ALGAL HYDROGEN PHOTOPRODUCTION

Maria L. Ghirardi and Michael Seibert

National Renewable Energy Laboratory, Golden, CO

Program Review Meeting Berkeley, CA

May 19-22, 2003

Relevance/Objectives

Develop Advanced, Renewable, Photolytic H2-Generation Technologies Based on Algal

Water-Splitting Processes

• Meet the technical challenges associated with the continuity of algal H2-production under aerobic conditions (Technical Barrier M);

• Contribute to reaching the mid-term target of continuous H2photoproduction at a utilization efficiency of 20% and a cost of $30/kg by 2010 (Table 4.1.6).

Renewable Hydrogen Production/Utilization

H2Fuelcell

Electricity

O2

Green algaeSunlight

H2O

Biochemical Pathways (light reactions)

PS I

FdH2ase

H2Chl antenna

CO2 starch

H+

Sun

ADP + P ATP

2H2O

PS II e-

Chl antenna

H+

O2

PQ-pool

OUTSIDE

e-MEMBRANE

INSIDE

H+H+

Approaches to Generate an O2-Tolerant, Algal H2-Producing

System

1. Separate O2 and H2-production either temporally or physically;

2. Engineer a hydrogenase that functions in the presence of O2.

FY00 01 02 03 04 05 10

1. Separate O2 and H2 production

Significant Past Results and Future Milestones

FY00-FY10

(1) 96 hours H2-production per batch operation cycle;(2) 240 hours of continuous H2-production at sustained rates;(3) 480 hours of continuous H2-production (Milestone) but at decreased end rates;(4) 500 hours of continuous H2-production at sustained rates;(5) 1500 hours of continuous H2-production at sustained rates.

(1) (2) (3) (4) (5)

1. Temporal Separation of O2 and H2 Production (batch system)

Phase I (growth)

Phase II (-S)

Light

Light

H2OStarch

O2

H2O

H2

O2

CO2

140120100806040200

400

300

200

100

0

Time, h

H2 gas

l

Physical Separation of O2 and H2Production (continuous system)

Time of hydrogen production, h0 50 100 150 200 250

Hyd

roge

n pr

oduc

ed, m

l

0

100

200

300

400

500

600

batch modechemostat mode

9 µg Chl/ml, 1.5 ml H2/ h

15 µg Chl/ml, 1.25 ml H2/h

Che

mos

tat

18 µg Chl/ml,9 ml H2/h

Limiting

sulfate

No sulfate

H2

• biomass

Photosynthetic growth Anaerobic H2

production

air in air out

• co-products (dyes, protein, antioxidants, nutritional supplements, pharmaceuticals, etc.)

• fermentation products (acetate, formate) This represents a major break-through!

Effects of Research Progress on Projected H2 Cost

Continuous photoproduction of pure H2 at a rate of 1.5 ml H2 gas/liter culture

•Current system hydrogen cost: $200/kg, down from $760/kg in FY00;•Projected hydrogen cost: $2.34/kg (land-based system), < $1.40/kg (ocean-based system).

FY00 01 02 03 04 05 10

Significant Past Results and Future Milestones

FY00-FY10

(1) (2) (3) (4) (5) (6)

(1) Cloned and sequenced the catalytic site of 2 algal hydrogenases;(2) Cloned and sequenced 2 algal hydrogenase genes;(3) Generated our first mutant with improved O2 tolerance (0.1% to 1% O2);(4) Generate 3 new mutants tolerant to O2;(5) Achieve 10% utilization efficiency in a theoretically integrated organism (with U.C.

Berkeley and ORNL);(6) Achieve 20% utilization efficiency in a physically integrated organism (with U.C.

Berkeley and ORNL).

2. Engineer the hydrogenase enzyme

2. Engineering the Hydrogenase for O2-Tolerance

Cloning of two algal hydrogenases

5'

Hyd A1PstI

3'

500 1,000 1,500 2,000 Bases

5' 3'

Coding sequence (Hyd A: 498aa, Hyd B: 505 aa)H-cluster cysteinesInsertion (Hyd A: 8 and 44 aa, Hyd B: 8 and 54 aa)

0

Chloroplast signal peptide (HydA: 56 aa, HydB: 61 aa)Hyd A enzyme’s NH2-terminus (previously described 24aa)

Hyd A2 NotI NotI

2,500

Engineering the Hydrogenase for O2-tolerance

Structural modeling of the two proteinsH2 channelH2 channel

C. reinhardtii HydA1 C. reinhardtii HydA2

Engineering the Hydrogenase for O2-tolerance

Generation of a H2-channel mutant

% O20 1 2 3 4

µmol

es H

2 x

mg

Chl

-1 x

h-1

0

20

40

60

80

100Wild-typeV240W

duc t

ion

Rat

es

HydA1

H2

Pro

% O20 1 2 3 4

Rel

ativ

e un

its

0

20

40

60Wild-typeV240WV240W

This represents a major break-through!

Interactions with Others• Papers: 7 published or in press; 3 submitted. Patents: 1 issued, 2 submitted.• A. Melis (UC Berkeley) and J. Lee (ORNL) – Team is coordinating efforts to

generate a small chlorophyll antenna mutant with non-limited rates of electron transport and an O2-tolerant hydrogenase.

• A. Rubin (Moscow State University) and A. Tsygankov (Russian Academy of Sciences, Pushchino, Russia) – collaborating on physiological and engineering studies of algal H2 production;

• T. Happe (U. Bochum, Germany) – collaborated on the cloning of the algal hydrogenases;

• D. Ahmann (Colorado School of Mines) – using gene shuffling techniques to generate O2-tolerant mutants;

• R. Schulz (Christian Albrechts University, Kiel, Germany) –studying the H2-photoproduction capability of different green algal species;

• W. Jacoby (University of Missouri) – examining photobioreactor engineering questions.

Plans and Future Milestones for FY04

• Separate O2 and H2 production:- maximize cell density in the continuous system to increase the volume of H2 gas collected (May 2004);- improve the efficiency of H2 photoproduction using immobilized algal cultures (July 2004).

• Engineer the algal hydrogenases:- Generate and test double mutants of the gas channel (August 2004)- Isolate algal hydrogenases for structural studies (September 2004)

Response to Last Year’s Panel• “Little evidence of practicality”; “not likely to be a source of reasonably price

hydrogen”; “mentions secret engineering/ economic study regarding practical (allegedly) application”; “it is difficult to know if biological hydrogen production will ever be a large scale source of hydrogen”; “see no useful purpose for this project”.

Economic analyses done for DOE by the NREL Analysis Team (Nov. 2002) indicate that, currently, the two-stage system is not economical. However, when operated with a small antenna size mutant (U.C. Berkeley), at the maximum theoretical electron transport rates (ORNL), and at optimized productivity (NREL), estimated prices of H2 are about $2.34/kg.

• “Results are not encouraging”; “the goal is not within sight and presumably unattainable in green algae”; “the holy grail is beyond reach”.

Our results this year serve as definitive proof that it is indeed possible to engineer an O2-tolerant hydrogenase by modifying O2 access to the catalytic site along the gas channel. This is a longer-term approach, and our milestone for the generation of an ideal organism is 2010.