AN INVESTIGATION OF MICROBIAL DIVERSITY AND MICROBIOLOGICALLY INFLUENCED CORROSION IN AUTOMOTIVE FUEL ENVIRONMENTS by Charles H.D. Williamson IV A thesis submitted to the Faculty and the Board of Trustees of the Colorado School of Mines in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Environmental Science and Engineering). Golden, Colorado Date ____________________________ Signed: ___________________________ _ Charles H.D. Williamson IV Signed: ____________________________ Dr. John R. Spear Thesis Advisor Golden, Colorado Date ____________________________ Signed: ____________________________ Dr. John McCray Professor and Director Department of Civil and Environmental Engineering ii ABSTRACT Microbial contamination of fuels can cause issues such as biofouling, fuel degradation and microbiologically influenced corrosion (MIC). The focus of the research presented in this thesis was characterizing the microbial diversity of automotive fuels and automotive fuel environments in the United States via both molecular-based techniques as well as cultivation- based methods in order to gain insight into how this diversity is impacting fuels and fuel system infrastructure. A field survey of fuels including biodiesel, diesel, E10, E85, fuel-grade ethanol and gasoline was conducted; and 454 pyrosequencing of both 16S/18S rRNA genes as well as 16S/18S rRNA (transcribed into cDNA) was applied to identify both total and active microbial communities in these environments. Microbial communities in all fuel types were broadly similar, and prevalent phylotypes included Halomonas spp., Pseudomonas spp., Shewanella spp., Corynebacterium spp. and Acetobacter spp. Pyrosequencing libraries generated from cDNA and DNA indicated that the active and total communities of the sampled environments show significant overlap. The microbial communities of storage tanks containing fuel-grade ethanol and water were also characterized by molecular and cultivation-based techniques. Industry personnel have reported corrosion issues (suspected to be microbial corrosion) impacting storage tanks and other infrastructure exposed to fuel-grade ethanol and water, and acetic-acid-producing microbes were prevalent in samples collected from these environments. Acetobacter spp. and sulfate-reducing microbes were cultivated from samples collected from these storage tanks for laboratory corrosion testing. These corrosion tests (reported elsewhere) indicated that Acetobacter spp. increased pitting and cracking of carbon steels and that sulfate-reducing iii microbes increased general corrosion rates as well as increased pitting and cracking of carbon steels. Additionally, a Bacillus sp. that produces spores that catalyze Mn(II) oxidation was isolated from an E10 fuel sample. The potential impact that these sorts of microbes may have on corrosion in fuel system infrastructure is discussed. Increased knowledge of the the microbial diversity associated with fuel system infrastructure will improve monitoring and prevention strategies and guide future research of issues such as microbial corrosion in fuel systems. iv TABLE OF CONTENTS ABSTRACT...................................................................................................................................iii LIST OF FIGURES........................................................................................................................ix LIST OF TABLES..........................................................................................................................xi LIST OF ACRONYMS.................................................................................................................xii ACKNOWLEDGEMENTS..........................................................................................................xiii CHAPTER 1 INTRODUCTION AND BACKGROUND.......................................................1 1.1 Microbes and Molecular Methods......................................................................2 1.2 Microbes and Fuel Environments.......................................................................5 1.2.1 Microbiologically Influenced Corrosion (MIC)..................................................6 1.2.1.1 Biofilms and Differential Concentration (Aeration) Cells..................................7 1.2.1.2 Microbial Sulfate Reduction and Corrosion.......................................................8 1.2.1.3 Microbial Production of Organic Acids............................................................10 1.2.1.4 Microbial Oxidation and Reduction of Metals..................................................10 1.2.2 Biofouling and Fuel Degradation......................................................................11 1.3 Methods for Studying Microbes in Fuel Environments....................................11 1.4 Research Motivation and Approach .................................................................14 1.5 Research / Thesis Outline..................................................................................16 CHAPTER 2 AN INVESTIGATION OF MICROBIAL DIVERSITY ASSOCIATED WITH AUTOMOTIVE FUELS IN THE UNITED STATES.......................................19 2.1 Abstract..............................................................................................................19 2.2 Introduction........................................................................................................20 2.3 Methods..............................................................................................................21 2.3.1 Sample Collection..............................................................................................21 v 2.3.2 Nucleic Acid Extraction, Sanger Sequencing and 454 Pyrosequencing.............22 2.3.3 Sequence Analyses.............................................................................................24 2.4 Results and Discussion.......................................................................................25 2.4.1 Initial screen of microbial diversity - Sanger sequencing of 16S rRNA genes..26 2.4.2 Pyrosequencing of 16S/18S rRNA genes (DNA)..............................................30 2.4.3 Comparison of Microbial Communities in Different Fuel Types via 16S rRNA Gene (DNA) Pyrosequencing............................................................................39 2.4.4 Active vs. Bulk Microbial Communities (DNA vs. cDNA)...............................43 2.4.5 Implications for Cultivation-based Monitoring.................................................50 2.5 Summary............................................................................................................52 CHAPTER 3 MICROBIAL COMMUNITIES ASSOCIATED WITH FUEL-GRADE ETHANOL ENVIRONMENTS: IMPLICATIONS FOR MICROBIOLOGICALLY INFLUENCED CORROSION ...............................54 3.1 Abstract..............................................................................................................54 3.2 Introduction........................................................................................................55 3.3 Materials and Methods.......................................................................................57 3.3.1 Sample Collection..............................................................................................57 3.3.2 DNA Extraction, PCR and 454 Pyrosequencing................................................58 3.3.3 Cultivation and identification of acetic-acid-producing and sulfate-reducing consortia.............................................................................................................60 3.4 Results................................................................................................................61 3.4.1 Sample Description............................................................................................62 3.4.2 Pyrosequencing Results.....................................................................................62 3.4.3 Cultivation of acetic-acid producing bacteria....................................................70 3.4.4 Cultivation of sulfate-reducing consortium.......................................................71 3.5 Discussion..........................................................................................................74 vi 3.6 Summary............................................................................................................79 CHAPTER 4 SPORE-INDUCED MANGANESE OXIDATION BY A BACILLUS SP. ISOLATED FROM E10 GASOLINE TANKS..................................................80 4.1 Abstract..............................................................................................................80 4.2 Introduction........................................................................................................80 4.3 Methods..............................................................................................................83 4.3.1 Sample Collection..............................................................................................83 4.3.2 Isolation and identification of manganese-oxidizing microbes.........................84 4.3.3 Spore separation and manganese oxidation tests...............................................87 4.3.4 Spore-Induced Manganese Oxidation and Metal Surfaces................................88 4.4 Results................................................................................................................89 4.4.1 Isolation and identification