NREL is a national laboratory of the U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, operated by the Alliance for Sustainable Energy, LLC.

Maria L. Ghirardi

Key personnel: 

M. Seibert, P. King, 

K. Ratcliff, 

S. Smolinski

National Renewable 

Energy Laboratory

2010 Annual Merit Review and Peer 

Evaluation Meeting, 10 June 2010, 

Washington, D.C.

NREL/PR-560-48068

Biological Systems for Hydrogen Photoproduction

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

Project ID # 37
National Renewable Energy Laboratory Innovation for Our Energy Future

Overview

Timeline

Budget

Barriers

Partners

Project start date: FY00

Project end date: FY18

Percent complete: N/A

Production barriers addressed

– Continuity of H2 production (AI)

– Feedstock cost in an integrated 

system (AT) 

– Rate of H2 production (AH)

Funding received in FY09: 

$800K

Funding allocated for FY10: 

$600K

Drs. Anatoly Tsygankov and Sergey 

Kosourov, Institute of Basic Biological 

Problems, RAS, Pushchino, Russia

Dr. Michael Flickinger, North Carolina State 

University

Dr. Eric Johnson, Johns Hopkins University

Drs. Iftach Yacoby and Shuguang Zhang, * New tasks in orange MIT
National Renewable Energy Laboratory Innovation for Our Energy Future

Objectives/Relevance

General: Develop photobiological and integrated 

photobiological/fermentative systems for large-scale H2

production.

• Task 1: Address the O2-sensitivity of hydrogenases, which prevents continuity of 

H2 photoproduction under aerobic, high solar-to-hydrogen (STH) conditions.

• Task 2: Utilize a limited STH H2-producing method (sulfur deprivation) as a 

platform to address other factors limiting commercial algal H2 photoproduction.

• Task 3: Integrate photobiological and fermentative systems in different 

configurations for less costly H2 production in the short term. 

Parameters Current Status 2013 Targets Maximum Potential

Duration of continuous

photoproduction

• Aerobic, high STH (O2-tolerant)

• Aerobic, limited STH (S-deprivation)

• Anaerobic, limited STH (S-deprivation)

0

10 days

90 days

30 min 12 hours

indefinite

indefinite

O2 tolerance (half-life in air)

• Oxidized conditions

• Reduced conditions

4 min

40 min

Cost ($/kg H2)

• Aerobic, high STH (O2-tolerant)

• Anaerobic, limited STH (S-deprivation)

• Integrated (photo + fermentative)

$2.99

$6.02

$3.21
Approaches:

• Use computational simulations to identify pathways by which O2 accesses 

the catalytic site and use site-directed mutagenesis to molecularly 

engineer the enzyme to prevent O2 access. 

• Use random methods to generate mutants with higher O2 tolerance.

• Introduce a more O2-tolerant bacterial hydrogenase into algae.

• Evaluate the feasibility of creating fusions between hydrogenases and 

ferredoxin to increase electron flux to the hydrogenase.

National Renewable Energy Laboratory Innovation for Our Energy Future

Task 1 – O2 Sensitivity/Rate of Hydrogenases

Objectives, Approaches, and Collaborations

Objectives: (1) Develop and optimize aerobic, high-STH photobiological systems 

for the production of H2 from water by engineering a H2-producing catalyst ([FeFe]-

hydrogenase) that has an extended half-life following exposure to O2. 

(2) Explore fusions between hydrogenase and ferredoxin to increase 

photosynthetic electron flow to the hydrogenase (this is unrelated to O2 sensitivity, 

but it addresses the rate of H2-production barrier). 

Collaborator: MIT (currently unfunded)
National Renewable Energy Laboratory Innovation for Our Energy Future

Task 1 – O2 Sensitivity of Hydrogenases

Accomplishments and Milestones 

1. Computational modeling: We 

extended analysis of pathways to 

[NiFe]-hydrogenases; identified 3 key 

residues as potential targets for 

mutagenesis to decrease O2 diffusion 

to catalytic site:Val67, Val110 and 

Leu115 in D. gigas. 

2. Site-directed mutagenesis: 

(a) We attributed the multiphasic kinetics of 

O2 inactivation to the existence of three 

states of [FeFe]-hydrogenases, each with 

different tolerance toward O2 (the reduced 

state is more O2-tolerant than the oxidized 

one; the third state is O2-insensitive); 

(b) we are re-assessing our strategy for 

controlling O2 diffusion to the catalytic site 

of [FeFe]-hydrogenases; a manuscript is in 

preparation (see future work).

Previous results showed that the clostridial 

H2ase is 100X more tolerant to O2 than the 

algal enzyme; re-directed resources toward 

expressing the clostridial hydrogenase in 

Chlamydomonas to assess the effect of a 

more O2-tolerant hydrogenase on H2

production in vivo (see next slide).

Task Due date Status

Use implicit ligand sampling method to map the 

pathways in [NiFe]-hydrogenases

January 2010 completed
National Renewable Energy Laboratory Innovation for Our Energy Future

Task 1 – O2 Sensitivity of Hydrogenases

Accomplishments and Milestones 

3. Random mutagenesis: No new results to report.

4. Expression of the clostridial hydrogenase in Chlamydomonas: Inconclusive 

activity results with one transformant; evaluation of additional transformants 

show expression in Chlamydomonas; activity is being evaluated.

5. Create fusions between hydrogenases and 

ferredoxin to improve reductant flux to the 

hydrogenase: We simulated the docking 

between the Ca1 H2ases with the algal ferredoxin

to guide MIT’s engineering efforts. Results 

suggest that the interaction could be facilitated if 

the clostridial hydrogenase were truncated, to 

reposition the N-terminus for fusion with Fd.

Task Due date Status

Use computational modeling to design fusions 

between [FeFe]-hydrogenases and ferredoxin

December 

2009

completed

Create genetic constructs of CaI and PetF (by MIT) March 2010 completed

N

C C

N

Models of docking between complete 

(left) or truncated (right) Ca1 H2ase 

with algal ferredoxin.

Task Due date Status

Demonstrate that CaI is active in C. 

reinhardtii

February 2010 Inconclusive;

postponed

Measure the O2 sensitivity of H2ase 

activity in C. reinhardtii transformants

April 2010 In progress
National Renewable Energy Laboratory Innovation for Our Energy Future

Task 1 – O2 Sensitivity of Hydrogenases

Future Work

1. Computational simulation: We will compare the geometry and energetics of the 

catalytic center and adjacent structures of [FeFe]-hydrogenases with different 

sensitivity to O2. We are re-assessing how O2 accesses the enzyme’s catalytic 

center and to what extent this depends on channel structure/configuration. 

2. Site-directed mutagenesis: A manuscript will be submitted summarizing current 

observations regarding the redox states effects on H2ase O2 inactivation; the 

approach involving gas channels is on hold until expression studies clarify whether 

higher hydrogenase O2 tolerance as measured in vitro translates into higher O2

tolerance in vivo. 

3. Random mutagenesis: New personnel are being hired to restart the research. We 

will determine a new strategy based on new results from Subtask 1. 

4. Expression of clostridial hydrogenase in Chlamydomonas: We will characterize 

additional constructs and, if required, design new Ca1 constructs or alternative 

approaches to increase H2 production in vivo. 

5. Hydrogenase/ferredoxin fusions: NREL will continue to provide guidance to MIT’s 

work and will test their transformants in house if additional funding is available.
National Renewable Energy Laboratory Innovation for Our Energy Future

Task 2 – Sulfur-Deprivation Platform

Objectives, Approaches, and Collaborations

Objectives: Further optimize and utilize an anaerobic, limited-STH 

working platform to study biochemical and engineering factors that affect 

H2 photoproduction by biological organisms; focus on the effect of an 

inactive, leaky ATP synthase on the rates.

Approaches: 

• Continue to improve the H2-production yields by alginate-immobilized algae 

RAS).

• Test and optimize the performance of immobilized, photoautotrophic cultures 

(RAS). 

• Generate inducible ATP synthase mutants and test them with the immobilized 

system. 

Collaborators: Johns Hopkins University, the Institute of Basic Biological Problems, 

Russian Academy of Sciences (RAS)

Photosynthesis

(light)

Anaerobic 

glycolysis

Aerobic 

respiration 

sugar

O2

Sulfur replete

Anaerobic 

glycolysis

respiration

sugar

CO2

Sulfur depleted

Formate, 

acetate

CO2 + H2O

Photosynthesis

(light)

H2

Aerobic O2

H2O
1. Improve H2 rates and yields using immobilized films: Lower 

thickness improves rates and yields; higher thickness improves protection 

against O2 inactivation under aerobic conditions and prevents acetate 

diffusion.

2. Test and improve the performance of photoautotrophic, immobilized 

cultures: No results to report; work just getting started.

National Renewable Energy Laboratory Innovation for Our Energy Future

Task 2 – Sulfur-Deprivation Platform

Accomplishments and Milestones

Film 

thickness, 

μm

Total Chl 

concentration, 

μg/cm2 film

Maximum 

specific rate of 

H2 production in 

argon, μmole mg 

Chl-1 h-1

Maximum 

specific rate of 

H2 production in 

air, μmole mg 

Chl-1 h-1 (% of

rate in argon)

Total yield of 

H2 gas in 

argon, mol m-2

Total yield of 

H2 gas in air, 

mol m-2 (% of 

rate in argon)

180 71.37 13.5 3.4 (25%) 0.55 0.094(17%)

260 101.6 7.8 2.8 (36%) 0.43 0.096 (22%)

290 117.74 6.1 2.6 (43%) 0.42 0.113 (27%)

310 110.89 5.9 2.3 (39%) 0.41 0.093 (23%)
National Renewable Energy Laboratory Innovation for Our Energy Future

Task 2 – Sulfur-Deprivation Platform

Accomplishments and Milestones

3. Design ATP synthase conditional mutants: A C-terminus-mutated εsubunit of the ATP synthase will be expressed in the chloroplast of 

Chlamydomonas behind a promoter that induces expression upon 

anaerobiosis. Specific mutations have been identified and transformants 

are being screened in an immobilized environment.

∆6

Site-directed alteration of the Cterminus to remove positive charges 

should further stimulate H2.

Task Due date Status

Design ATPase conditional 

mutants

December 2009 completed

Test immobilized ATPase 

mutants under sulfur-deprived 

conditions

August 2010 completed
National Renewable Energy Laboratory Innovation for Our Energy Future

Task 2 – Sulfur-Deprivation Platform

Future Work

1. Improve H2 rates and yields using immobilized films: Test the effect of 

the volume of the photobioreactor’s headspace on the H2-production 

properties of algal cultures.

2. Test and improve the performance of photoautotrophic, immobilized 

cultures: Adapt and improve on the methods previously used to induce 

photoautotrophic cultures to produce H2 in the absence of added acetate.

3. Construct and test the performance of Chlamydomonas inducible 

transformants carrying a leaky ATP synthase ε-subunit gene: 

Transformants will be tested for growth, photosynthetic activity ,and H2

production capability.
National Renewable Energy Laboratory Innovation for Our Energy Future

Task 3 – Integrated Systems

Objectives, Approaches, and Collaborations

Objectives: Integrate photobiological with fermentative organisms to more 

efficiently utilize the solar spectrum and the substrates/products from each 

reaction for H2 production.

Green algae

Photosynthetic bacteria

H2

Fermentative

bacteria

CO2 acetate

and other organic acids

algal biomass

H2

H2O

CO2

Approaches: 

• Integrate sulfur-deprived, alginateimmobilized algal H2 production to 

fermentative H2 production by an anaerobic 

consortium isolated from wastewater sludge.

• Integrate fermentative H2 production from 

potato waste to photosynthetic H2 production 

by anaerobic, purple non-sulfur bacteria 

(RAS).

Collaborator: Institute of Basic Biological Problems, RAS
National Renewable Energy Laboratory Innovation for Our Energy Future

Task 3 – Integrated Systems

Accomplishments and Milestones

1. Complete small-scale experiments on fermentability of algal biomass 

feedstock by the anaerobic consortium: The consortium ferments algal biomass with 

a molar yield >4 , which suggests that other cell components are being utilized.

2. Optimize fermentative H2 production

from potato waste.

Factors that increase rates/yields: exclusion 

of ammonium, addition of Fe ions, peptone 

and zinc; high phosphate buffering capacity; 

best yield: 1.6 mol H2/mol glucose. 

3. Demonstrate sequential H2

production from integrated dark 

and light-driven processes.

Maximum demonstrated yield from 

sequential process using potato waste 

as feedstock is 5.6 mol H2/mol 

glucose.

Task Due date Status

Determine the fermentability of 

alginate films

March 2010 completed

Design and test connections 

between fermentors and 

photobioreactors

March 2010 completed

Report on the carbon mass 

balance and H2 yields of a scaledup fermentative system

September 2010 In progress

Biomass mol H2/mol glucose

(from starch)

mg glucose (from 

starch)/100 mg 

biomass dry wt

μmol H2/mg 

biomass dry wt

142h-S (fresh) 1.86 8.7 0.60

142h-S (frozen) 2.11 3.5-8.7 0.64

+S (frozen) 6.30 1.9 0.52

Feedstock mol H2/mol feedstock μmol H2/mg 

feedstock

Lipid 0.09 0.20

Protein 6.56 0.10
National Renewable Energy Laboratory Innovation for Our Energy Future

Task 3 – Integrated Systems

Future Work

1. Scale up and further optimize fermentation of suspended 

and immobilized algal biomass by the fermentative 

consortium using new fermenters.

2. Optimize the integration of the fermentative/photobiological 

H2-production system using potato waste as the feedstock.
National Renewable Energy Laboratory Innovation for Our Energy Future

Summary

Task 1: 

• Extended the computational modeling techniques used to identify gas diffusion to the 

Desulfovibrio gigas [NiFe]-hydrogenase.

• Confirmed that the reduced state of the [FeFe]-hydrogenase is more tolerant to O2 in 

vitro than the oxidized state.

• Identified positive Chlamydomonas transformants containing the Ca1 hydrogenase 

gene.

• Simulated fusions between the petF ferredoxin and algal/clostridial hydrogenases to 

test optimal interactions.

Task 2:

• Observed that increased thickness of the alginate film improves O2 tolerance but 

decreases H2-production rates.

• Designed ATP synthase inducible mutants.

Task 3:

• Demonstrated that an anaerobic clostridial consortium ferments algal biomass, pure 

algal lipid,s and pure proteins.

• Optimized fermentative H2 production from potato waste.

• Demonstrated sequential H2 production from dark- and light-driven processes.
National Renewable Energy Laboratory Innovation for Our Energy Future

Supplemental Slides
Responses to Previous Year’s 

Reviewer Comments

Reviewer’s comment: “The project milestones should be better defined; The near term milestones are 

inadequate for this project. The milestones should include some performance targets; there are no milestones 

for the high-volume screening process development; milestones should be added.”

Response: The new milestones (FY 2010) are now better defined, but it is much harder to set specific, 

quantitative performance targets for longer-term research projects, given their discovery nature. Actually, 

discoveries made in longer-term projects lead to redirection of projects. Regarding the high-volume screening, 

see next comment. 

Reviewer’s comment: “The team has been developing the high-volume throughput screening tests since 

2007. More details on their progress are needed; they have been working on developing high-volume 

throughput screening for several years and it is not clear what progress has been made. The challenges and 

progress should be better identified.”

Response: Due to budget uncertainties in the last two years, we elected to focus our work on the site-directed 

mutagenesis/expression approach. Moreover, the proposed new computational simulations may redirect the 

particular localized random mutagenesis approach that we chose to take.

Reviewer’s comment: “The team’s antenna and sulfur deprivation work seem very similar to what was done 

by Professor Melis at UC Berkeley.”

Response: The team collaborated with Professor Melis in the discovery of the sulfur deprivation approach and 

NREL subsequently optimized the process. We are now testing truncated antenna mutants generated at UC 

Berkeley in an attempt to integrate the different research areas in photobiology. Prof. Melis had not reported 

on the H2-production properties of his truncated antenna mutants before.

National Renewable Energy Laboratory Innovation for Our Energy Future
Responses to Previous Year’s 

Reviewer Comments

Reviewer’s comment: “...more details about pretreatments regarding the integrated system are needed.”

Response: No pre-treatment was required. The algal biomass was previously frozen or fed directly to the 

fermentor. We will be addressing the effect (if any) of pre-treatment next.

Reviewer’s comment: “The PI should identify areas that the partners collaborated more clearly. It is difficult to 

determine what was done by partners and what was done by NREL.”

Response: The collaborator’s contribution was more clearly delineated in this presentation.

Reviewer’s comment:”The project team’s future plans are the same as in previous years. Since progress has 

been made, it would seem reasonable to adjust the plans.”

Response: Two new projects were added in FY2010: examine fusions between hydrogenase and ferredoxin, 

and study the effects of a “leaky” ATP synthase on H2 production rates under sulfur deprivation. 

Readjustments in the direction of the molecular-engineering project have also been made to reflect results on 

the heterogeneity of redox states of hydrogenases.

Reviewer’s comment: “There is no indication, at this point, how this work could be scaled and no real 

understanding of how to increase the hydrogen production to a point that will be useful at scale.”

Response: The technoeconomic analysis performed by DTI has addressed some of those issues and did come 

up with a concept of a process for large-scale H2 production.

National Renewable Energy Laboratory Innovation for Our Energy Future
Reviewer’s comment: “The PI has reported making an impressive number of 

presentations – 19. This is over one conference a month, which is a lot of travel. Their 

resources and time would be better utilized if they limited their conference attendance to 

only the premiere conferences.”

Response: The team consists of 3 PIs; the presentations made this year were divided 

among them as follows: 7 presentations by Seibert, 2 by King, and 6 by Ghirardi. The 

number of invitations reflects the high standing of the PIs in the research community. 

National Renewable Energy Laboratory Innovation for Our Energy Future

Responses to Previous Year’s 

Reviewer Comments
National Renewable Energy Laboratory Innovation for Our Energy Future

Publications

Belokopytov, B.S., K.S. Laurinavichius, T.V. Laurinavichene, M.L. Ghirardi, M.Seibert, and A.A. Tsygankov. 

2009. “Towards the integration of dark- and photo-fermentative waste treatment. 2. Optimization of 

starch-dependent fermentative hydrogen production". Int. J. Hydrogen Energy, 34: 3324-3332.

Maness, P.C., J. Yu, C. Eckert and M.L. Ghirardi. 2009. “Photobiological hydrogen production – prospects 

and challenges”. Microbe 4: 275-280.

Seibert, M. 2009. "Applied Photosynthesis for Biofuels Production", in Photobiological Sciences Online (K. 

C. Smith, Ed.) Am. Soc. Photobiol. Website: http://www.photobiology.info/Seibert.html#TOP

Ghirardi, M.L., S. Kosourov, P.C. Maness, S. Smolinski and M. Seibert. 2009. “Algal H2 Production” in 

Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation and Cell Technology (ed. M.C. 

Flickenger), John Wiley and Sons, Inc., xx-xxx. 

Long H., P.W. King, M.L. Ghirardi and K. Kim. 2009. “Hydrogenase/ferredoxin charge-transfer complexes: 

effect of hydrogenase mutations on the complex associatio”n. J. Phys. Chem. A. 113:4060-7. 

English C.M., C. Eckert, K. Brown, M. Seibert and P.W. King. 2009. “Recombinant and in vitro expression 

systems for hydrogenases: new frontiers in basic and applied studies for biological and synthetic H2

production”. Dalton Trans. 45:9970-78. 

Ghirardi, M.L. and Mohanty, P. 2010.” Oxygenic hydrogen photoproduction – current status of the 

technology”. Current Science India, in press.

Laurinavichene, T.V., B.F. Belokopytov, K.S. Laurinavichius, D.N. Tekucheva, M. Seibert and A.A. 

Tsygankov. 2010. “Towards the integration of dark- and photo-fermentative waste treatment. 3. Potato 

as substrate for sequential dark fermentation and light-driven H2 production.” Int. J. Hydrogen Energy, 

in press.

Smolinski, S., S.N. Kosourov, P.C. Maness and M.L. Ghirardi. “Hydrogen production from the fermentation 

of algal biomass by a bacterial consortium isolated from wastewater sludge”. Submitted.

Tekucheva, D.N., T.V. Laurinavichene, M.L. Ghirardi, M. Seibert, A.A. Tsygankov (2010) "Immobilization of 

purple bacteria for light-driven H2 production from starch and potato fermentation effluents." 

Submitted.
National Renewable Energy Laboratory Innovation for Our Energy Future

Presentations

• Invited seminar at the CSIC Spanish National laboratory in Zaragoza, Spain, Apr 09 (Seibert).

• Presentation to the group of Dr. X. Zhang at MIT, Apr 2009 (King).

• Invited plenary talk at the Great Lakes Bioenergy Research Center (GLBRC) Hydrogenase Forum, May 

09 (Seibert). 

• Presentation at the American Society for Plant Biology meeting in Hawaii, Jul 09 (Ghirardi).

• Invited update on EERE BioHydrogen research at the U.S. Air Force Office of Scientific Research 

Annual Review Meeting, Aug 09 (Seibert).

• Presentation of the USA country report at the IEA Annex 21 Biohydrogen Experts Meeting in Jyväskylä, 

Finland, Sep 09 (Seibert).

• Invited presentation at the University of Washington, St. Louis, Sep 09 (Ghirardi).

• Invited presentation at the Rocky Mountain American Vacuum Society meeting, Denver, Sept 09 

(Ghirardi).

•Invited presentation at the Center for Revolutionary Solar Photoconversion meeting in Denver, Oct 09 

(Ghirardi).

• Invited presentation at the Fall Rocky Mountain Branch of the American Society for Microbiology in 

Denver, Nov 09 (Ghirardi).

• Oral presentation to the Solar Fuels 2009 Meeting, Sigtuna, Sweden, Oct 2009 (King).

•Invited presentation to the Microbiology Department, Colorado State University, Nov. 09 (Seibert). 

• Presentation at NREL’s Energy Bioscience Center monthly seminar, Jan 10 (Ghirardi).

• Presented USA country report at the IEA Annex 21 Biohydrogen Experts Meeting in Florence, Italy, 

March, 2010 (Seibert).

• Invited talk at the DiBA-UNIFI & ISE-CNR Workshop on BioHydrogen in Florence, Italy, March, 2010 

(Seibert). 
National Renewable Energy Laboratory Innovation for Our Energy Future

Other Activities

Ghirardi reviewed articles for Nature, Journal of Photobiology, Applied Environmental 

Microbiology, Resources, Energy and Development, International Journal of Hydrogen 

Energy, Bioresource Technology. 

Ghirardi reviewed proposals for EPSCoR, University of Padua, National Science Foundation, 

the Joint Genome Institute, ARPA, USDA, and DOE.

Ghirardi was nominated a Fellow of the Renewable and Sustainable Energy Institute (RASEI).

Ghirardi hosted Dr. Alex Bradel, Chris Yeager (SRNL), Patrice Hamel (OSU), members of the 

European Commission, Dr. Glaucia Souza (Brazil’s FAPESP).

Ghirardi serves as an advisor for the University of Tennessee’s, NSF-funded STAIR 

(Sustainable Technology through Advanced Interdisciplinary Research) Program.

Seibert was elected the new Operating Agent for the IEA/HIA Task 21 (Biohydrogen).
National Renewable Energy Laboratory Innovation for Our Energy Future

Critical Assumptions

Assumptions:

Molecular engineering of O2 accessibility to the [FeFe]- and [NiFe]-hydrogenases’ catalytic 

sites will improve O2 tolerance. This assumption was based on published reports 

demonstrating that significant changes in the O2 sensitivities of other FeS proteins 

(ferredoxin and the H2-sensing NiFe hydrogenase) were achieved solely by changing the 

accessibility of the FeS centers or catalytic sites to O2 by substitution of critical amino-acid 

residues. 

Since the clostridial hydrogenase is already more tolerant to O2 than the algal enzyme, we 

expect to see increased activity under aerobic conditions when we express it in 

Chlamydomonas. Successful expression will also allow us to start addressing other 

barriers (such as competition for reductant) in a more effective manner.

Immobilizing dense algal cultures in very inexpensive matrices will drive the light-conversion 

efficiency up, while contributing very little to (and perhaps lowering) the cost of the H2

produced by the system. 

Integrating photobiological with fermentative systems will contribute beneficially to the overall 

cost of biological H2 production if parameters such as biomass disposal are taken into 

consideration. This will be considered in a new technoeconomic analysis to be performed 

at the end of FY10.
Other Issues

Issues:

Inconsistent funding and the lack of understanding regarding the nature of the 

different O2-tolerant states of the hydrogenase has prevented us from further 

developing the site-directed mutagenesis approach. The expression of a more 

O2-tolerant clostridial hydrogenase in Chlamydomonas will give us a better 

handle on which enzyme characteristics are important for hydrogenase activity 

under O2-evolving conditions, thus allowing us to redesign our strategy and 

screening assays.

Preliminary evidence gained by comparing the structure of different hydrogenases 

suggests that factors beyond gas diffusion may contribute significantly to O2

sensitivity. We will further examine this through computational simulations, 

which may lead to a change in our mutagenesis strategy.

National Renewable Energy Laboratory Innovation for Our Energy Future