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THE MICROBIAL COMMUNITY ECOLOGY OF VARIOUS SYSTEMS FOR THE CULTIVATION OF ALGAL BIODIESEL by Tisza Ann Szeremy Bell A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology MONTANA STATE UNIVERSITY Bozeman, Montana January 2017 ©COPYRIGHT by Tisza Ann Szeremy Bell 2017 All Rights Reserved ii DEDICATION This body of work is dedicated to my beloved best friend, the epitome of compassion and strength, the wild thing I never saw sorry for itself, who lived in love, and never let me quit - even in her absence. Ryan Marie Patterson January 6, 1983 – October 9, 2011 i carry your heart with me(i carry it in my heart)i am never without it(anywhere i go you go,my dear;and whatever is done by only me is your doing,my darling) here is the deepest secret nobody knows (here is the root of the root and the bud of the bud and the sky of the sky of a tree called life;which grows higher than soul can hope or mind can hide) and this is the wonder that’s keeping the stars apart i carry your heart(i carry it in my heart) -EE Cummings iii ACKNOWLEDGEMENTS I would like to thank family and friends for their love, support, and encouragement. I would be nowhere in this world without my amazing parents, Susi and Richard, my brother Devon, and my partner Brian Guyer, and my faithful Puli, Jack, who have loved me, commiserated with me, and tolerated me on my worst days. I am indebted to my late great-uncle, Tom Bell, the first black sheep, for his encouragement, faith, and inspirational life’s work through High Country News. I have had endless love and support from my aunt and uncle, Tunde and Dick Dwyer, my cousins Chris & Andy Szeremy, Ajna Akantisz, everyone in Hungary, and the whole of the Patterson clan especially, Uncle Billy, Mike, Marie, and Danielle. My dear friends Tracy Thomas, Flynn Murray, Karen Moll, Emily Wellington, Leila Sterman, Josiah Pisel, Julia Mead, Katie & Steve Breston, Andrea Hobbs, Kevin Clark, Mason Griffith, and Maria Blevens have given me endless strength and support. I am in debt to Julie and Jim Wellington for providing me sanctuary and solitude to write at one of my favorite places in the world, Longhorn Ranch. I am forever indebted to my lab-mates and my advisers, Brent Peyton and Matthew Fields, for their guidance and mentorship, and my committee members Anne Camper and Christine Formeman. I am especially thankful to Christine Foreman for convincing me not to quit my PhD on more than one occasion, to Sarah Codd for her mentorship, and Ann Willis for support that knew no bounds (ex: dumpster diving for samples at 11pm on a Sunday). I am grateful for the formatting prowess of Robin Kelley and to my lab mates past and present especially Kara DeLeon, Karen Moll, Muneeb Rathore, Todd Pedersen, Matthew Jackson, Jackson Herron, Katie Davis, Anna Zalaya, Chiachi Hwang, Heidi Schoen, Lakotah Doig, Emel Sen, Sarah Altenburg, and Jacob Valenzuela. iv TABLE OF CONTENTS 1. INTRODUCTION ...........................................................................................................1 Renewable Energy and Role of Algal Biodiesel ..............................................................1 Current Algal Cultivation and Lipid Augmentation Approaches and Challenges ..........4 Strain Selection and Biomass ...................................................................................4 Stress and Nutrient Deprivation ...............................................................................4 Photobioreactors ..............................................................................................................5 Genetic Engineering .........................................................................................................6 Prospective Methodologies to Further Maximize Lipid Potential ...................................8 Identification of Promising Regional Systems and Strains......................................8 Employing Ecologic Principals to Microbial Systems ............................................8 Diversity and Productivity ...........................................................................8 Niche Occupancy and Stability ....................................................................9 The Phycosphere ........................................................................................10 Improving Stability by Utilizing Alkalinity ...............................................12 Mass Production.............................................................................................................13 Water Limitations for Mass Production .................................................................14 Viability of Wastewater .........................................................................................15 Wastewater Nutrients .............................................................................................16 Conclusion .....................................................................................................................19 2. A LIPID-ACCUMULATING ALGA MAINTAINS GROWTH IN OUTDOOR, ALKALIPHILIC RACEWAY POND WITH MIXED MICROBIAL COMMUNITIES ...................................................................................22 Contribution of Authors and Co-Authors ......................................................................22 Manuscript Information Page ........................................................................................23 Abstract ..........................................................................................................................24 Introduction ....................................................................................................................25 Methods..........................................................................................................................27 Site Description and Raceway Pond Conditions ...................................................27 Sample Collection ..................................................................................................28 DNA Extraction and Sequencing ...........................................................................29 DNA Extraction .........................................................................................29 Bar-Coded Pyrosequencing .......................................................................29 Bioinformatic Sequence and Community Analysis ...............................................30 Statistical Analysis .................................................................................................32 Results and Discussion ..................................................................................................33 Environmental Variables .......................................................................................33 Community Composition and Interaction ..............................................................34 Correlation of Environmental Variables and Community Structure .....................40 v TABLE OF CONTENTS CONTINUED Correlations between Community Members .........................................................43 Conclusions ....................................................................................................................47 Funding ..........................................................................................................................48 Acknowledgements ........................................................................................................48 3. BACTERIA AND EUKARYA COMMUNITY DURING EUTROPHICATION AND TOXIC CYANOBACTERIAL BLOOMS IN THE ALKALINE LAKE VELENCE, HUNGARY ............................ 49 Contribution of Authors and Co-Authors .................................................................... 49 Manuscript Information Page ...................................................................................... 50 Abstract ........................................................................................................................ 52 Introduction .................................................................................................................. 53 Materials and Methods ................................................................................................. 54 Site Description .................................................................................................... 54 Sample Collection ................................................................................................ 55 MALDI-TOF MS Analysis .................................................................................. 57 Chemical Analyses............................................................................................... 58 DNA Extraction and Sequencing ......................................................................... 59 DNA Extraction ....................................................................................... 59 Bar-Coded Pyrosequencing ..................................................................... 59 Processing of Pyrosequencing Data and Community Analysis ........................... 61 Statistical Analysis ............................................................................................... 62 Results and Discussion ................................................................................................ 63 Chemical Analyses............................................................................................... 64
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