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Biological Systems for Hydrogen 

Photoproduction

2012 Annual Merit Review and 

Peer Evaluation Meeting

Maria L. Ghirardi, PI

National Renewable Energy Laboratory

May 16, 2012

NREL/PR-5200-54467

PD037

This presentation does not contain any proprietary, confidential, or otherwise restricted information
2 

Overview

Timeline

• Project start date: FY00

• Project end date: 9/30/2012*

• Percent complete: 80%

 *Project continuation and direction

 determined annually by DOE

Budget

• Total project funding: $9,951K

• Funding received in FY11: $750K

• Planned funding for FY12: $600K

Barriers 

• Barriers addressed:

 - Rate of H2 production (AH)

 - Continuity of H2 production (AI)

 - Engineering issues (AJ)

• Targets (see next page)

light conversion efficiency

rates of production

duration of production

Partners

• Dr. Sergey Kosourov, Institute of Basic 

Biological Problems, RAS, Pushchino, 

Russia 

• Dr. Eric Johnson, Johns Hopkins University 
3 

Relevance/Objectives

• General goal: Develop photobiological systems for large-scale, low 

cost and efficient H2 production from water (barriers AH, AI and AJ).

• Specific tasks:

Task 1: Address the O2 sensitivity of hydrogenases that prevent 

continuity of H2 photoproduction under aerobic, high solar-tohydrogen (STH) light conversion efficiency conditions.

Task 2: Utilize a limited STH H2-producing method (sulfur 

deprivation) as a platform to address or test other factors limiting 

commercial algal H2 photoproduction, including low rates due to 

biochemical and engineering mechanisms. 

Characteristics Units 2003 2006 2013 

Target

2018 

Target

Utilization efficiency of incident solar energy % 10 15 15 20 

Efficiency of incident light energy conversion 

of water to hydrogen

% 0.1 0.1 2 5 

Duration of continuous H2 photoproduction Time units NA NA 30 min 4 h

O2 tolerance (half-life in air) Time units 1 sec 1 sec 10 min 2 h
4 

Approach/Milestones – Task 1

Task 1: Address the O2 sensitivity of hydrogenase by (a) using targeted 

random mutagenesis to generate O2-tolerant hydrogenases; and (b) 

introducing the gene encoding for a more O2-tolerant hydrogenase from 

Clostridium acetobutylicum into the photosynthetic alga Chlamydomonas 

reinhardtii; measure its linkage to water oxidation and in vivo O2

tolerance.

Dr. Paul King, NREL Dr. Kath Ratcliff, NREL Dr. David Mulder, NREL

Task 1 Milestone Due date Status

3.3.2 Go/NoGo: Assess progress in the random 

mutagenesis approach to evaluate whether to further 

pursue this approach in FY12.

12/31/11 Completed: NoGo

3.3.5 Demonstrate expression of an active Ca1 in a C. 

reinhardtii hydrogenase-less background and 

characterize O2-sensitivity of light-driven H2

production.

9/30/12 80% completed
5 

Task 1 – Random mutagenesis – completion of milestone 3.3.2 – NoGo. 

 

We re-evaluated the effects of O2 on the H2-production activity of the more O2-tolerant 

Clostridium acetobutylicum Ca1 hydrogenase and concluded that (a) a targeted random 

mutagenesis effort would require more funding than currently available through the FCT 

Program; (b) we need to first validate that higher in vitro O2 tolerance translates into 

higher in vivo O2 tolerance. We decided, instead, to focus on the expression of Ca1 in 

Chlamydomonas reinhardtii (see next slide). 

 

Progress before the Go/NoGo milestone included: 

 (a) Development of a chemochromic

 high-throughput assay for H2 production in micro-wells, based on methyl viologen reduction by H2. 

(b) Selection of a E. coli strain lacking native

hydrogenase activity and expressing Ca1 at

high levels.

 

Accomplishments and Progress – Task 1

Strain

Ca1 whole cell activity (nmol

H2/min/mL)

Rosetta-2 (DE3) 1625

MC4100 (DE3) 1194

MW1001 (DE3) 173

Background control rate: 16 nmol H2/min/mL
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Accomplishments and Progress – Task 1

Task 1 – Expression of Ca1 in C. reinhardtii and measurement of in vivo O2

tolerance – towards completion of milestone 3.3.5.

Ca1

algal

Justification: the Ca1 hydrogenase is 2 orders of magnitude 

more O2-tolerant than the algal hydrogenase in vitro. 

Previous work: we successfully expressed Ca1 in a wildtype strain of C. reinhardtii that contained native 

hydrogenase activity; O2-inactivation kinetics were 

biphasic, suggesting the presence of a more O2-tolerant 

enzyme in combination with the native hydrogenases. 

PCR (genomic DNA) RT-PCR (mRNA) 
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Figure 1. PCR of genomic DNA from various 

Ca1transformants

Ca1 transformants

Ca1

Accomplishments and Progress – Task 1

Task 1 (cont.) – Expression of Ca1 in C. reinhardtii and measurement of in 

vivo O2 tolerance – towards completion of milestone 3.3.5.

 

Current Progress: A double hydrogenase knock-out mutant was isolated under BES 

funding and served as a host for expression of Ca1 behind the psaD promoter.

Meuser et al., Bioch. Biophys. 

Res. Comm. 417, 704

PCR (genomic DNA)

RT-PCR (mRNA) 

Light-dependent H2 

production was 

detected!

seconds

Relative units
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Task 2: Utilize the limited STH sulfur-deprivation method to test (a) the rates of 

H2 production by inducible ATP synthase mutants that are not limited by the nondissipation of a proton gradient; and (b) the long-term performance of 

immobilized algal cultures.

Approach/Milestones – Task 2

Dr. Eric Johnson Dr. Sergey Kosourov

JHU RAS

Task 2 Milestone Due date Status

3.3.3 Physiologically characterize mutant strains with a 

defective AtpE gene under the regulation of an 

inducible promoter

3/30/12 Completed

3.3.4

(CSP 

Agreement 

milestone 

51536)

Demonstrate continuous operation of the sulfurdeprivation process for a total of 2 months upon 

addition of phosphate/sulfate to alginateimmobilized cultures

9/30/12 50% completed
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Accomplishments and Progress – Task 2

Task 2 – Generate, physiologically characterize and test the rate of inducible 

ATP synthase mutants that are defective in maintaining the proton gradient –

completion of milestone 3.3.3.

 Justification: electron transport from water to 

 ferredoxin is accompanied by the formation of a 

 proton gradient that drives the 

 production of ATP (essential for CO2 fixation). 

 During H2 photoproduction, ATP demand 

 drops, impeding the efficient dissipation of the 

 proton gradient and impairing electron transport 

 (and thus of H2 production). 

 Previous results: C. reinhardtii mutants 

defective in one of the ATP synthase

subunits, AtpE (∆6, dark bars), grow 

more slowly but produce H2 at higher 

rates than the corresponding wild-type 

(cc124, light bars) strains, particularly at 

high light intensities.
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Task 2 (cont.) – Generate, physiologically characterize and test the H2-

production rate of inducible ATP synthase mutants that are defective in 

maintaining the proton gradient – milestone 3.3.3 completed.

Current progress: 

 (a) Generated six mutants in the C-terminal of the ATP 

 synthase subunit ε ; four showed similar rates of H2

 production as their parental strain but two, EJ13 and 

EJ17, were unable to grow constitutively.

(b) The defective EJ17 gene was transformed behind the PsbD promoter into strain 

Ind41_15D. The transformed gene was shown to be stably incorporated into the chloroplast 

genome and to allow growth of the transformant under photo or heterotrophic conditions in 

the presence of spectinomycin - completion of milestone 3.3.3. 

 

(c) The inducibility of the Ind41_15D/psbD system under selective pressure was positively 

demonstrated by using the expression of an orange fluorescent protein as a marker. 

 

 

Accomplishments and Progress – Task 2
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Accomplishments and Progress – Task 2

Task 2 – Demonstrate continuous H2 production for 2 months by sulfurdeprived, alginate-immobilized algae – towards completion of milestone 3.3.4.

 

Previous results: alginate-immobilized algae photoproduce H2 at higher specific rates and 

light conversion efficiencies than cultures in suspension upon sulfur deprivation, and they 

show higher tolerance to aerobic environments; cycles of +S/-S resulted in prolongation of 

H2 production for an additional 6 cycles of about 500 hours each.

 

 

Current results: preliminary results by using continuous flow of S+ medium resulted in low 

rates of H2 production; the experiment is being repeated under different operational 

conditions.

 

+S

-S 
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Accomplishments and Progress – Task 2

Task 2 – Demonstrate the effect of headspace volume on the H2

photoproduction yield.

Current results: In suspension cultures, a 4x 

increase (from ~0.5 to ~2) in the ratio of 

gas/liquid volume results in a 2x increase in 

the total yield of H2 gas. Similar results were 

shown with immobilized cultures.

Remarkably, 565 ml of H2 gas per liter of the suspension culture is the highest yield ever 

reported for a wild-type strain in a time period of less than 180 hours. A control PhBR

with a historically small gas phase volume of ~5 – 10 ml only produced up to 120 ml L-1 of 

H2 gas.
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Collaborations

Partners (subcontractors):

 - Dr. Sergey Kosourov, Russian Academy of Sciences – applies sulfur 

deprivation to sulfur-immobilized C. reinhardtii cultures and tests their H2-

production capabilities (Task 2).

 - Dr. Eric Johnson, Johns Hopkins University – generates ATP synthase 

mutants, develops transformation protocols and transforms 

Chlamydomonas reinhardtii; tests physiological properties of 

transformants (Task 2).
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Proposed Future Work

2012

Task 1 – Complete milestone 3.3.5: Demonstrate expression of an active 

Ca1 in a C. reinhardtii hydrogenase-less background and characterize O2

sensitivity of light-driven H2 production.

Task 2 – Complete milestone 3.3.4: Demonstrate continuous operation 

of the sulfur-deprivation process for a total of 2 months upon addition of 

phosphate/sulfate to alginate-immobilized cultures.

Work beyond FY12: Start to genetically express mutated ATP synthase

and truncated antenna regulatory genes into a strain expressing the Ca1 

hydrogenase. Develop photobioreactor systems for cyclic or continuous H2

production based on current optimization of gas space composition (not 

shown), alginate immobilization, and others.

 
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Summary Slide

Relevance: Photobiological water splitting coupled to hydrogenase-mediated H2 

production has the potential to convert about 10% of incident solar energy into H2. 

Various barriers have been identified as currently limiting green algal H2

production, including the O2 sensitivity of the hydrogenase enzyme, and

down-regulation of photosynthesis due to non-dissipation of the proton gradient. 
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Approach: NREL is expressing a more O2-tolerant bacterial hydrogenase in green algae 

and generating ATP synthase mutants that prevent down-regulation of photosynthesis 

with JHU. NREL and RAS use the low STH sulfur-deprivation process to test candidates 

that have been generated to address various barriers, as well as optimizing 

photobioreactor conditions for efficient and sustainable production of H2 gas.

Technical Accomplishments and Progress: 

1. Successfully expressed a bacterial hydrogenase in a Chlamydomonas strain lacking 

native hydrogenase activity and detected photoproduction of H2. 

2. Generated inducible ATP synthase mutants , tested for growth, and are testing their 

phenotype with respect to H2 photoproduction. 

3. Continues to optimize long-term H2 photoproduction by alginate-immobilized 

Chlamydomonas.

Collaborations: Drs. Sergey Kosourov (RAS) and Eric Johnson (JHU).

Proposed Future Research: For 2012: complete milestones 3.3.4 (demonstrate 

continuous H2 photoproduction by addition of sulfate) and 3.3.5 (measure the in vivo 

O2 tolerance of mutants expressing Ca1); measure the inducible H2 photoproduction

capability of the ATP synthase mutant. After 2012: genetically combine useful 

phenotypes into a single organism expressing Ca1.

Summary Slide (cont.)