Improving Cyanobacterial Hydrogen Productionthrough Bioprospecting of Natural Microbial Communities by Ankita Kothari A Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Approved November 2013 by the Graduate Supervisory Committee: Ferran Garcia-Pichel, Chair Willem F. J. Vermaas Bruce Rittmann Cesar I. Torres ARIZONA STATE UNIVERSITY December 2013 ABSTRACT Some cyanobacteria can generate hydrogen (H2) under certain physiological conditions and are considered potential agents for biohydrogen production. However, they also present low amounts of H2 production, a reaction reversal towards H2 consumption, and O2 sensitivity. Most attempts to improve H2 production have involved genetic or metabolic engineering approaches. I used a bio-prospecting approach instead to find novel strains that are naturally more apt for biohydrogen production. A set of 36, phylogenetically diverse strains isolated from terrestrial, freshwater and marine environments were probed for their potential to produce H2 from excess reductant. Two distinct patterns in H2 production were detected. Strains displaying Pattern 1, as previously known from Synechocystis sp. PCC 6803, produced H2 only temporarily, reverting to H2 consumption within a short time and after reaching only moderately high H2 concentrations. By contrast, Pattern 2 cyanobacteria, in the genera Lyngbya and Microcoleus, displayed high production rates, did not reverse the direction of the reaction and reached much higher steady-state H2 concentrations. L. aestuarii BL J, an isolate from marine intertidal mats, had the fastest production rates and reached the highest steady-state concentrations, 15-fold higher than that observed in Synechocystis sp. PCC 6803. Because all Pattern 2 strains originated in intertidal microbial mats that become anoxic in dark, it was hypothesized that their strong hydrogenogenic capacity may have evolved to aid in fermentation of the photosynthate. When forced to ferment, these cyanobacteria display similarly desirable characteristics of physiological H2 production. Again, L. aestuarii BL J had the fastest specific rates and attained the highest H2 concentrations during fermentation, which proceeded via a mixed-acid i pathway to yield acetate, ethanol, lactate, H2, CO2 and pyruvate. The genome of L. aestuarii BL J was sequenced and bioinformatically compared to other cyanobacterial genomes to ascertain any potential genetic or structural basis for powerful H2production. The association hcp exclusively in Pattern 2 strains suggests its possible role in increased H2production. This study demonstrates the value of bioprospecting approaches to biotechnology, pointing to the strain L. aestuarii BL J as a source of useful genetic information or as a potential platform for biohydrogen production. ii ACKNOWLEDGMENTS This work would have not been possible without the mentoring of Dr Ferran Garcia- Pichel. I owe him my foundation and core values in science. His passion for science and critical thinking has inspired me tremendously. I am very thankful to him for his belief in me all through my PhD. I would like to express my deepest gratitude to my committee members; Dr Willem F. J. Vermaas, Dr Bruce Rittmann andDr Cesar I. Torres, for their immense support and guidance in helping me shape my project.I am also grateful to Dr Marty Wojciechowski and Dr Anne Jones for their comments and guidance on my project. I would like to thank all the past and present members of the Garcia-Pichel lab, who have played an important part in my scientific development, as peers, mentors and friends. I would like to specially thank Ipsita Dutta, my friend and part of the Biohydrogen project, who has been extremely helpful throughout the project. I would like tothank people associated with the Biohydrogen project, Cosmin Sicora, Doerte Hoffmann, Daniela Ferreira, Prathap Parmeswaran, Michael Vaughn and Juan Maldonaldowith whom I could discuss specific aspects of my project.I am also thankful to my labmates, Estelle Couradeau, BrandonGuida, Ana Giraldo,Ruth Potrafka, Yevgeniy Maruseko, Natalie Myers andEdgardo Ramirez who have been of great help with my project and also made the work atmosphere a lot of fun.I would like to thank Yvonne Delgado and Wendi Simonson for administrative help. And to all those that I have failed to mention, who made the manyyears of graduate school a gratifying journey, thank you! iii I am grateful to Brian Swetteand his family for supporting this research through the ASU President‟s Fusion Fund. I would also like to thank Dr Garcia-Pichel, Dr Rittmann and School of Life Sciences for financial support. Above all, I am thankfulto my father, Dr Sushil Kumar Kothari, whose hardwork and dedication to science continues to inspire me, my mother, Shashi Kothari, and sister, Snehita Kothari, for their immense love and support.I am thankful to all my friends who have been my pillar of support through my PhD, especially Nilotpal Chakravarty, Sriya Sanyal, Mayur Agarwal and Arpan Deb. iv TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES ............................................................................................................ x ABBREVIATIONS...........................................................................................................xii I. INTRODUCTION ........................................................................................................ 1 1. Biological Hydrogen Production ................................................................................ 1 1.1 Methods for hydrogen production......................................................................... 2 1.2 Methods for biological hydrogen production........................................................ 4 1.3 Promising methods: Rationale for choosing suitable method ............................. 14 2. Cyanobacterial Hydrogen Production ....................................................................... 17 2.1 Cyanobacteria as model for hydrogen production .............................................. 17 2.2 Cyanobacterial enzymes involved in hydrogen metabolism .............................. 18 3. Cyanobacterial Hydrogenases ................................................................................ 26 3.1 Cyanobacterial uptake hydrogenase ................................................................... 26 3.2 Cyanobacterial bidirectional hydrogenase .......................................................... 28 4. Approach Used in this Study .................................................................................... 49 Tables/Figures ............................................................................................................... 52 References ..................................................................................................................... 58 II. DIVERSITY IN HYDROGEN EVOLUTION FROM BIDIRECTIONAL HYDROGENASES IN CYANOBACTERIA FROM TERRESTRIAL, FRESHWATER AND MARINE INTERTIDAL ENVIRONMENTS. ....................................................... 87 Abstract ......................................................................................................................... 88 v CHAPTER ................................................................................................................ Page 1. Introduction ............................................................................................................... 88 2. Material and Methods ............................................................................................... 92 2.1 Sampling and isolation of strains. ....................................................................... 92 2.2 Cultivation and maintenance ............................................................................... 94 2.3 Molecular analyses .............................................................................................. 95 2.4 Standard assay for hydrogen production ............................................................. 96 3. Results ....................................................................................................................... 97 3.1 Diversity of the set of strains surveyed ............................................................... 97 3.2 Identity of the isolates and phylogenetic placements. ........................................ 98 3.3 Patterns of hoxH detectability ............................................................................. 99 3.4 Physiology of hydrogen production .................................................................. 100 4. Discussion ............................................................................................................... 102 Tables/Figures ............................................................................................................. 107 Supplementary Information ........................................................................................ 115 References ................................................................................................................... 116 III. POWERFUL FERMENTATIVE HYDROGEN EVOLUTION OF PHOTOSYNTHATE IN THE CYANOBACTERIUM LYNGBYA AESTUARII BL J MEDIATED BY A BIDIRECTIONAL HYDROGENASE. ......................................... 123 Abstract ......................................................................................................................