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The Biofilm Matrix in Sulfate-Reducing Bacterial Biofilms

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The Biofilm Matrix in Sulfate-Reducing Bacterial Biofilms THE BIOFILM MATRIX IN SULFATE-REDUCING BACTERIAL BIOFILMS: POTENTIAL ROLES FOR ELECTRON MEDIATORS AND LARGE PROTEINS by Gregory Peter Krantz A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Immunology MONTANA STATE UNIVERSITY Bozeman, Montana November 2019 ©COPYRIGHT by Gregory Peter Krantz 2019 All Rights Reserved ii ACKNOWLEDGEMENTS Many thanks to my mentor, Dr. Matthew Fields for allowing me the opportunity to grow into the scientist that I am in your laboratory. It has been a privilege to work with an advisor who respects the overall wellbeing of their students in addition to their professional progress. I am grateful to my committee members Dr. Robin Gerlach, Dr. Gill Geesey, Dr. Jim Wilking, and Dr. Zbigniew Lewandowky for taking the time to meet and review my work and provide your feedback. The Center for Biofilm Engineering has been an incredible place to work. From exposure to industrial associates, to highly organized and well maintained equipment resources, these attributes grease the wheels of a career in science. While getting a Ph.D can be intense at times, one thing that stood out to me was the gift of having time to wonder about things, and to have that be my job. The freedom to let my mind be curious about bacteria and to have the time to answer the questions that arose, has been an incredible and memorable experience. Most importantly, I would like to thank my family, specifically my parents Jan and Ken, and my siblings Keegan and Lendy, because without their loving and articulate support, this would not have come to fruition. I am deeply grateful to the Molecular Biosciences Fellowship Program and to ENIGMA for funding. The research community, funding resources, and vast array of collaboration opportunities have been breath-takingly impressive. I would also like to thank all of my Bozeman friends outside of science that have supported me and enriched my life during my time in graduate school. iii TABLE OF CONTENTS 1. INTRODUCTION ...........................................................................................................1 Sulfate-Reducing Microorganisms ..................................................................................1 Model Organisms .............................................................................................................2 Biofilm Matrix .................................................................................................................3 Type 1 Secretion Systems ................................................................................................5 Shear Force on the Biofilm Matrix ..................................................................................6 Microbially Induced Corrosion ........................................................................................6 Corrosion Minerals ..........................................................................................................9 Nutrient Limitation ........................................................................................................10 Thesis Objectives ...........................................................................................................12 References ......................................................................................................................15 2. BULK PHASE RESOURCE RATIO ALTERS CARBON STEEL CORROSION RATES AND ENDOGENOUSLY PRODUCED EXTRACELLULAR ELECTRON TRANSFER MEDIATORS IN A SULFATE-REDUCING BIOFILM .........................................................................19 Contributions of Authors ...............................................................................................19 Manuscript Information .................................................................................................21 Abstract ..........................................................................................................................22 Introduction ....................................................................................................................23 Materials and Methods ...................................................................................................26 Microorganism .......................................................................................................26 Planktonic Growth .................................................................................................26 Biofilm Growth ......................................................................................................27 Biofilm and Coupon Harvesting ............................................................................27 Preparation of Samples for Imaging ......................................................................28 Microscopy of Biofilm Cross Sections ..................................................................29 Growth Parameter Measurements ..........................................................................29 Protected 1018 Steel Coupons ...............................................................................30 Metabolomics and Data Processing .......................................................................30 Results and Discussion ..................................................................................................30 Planktonic Growth Under Different Resource Ratios ...........................................31 Biofilm on Metal and Glass ...................................................................................32 Biofilm Biomass ....................................................................................................34 Lactate/Acetate Levels ...........................................................................................38 Sulfate Levels .........................................................................................................39 Sulfide Levels ........................................................................................................39 iv TABLE OF CONTENTS CONTINUED Corrosion Rates ......................................................................................................41 Biofilm Contact ......................................................................................................42 Metabolomic Analysis on Biofilm .........................................................................43 Sulfur Cycling ........................................................................................................45 Role of Electron Shuttles .......................................................................................46 Conclusions ....................................................................................................................51 References ......................................................................................................................53 Supplemental Information .............................................................................................56 3. LARGE EXTRACELLULAR PROTEINS ARE ESSENTIAL FOR BIOFILM FORMATION AND SHEAR FORCE RESPONSE IN DESULFOVIBRIO VULGARIS .........................................................................................59 Contributions of Authors ...............................................................................................59 Manuscript Information .................................................................................................60 Abstract ..........................................................................................................................61 Introduction ....................................................................................................................62 Materials and Methods ...................................................................................................64 Microorganism and Growth Medium ....................................................................64 Measurement of Growth ........................................................................................64 Biofilm Growth ......................................................................................................64 Growth Parameter Measurements ..........................................................................65 Preparation of Samples for Scanning Electron Microscopy ..................................66 Structural Predictions .............................................................................................66 Structure Analysis ..................................................................................................66 Single Particle Analysis of DVU1012 ...................................................................67 Thin-Section Imaging of Biofilms .........................................................................68 Multiple Sequence Alignment ...............................................................................68 Results ............................................................................................................................69 Planktonic Growth .................................................................................................69 Biofilm Growth and Composition .........................................................................70 Substrates and Products .........................................................................................73 Biofilm Electron Microscopy ................................................................................73
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