. (2005)


PSII Variable Fluorescence at 100Hz flash repetition rate; Arthrospira p. whole cells

0 500 1000 1500 20000.46

0.48

0.50

0.52

0.54

0.56

0.58

Fv/F

m

flash number

B

Pe

Kautsky curve demonstrates transition from single turnover eventsin PSII to multiple electrontransfer in whole electrontransfer chain

Arthrospira sp. have a larger PQ pool capacity3-5X vs. green algae!

PSII-WOC PQ pool PSI

NADP+Rubisco

[CO2]

dark preincubation period

60 μs

single turnover flash cluster

10 msdark

20 sec


0 10 20 30 40 50

0.50

0.52

0.54

0.56

0.58

Fv/F

m

STF number

5 6 7 8 9 10

X5

Experiment

Fitting

1 10 100 10002

3

4

5

6

7

8

Q

ualit

y fa

ctor

, 1/(α

+β)

dark time between STF, ms

B

Chlorella

Spirulina

Turnover Frequency of PSII is highest by 5x for Arthrospira maxima

Kok parameters:photochemical misses (α) & double hit/misses (β)

PSII quantum efficiency ~54%

* Ananyev & Dismukes, Photosyn Res. (2005)

Kok α & β


0 2 4 6 8 10 12

0

100

200

300

400

hydr

ogen

con

cent

ratio

n, re

l.u.

time, hours

3

4

5

6

7

8

•Ni2+ addition to standard cyanobacteria growth media (Zarrouk) is essential for H2ase↑•3-4 days lag to fermentative H2 , yields transient reductant pool ( 2 or 3 peaks)•Second lag phase 1-2 days later, oscillations in H2•Culture survives for 10-11 days in batch culturing no additives•Microscopy shows that most active dark-H2 producing cells have no gas vacuoles

0 10 20 30 40 50

0.20

0.25

0.30

0.35

0.40

0.45

Fv/F

m

STF number

3

4

5 7

8

( ) g

Induction of dark H2 ↑(Left) & PSII quantum efficiency (Right) during photoautotrophic growth at 1.7 μM Ni2+ of A. maxima

Days of photoautotrophic growth prior to anaerobisis PQ pool

oxidized

reduced

Transient reductant pool

PSI + PSII Illumination by 640 nm red light produces additional H2

0 2 4 6 8 10 12

0

100

200

300

400

500

600

700

hydr

ogen

, mV

time, hours

0 - 12 h

12 - 24 h

Conclusions

•Dark (fermentative) and a light-induced pathways to H2 are present

These pathways share a common intermediate that is precursor to H2

Pulseslight/dark1s/99s

dark


PSI Illumination by IR 735 nm stops fermentative H2↑ & stores H2 precursor

0 2 4 6 8 10 120

200

400

600

800

hydr

ogen

, mV

time, hours

735 nm

0 - 12 h

12 - 24 h

delay 32 min

Fermentative H2 restoredin dark after IR light stoppedat 2x higher rate

0 2 4 6 8 10 120

100

200

300

400

hydr

ogen

, mV

time, hours

735 nm

1 s light/ 99 s dark 5 s light/95 s dark

cycle of dark H2

On Off

Dark H2

Light H2

H2 recovers

Dark H2 reductant is stored under IR illumination

2 x greater yield

IR Light suppresses dark H2


0 20 40 60 800

100

200

300

400

hy

drog

en, m

V

time, min

640 nm

735 nm

PSI illuminationSuppresses fermentative H2Slow photo-H2

PSI+PSII illuminationAdds to fermentative H2Fast photo-H2

A. maxima


Model for Dark and Light H2production by Arthrospira m.

PSIIH2O

O2

PQH2

PQPSI

FdFNR

NADP+

NAD-dependentNiFe-H2ase

glycogen

glucose

pyruvate

NAD+

NDHComplex I

H2

Sustained H2 production rates: representative reports of cyanobacteriausing water as electron donor

Synechocystissp 6803 mutant M55

Arthrospiraplatensis

Oscillatoriasp Miami BG7

Arthrospiramaxima

CyanothecePCC 7822

Our current results[nitrate in media]

In Dark

In lightvia hydrogenase

probably via nitrogenase

Est. rate of H2photoproductionin green algae C. reinhardtii under S deprivation(2005) NREL, UQ/UB

Removal of nitrate from media

30°C

23°


-BioSolarH2

Conclusions

•New tools for measuring H2, O2 and redox status provide reliable quantitative data on concentrations and fluxes

•greatly simplify and clarify the fundamental mechanisms of H2 production in microalgae and cyanobacteria

•Cyanobacteria & microalgae have different pathways to H2

•Ideal temporal separation of H2 and O2 production in cyanobacteria solves gas separation problem


-BioSolarH2

Eric Hegg, UUTyler Brown, UG06

Nick Benette, GS1Robert Bidigare, UHKelsey McNeely,GS1

Damian Carrieri,GS2Donald A. Bryant, PSUDr. Tuan Nguyen

Dr. Derrick KollingMatthew Posewitz, CSMDr. Gennady Ananyev

Edward Stiefel, co-ICo-InvestigatorsPrinceton

G. C. Dismukes - Princeton University

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