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Progress Report for Hydrogen, Fuel Cells, and Infrastructure

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3 Efficient Hydrogen Production Using Enzymes of the Pentose Phosphate Pathway Barbara R. Evans (Primary Contact), Jonathan Woodward (Retired), Hugh M. O'Neill (University of Tennessee, Knoxville), Eugene Pinkhassik (University of Memphis), and John P. Getty, Jr. P. , Oak Ridge, Tennessee Objectives • • • Cloning and expression of thermophilic pentose phosphate pathway enzymes Immobilization and stabilization of enzymes and cofactors for bioreactor development High stoichiometric yields of hydrogen from sugars derived from renewable resources using the optimized pentose phosphate pathway enzymes Approach • • • Isolate genes for thermophilic enzymes by polymerase chain reaction (PCR) and subclone into expression vectors for production in mesophilic host Employ and analyze immobilization methods for optimal performance Determine enzyme kinetics separately and in combination to optimize hydrogen production Accomplishments • • • Demonstration of the production of hydrogen by combination of enzymes from different thermophiles Isolation and subcloning of two key enzymes, glucose 6-phosphate dehydrogenase and 6phosphogluconate dehydrogenase, from the thermophile Thermotoga maritima Production of hydrogen by enzymes encapsulated in lipsomes Future Directions • • • Isolate, clone, and express the remaining pentose phosphate pathway enzymes Demonstrate hydrogen production with thermophilic pentose phosphate pathway enzymes and compare with mesophilic counterparts Improve liposome encapsulation and investigate additional immobilization methods Introduction another common source of glucose.

These two rates cannot be distinguished from each other at the 95% confidence level. FY 2002 Publications/Presentations 1. -C. F. " Applied Microbiology and Biotechnology, 57, pp. 751-756 (2001). 2. F. " Applied and Environmental Microbiology, 68: 2633-2636 (2002). 3. -C. F. " International Journal of Hydrogen Energy. In press (October 2002). 4. Wolfrum, Edward J. and Watt Andrew S. " Applied Biotechnology and Bioengineering, 98-100, pp. 611-625 (2002). 5. -C. " Lecture to the Graduate Program in Plant Physiology, Institute of Biological Chemistry, Washington State University (March 15, 2002) (Honorarium).

Photo-Oxidative Killing of Rx. gelatinosus 24 Hydrogen, Fuel Cells, and Infrastructure Technologies FY 2002 Progress Report Figure 6. Effect of inlet CO concentration on CO conversion in a 1-L bioreactor. Curve is model prediction (Wolfrum 2002). Figure 7. Hydrogen Production and Carbon Monoxide Uptake Rates vs. Total Cell Mass for Photosynthetic Bacterium Rhodocyclus gelatinosus CBS apparent rate constant increases up to approximately 80 pounds per square inch absolute (psia), after which no further increase in reactor productivity occurs.

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