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43093-AEF
Solar Biohydrogen Production by Means of Biodiversity: A Biophysical Approach

Derrick R.J. Kolling, Princeton University

Impact of research

The current project is focused on using biophysical tools to capitalize on biodiversity in the search for novel photosynthetic hydrogen producers. The approach is two-tiered and consists of locating novel hydrogen-producing phototrophs and then using molecular spectroscopy to investigate the unique properties of the organisms, specifically focusing on the two enzymes central to solar biohydrogen production, Photosystem II (PSII) and hydrogenase. In addition to this work, biophysical techniques have been developed to answer fundamental questions about the chemical mechanism of biological water oxidation; specifically, the reversibility of the terminal reaction of water oxidation has been addressed

Bioprospecting

Screening of cyanobacteria and algae from culture collections and field samples for high photosynthetic efficiency and hydrogen production is ongoing. A high-throughput screen has been implemented using a time-resolved fluorescence imager to determine photosynthetic efficiency and a Pd/WO film is being used to colorimetrically detect hydrogen production (1). To date, 350 strains have been screened and 32 were found to have relatively high relative variable fluorescence yield (F_v/F_m > 0.5), which is proportional to quantum efficiency of charge separation. These organisms are currently being screened for hydrogen production. A candidate of particular interest is Arthrospira maxima, which has the fastest water-splitting rates observed in vivo (using fast-repetition-rate fluorometry) and evolves hydrogen as a by-product of fermentation (2).

Molecular Studies

The role that bicarbonate plays in water splitting is being investigated with high-resolution electron paramagnetic resonance (EPR) spectroscopy. Answering this question is important not only for understanding the fundamental chemistry of water splitting, but also because, depending on the interaction, one may use removal or addition of bicarbonate as a molecular "switch" to turn photosynthesis off or on, respectively. It is believed that this will be particularly fruitful in A. maxima, in which PSII activity has been shown to be dependent on bicarbonate presence by FRRF (3). Two-pulse electron spin echo envelope modulation (ESEEM) was used to probe for the presence of bicarbonate. This work was completed using PSII-enriched membranes from Spinacia oleracea. Similar experiments are planned for PSII isolated from A. maxima. Several approaches were undertaken to deplete bicarbonate from the sample medium and/or exchange bound bicarbonate with isotopically labeled bicarbonate (NaH¹³CO³). To look for possible magnetic coupling between the manganese cluster and ¹³C of the isotopically labeled bicarbonate in the samples, two-pulse ESEEM was performed. When compared to simulations, no detectable modulations from ¹³C were found in the spectra. This finding helps limit the possible role of bicarbonate in S. oleracea PSII function to proton shuttling and/or stabilization of the protein matrix. PSII centers from A. maxima have been isolated, but show little, if any, activity. Trials in which the centers are isolated in the presence of protein-stabilizing agents, such as glycerol and glycine betaine, are ongoing.

Biophysical Studies

The effects of elevated O₂ pressure on the production of O₂ in photosynthetic organisms and on damage to the two photosystem complexes were investigated in several examples of intact algae, cyanobacteria, and plants. We determined that the O₂-evolving reaction of PSII occurs unabated at O₂ pressures at least 50-fold above current atmospheric pressure, in contradiction to recent reports using isolated PSII enzymes and PSII-enriched membranes (4,5). Hence, O₂ pressure does not prevent O₂ production by photosynthesis. Rather, increasing O₂ pressure is shown to accelerate damage to PSII caused by light-induced production of reactive-oxygen species (ROS) formed upon photoreduction of dissolved O₂ by Photosystem I (PSI). This PSI-mediated damage causes loss of O₂ production by PSII.

Publications and Future Plans

The work funded by this grant has strengthened my desire to pursue an academic career with a focus on research that will contribute to the realization of solar-based alternative energies. In addition, I have mentored or co-mentored six undergraduate students on projects related to the research discussed here. This has given me the chance to hone my teaching and advising skills, which will be invaluable for managing a laboratory. The biophysical work reported herein has been compiled into two manuscripts, which will be submitted by the year's end. Further work on bioprospecting and the role of bicarbonate in photosynthetic water oxidation will be submitted by the end of the academic year.

References

1. Carrieri, Kolling, Ananyev, and Dismukes. Prospecting for biohydrogen fuel. Industrial Biotechnology (2006) 2: 40--45.
2. Ananyev and Dismukes. How fast can Photosystem II split water? Kinetic performance at high and low frequencies. Photosynthesis Research (2005) 84: 355-365.
3. Carrieri, Ananyev, Brown, and Dismukes. In vivo bicarbonate requirement for water oxidation by Photosystem II in the hypercarbonate requiring cyanobacterium Arthrospira maxima. Journal of Inorganic Biochemistry (2007) (In Press)
4. Clausen and Junge. Detection of an intermediate of photosynthetic oxygen evolution. Nature (2004) 430: 480-484.
5. Clausen, Junge, Dau, and Haumann. Photosynthetic water oxidation at high O₂ backpressure monitored by delayed chlorophyll fluorescence. Biochemistry (2005) 44: 12775-12779.