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The Effects of Long-Term Exposure of an Artificially Assembled Microbial Community to Uranium Or Low Ph

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The Effects of Long-Term Exposure of an Artificially Assembled Microbial Community to Uranium Or Low Ph MIAMI UNIVERSITY THE GRADUATE SCHOOL CERTIFICATE FOR APPROVING THE DISSERTATION We hereby approve the Dissertation of Ryann Michelle Brzoska Candidate for the Degree: Doctor of Philosophy ______________________________________ Dr. Annette Bollmann, Director ______________________________________ Dr. Mitchell F. Balish, Reader ______________________________________ Dr. Rachael M. Morgan-Kiss, Reader ______________________________________ Dr. Donald J. Ferguson ______________________________________ Dr. Melany C. Fisk Graduate School Representative ! ABSTRACT THE EFFECTS OF LONG-TERM EXPOSURE OF AN ARTIFICIALLY ASSEMBLED MICROBIAL COMMUNITY TO URANIUM OR LOW PH by Ryann Michelle Brzoska Uranium-contaminated environments contain microbial communities capable of immobilizing uranium. There is evidence that some bacterial communities are capable of immobilizing higher concentrations of uranium than individual species. However, the interactions between bacterial species contributing to increased uranium immobilization are not understood. Therefore, the goal of this study was to identify interactions between species in an artificial community and assess their influence on uranium immobilization. Bacterial species previously isolated from the uranium-contaminated subsurface at Oak Ridge, Tennessee, were characterized both individually and in a mixed community in the presence and absence of uranium or low pH. Growth of individual bacterial strains in the presence of uranium or low pH revealed two Sediminibacterium strains, Sediminibacterium spp. OR43 and OR53, with different degrees of tolerance towards uranium. The main physiological or genomic difference between the Sediminibacterium strains was the sensitivity of Sediminibacterium sp. OR43 to uranium concentrations ≥ 200 µM uranium. Sediminibacterium sp. OR53 was chosen along with Caulobacter sp. OR37, Ralstonia sp. OR214, and Rhodanobacter sp. OR444 to be a part of an artificially assembled community. The productivity of the community was assessed by monitoring the optical density and the abundance of each species in the community in the presence or absence of 200 µM uranium at pH 4.5 (pH4.5) or 7 (pH7) for 30 weeks (~300 generations). At pH7 and pH4.5, all strains were present, but the strain tolerant to the lowest pH, Ralstonia sp. OR214, was the most abundant. The presence of uranium selected for the uranium- tolerant strains, Sediminibacterium sp. OR53 and Caulobacter sp. OR37. Moreover, when a subculture of the consortium at pH 7 with uranium (pH7U) was transferred into pH7 without uranium, the uranium-sensitive strains did not recover. Re-isolated strains of Caulobacter spp. OR37 and Sediminibacterium spp. OR53 from the pH7U condition showed increased growth in coculture that did not correlate with an increase in uranium immobilization when compared to individual strains. Taken together, our results indicate that commensalistic and competitive interactions may develop between microbial species and impact growth and uranium immobilization, which are important for microbial communities considered for the purpose of bioremediation. THE EFFECTS OF LONG-TERM EXPOSURE OF AN ARTIFICIALLY ASSEMBLED MICROBIAL COMMUNITY TO URANIUM OR LOW PH A Dissertation Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology by Ryann Michelle Brzoska Miami University Oxford, OH 2015 Dissertation Director: Annette Bollmann, Ph.D. TABLE OF CONTENTS List of Tables iii List of Figures v Acknowledgements vii Introduction 1 Chapter 1. Physiological and genomic analysis of two novel Bacteroidetes strains Sediminibacterium spp. OR43 and OR53. 21 Chapter 2. The effects of uranium and low pH on the composition and productivity of an artificial community in a long-term growth experiment. 60 Chapter 3. Physiological and genomic comparison of strains re-isolated from an artificial community in the presence of uranium. 91 Summary 149 References 162 ! ! ! ii! LIST OF TABLES Table Page 1. Genome sequencing project information for Sediminibacterium spp. OR43 and OR53. 29 2. Differential physiological characteristics of strains Sediminibacterium spp. OR43 and OR53T in comparison with type strains of closely related species. 36 3. Fatty acid profiles of Sediminibacterium spp. OR43 and OR53 strains in comparison with type strains of closely related species. 38 4. Influence of the nitrate concentration (mM) in the medium on the growth -1 rate (day ) and maximum biomass (max OD600) of Sediminibacterium spp. OR43 and OR53 after two days. 40 5. Influence of the pH in the medium on the growth rate (day-1), maximum biomass (max OD600), and lag phase (days) of Sediminibacterium sp. OR43 after two days. 44 6. Influence of the pH in the medium on the growth rate (day-1), maximum biomass (max OD600), and lag phase (days) of Sediminibacterium sp. OR53 after two days. 45 7. Influence of the uranium concentration (µM) in the medium on the growth rate -1 (day ), maximum biomass (max OD600), and lag phase (days) of Sediminibacterium sp. OR43 after two days. 46 8. Influence of the uranium concentration (µM) in the medium on the growth rate -1 (day ), maximum biomass (max OD600), and lag phase (days) of Sediminibacterium sp. OR53 after two days. 47 9. Genome properties of Sediminibacterium spp. OR43 and OR53. 48 10. Number of genes associated with the general COG functional categories. 49 11. Presence (+) and absence (-) of genes involved in nitrate metabolism in Sediminibacterium spp. OR43 and OR53. 50 12. Enumeration of genes potentially involved in the adaptation to low pH in Sediminibacterium spp. OR43 and OR53. 52 13. Heavy metal resistance genes in the genome of Sediminibacterium sp. OR53. 53 14. Membrane associated phospholipid phosphatases in the genome of Sediminibacterium sp. OR53 and the percent identity (%) of orthologous sequences in Sediminibacterium spp. OR43, C3, and S. salmoneum. 56 ! iii! 15. Primers used for measuring the relative abundance of Caulobacter sp. OR37, Sediminibacterium sp. OR53, Ralstonia sp. OR214, and Rhodanobacter sp. OR444. 67 16. PCR conditions (temperature (°C)/time (s)) and validation of qPCR. 68 17. Maximum uranium concentration and minimum pH value in the MS medium that allowed growth of the bacterial species as pure cultures. 70 18. Influence of 0, 200, and 300 µM uranium on the growth rate (h-1) of the re-isolated and ancestral strains after two days of exposure. 104 19. Influence of 0, 200, and 300 µM uranium on the max OD600 of re-isolated and ancestral strains of Caulobacter spp. OR37 and Sediminibacterium spp. OR53 after two days of exposure. 105 20. Influence of 0, 200, and 300 µM uranium on the length of lag phase (hours) of the re-isolated and ancestral strains of Caulobacter spp. OR37 and Sediminibacterium spp. OR53 after two days of exposure. 106 21. Growth rate and maximum OD600 (biomass yield) of ancestral and re-isolated Caulobacter spp. OR37 and Sediminibacterium spp. OR53 as a coculture in the presence of 300 µM uranium. 110 22. Uranium concentration (µM) in the presence of re-isolated and ancestral strains of Caulobacter spp. OR37 and Sediminibacterium spp. OR53 either individually or in coculture after seven days exposure to 300 µM uranium. 111 23. Read mapping coverage of re-isolated strains to the ancestral genomes. 132 24. Genetic variations in the re-isolated strains of Sediminibacterium spp. OR53 compared to the ancestral strain. 133 25. Location of amino acid changes in re-isolated Sediminibacterium spp. OR53 strains. 134 26. Genetic variations in the re-isolated strains of Caulobacter spp. OR37 compared to the ancestral strain. 135 27. Location of amino acid changes in re-isolated Caulobacter spp. OR37 strains. 137 28. Positive effects (+) of the OD600 (growth) in cocultures of ancestral and re-isolated Caulobacter spp. OR37 and Sediminibacterium spp. OR53 in the presence of 300 µM uranium after two days. 139 ! iv! LIST OF FIGURES Figure Page 1. Uranium speciation as a function of pH. 2 2. The S-3 ponds and the surrounding contaminated areas at Oak Ridge. 6 3. Schematic of efflux transporters involved in heavy metal resistance. 11 4. Mechanisms used by bacteria for immobilizing uranium. 13 5. Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences of Sediminibacterium spp. OR43 and OR53. 32 6. Transmission electron micrographs of (A) Sediminibacterium sp. OR43; and (B) Sediminibacterium sp. OR53. 34 7. Growth rate (day-1) of Sediminibacterium spp. OR43 and OR53 in the presence of (A) different pH values; and (B) uranium concentrations. 41 8. Influence of 0-200 µM uranium on the growth rates (h-1) and length of lag phase (h) of Caulobacter sp. OR37, Sediminibacterium sp. OR53, Ralstonia sp. OR214, and Rhodanobacter sp. OR444. 71 9. Influence of pH on the growth rate (h-1) of Caulobacter sp. OR37, Sediminibacterium sp. OR53, Ralstonia sp. OR214, and Rhodanobacter sp. OR444. 73 10. OD600 (as a measure of the total biomass) at the end of each week during the long- term growth experiment. 75 11. Relative abundance (%) of the 16S rRNA genes of Caulobacter sp. OR37, Sediminibacterium sp. OR53, Ralstonia sp. OR214, and Rhodanobacter sp. OR444 in the artificial community at (A) pH7; (B) pH7->pH 4.5; (C) pH4.5; and (D) pH4.5->pH7. 78 12. Relative abundance (%) of the 16S rRNA genes of Caulobacter sp. OR37, Sediminibacterium sp. OR53, Ralstonia sp. OR214, and Rhodanobacter sp. OR444 in the artificial community at (A) pH7; (B) pH7->pH7U; (C) pH7U; and (D) pH7U->pH7. 80 13. Relative abundance (%) of the 16S rRNA genes of Caulobacter sp. OR37, Sediminibacterium sp. OR53, Ralstonia sp. OR214, and Rhodanobacter sp. OR444 in the artificial community under all conditions at the beginning of the ! v! experiment (week 0), at week 12, and at the end of the experiment (week 30). 82 14. Concentration of uranium (µM) in medium at pH7U and pH7->pH7U communities of the long-term growth experiment. 85 15. Influence of 0, 200, and 300 µM uranium on the change in OD600 over time (growth) of the ancestral strains of Caulobacter sp. OR37 (OR37A) and Sediminibacterium sp. OR53 (OR53A) and the re-isolated (OR37 Is8, OR37 Is14, OR37 Is28, OR53 Is7, OR53 Is18, OR53 Is70) strains.
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