MIAMI UNIVERSITY THE GRADUATE SCHOOL CERTIFICATE FOR APPROVING THE DISSERTATION We hereby approve the Dissertation of Elizabeth Aileen French Candidate for the Degree: Doctor of Philosophy ______________________________________ Dr. Annette Bollmann, Director ______________________________________ Dr. Donald J. Ferguson, Reader ______________________________________ Dr. Rachael M. Morgan-Kiss, Reader ______________________________________ Dr. Gary R. Janssen ______________________________________ Dr. Michael J. Vanni Graduate School Representative ABSTRACT INVESTIGATION OF FACTORS INFLUENCING NICHE DIFFERENTATION OF AMMONIA-OXIDIZING ARCHAEA AND BACTERIA IN FRESHWATER ENVIRONMENTS by Elizabeth Aileen French Nitrification, the transformation of nitrogen from its most reduced form (ammonia) to its most oxidized form (nitrate) is a component of the nitrogen cycle in aquatic habitats. The first step of this process, the oxidation of ammonia to nitrite, is performed by two distinct groups of microorganisms in the environment, the ammonia-oxidizing archaea (AOA) and bacteria (AOB). Here we present a study using cultivation techniques to investigate environmental factors that influence the niche differentiation of AOA and AOB from freshwater environments. In order to investigate factors that drive the diversity of freshwater AOB communities, we enriched AOB from lakes representing a range of trophic states. The resulting enrichments were not influenced by the ammonium concentrations used in enrichment medium, but were comprised of AOB species commonly detected in freshwater environments. The AOB communities of the sediment and enrichments were strongly influenced by the watershed land use of the lake, and ammonium and nitrate concentrations within the sediment. Further enrichment efforts yielded cultures of three species of AOA, two of which represent a previously undescribed genus. These AOA grew more slowly than a freshwater AOB enrichment in all conditions tested (ammonium concentration, oxygen concentration, pH, and light exposure). Data from these experiments indicated that ammonium and oxygen concentrations and light exposure, would be the strongest factors driving niche separation of AOA and AOB. Chemostat competition experiments were conducted using one representative AOA and AOB under ammonia-limiting, high oxygen (21%) and ammonia-limiting, low oxygen (1%) conditions. In all cases, the AOA outcompeted the AOB for ammonia, and the AOB was washed from the chemostat. AOA and AOB were also investigated with respect to their starvation tolerance; both AOA and AOB survived nearly two months of starvation while maintaining amoA mRNA and 16S rRNA, however the AOB was able to recover from starvation faster. The results from these experiments suggest that, within the ammonia-oxidizers, AOB represent a copiotrophic lifestyle and will thrive in conditions of pulses of high ammonia availability, while AOA represent an oligotrophic lifestyle and will thrive in conditions of very low but constant ammonia availability. Investigation of Factors Influencing Niche Differentiation of Ammonia-oxidizing Archaea and Bacteria in Freshwater Environments 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 Elizabeth Aileen French Miami University Oxford, OH 2013 Dissertation Director: Annette Bollmann, Ph.D. TABLE OF CONTENTS List of Tables iii List of Figures v Acknowledgements vii Introduction 1 Chapter 1. Influence of Ammonium Concentration on the Enrichment of Ammonia-oxidizing Bacteria from Freshwater Sediments in Ohio. 29 Chapter 2. Ecophysiological Characterization of Ammonia-Oxidizing Archaea and Bacteria from Freshwater. 48 Chapter 3. Starvation and Competition: Survival Capabilities of Ammonia-oxidizing Archaea and Bacteria 90 Summary 130 References 144 ii LIST OF TABLES Table Page 1. Primers used for identification and DGGE separation of sequences from enriched AOB based on 16S rRNA gene sequence 35 2. Characteristics of lakes/sediments from which AOB were enriched 37 3. Closest cultured relative of enriched AOB 38 4. Primers used to identify AOA and AOB based on amoA and 16S rRNA gene sequence 58 5. Oligonucleotide probes used for CARD-FISH 59 6. Identities [%] of AOA in the enrichment cultures AOA-AC2, AOA-AC5, and AOA-DW in comparison with previously cultivated AOA. 63 7. Quantitative analysis of the composition of the enrichment cultures AOA-AC2; AOA-AC5 and AOA-DW. 64 + -1 8. Influence of the NH4 concentration on the growth rates [h ] of the enrichment culture AOA-AC2, AOA-AC5, AOA-DW and AOB-G5-7 68 + 9. Influence of NH4 concentration on the lag phase [h] before onset of logarithmic growth in the enrichment cultures AOA-AC2; AOA-AC5, AOA-DW, and AOB-G5-7 71 10. Influence of the calculated O2 concentrations in the headspace of the bottle on the growth rates [h-1] of the enrichment cultures AOA-AC2, AOA-AC5, AOA-DW, and AOB-G5-7 74 11. Influence of the pH value on the growth rates [h-1] of the enrichment cultures AOA-AC2, AOA-AC5, AOA-DW, and AOB-G5-7 77 12. Influence of white, red, and blue light with the intensity of 30 µmol photons m-2 s-1 and blue light with the intensity of 3 µmol photons m-2 s-1 on the growth rates [h-1] of the enrichment culture AOB-G5-7 81 13. Influence of white, red, and blue light with the intensity of 30 µmol photons m-2 s-1 and blue light with the intensity of 3 µmol photons m-2 s-1 on the growth rates [h-1] of the enrichment culture AOA-DW 82 14. Influence of white, red, and blue light with the intensity of 30 µmol photons m-2 s-1 and blue light with the intensity of 3 µmol photons m-2 s-1 iii on the growth rates [h-1] of the enrichment culture AOA-AC2 83 15. Influence of white, red, and blue light with the intensity of 30 µmol photons m-2 s-1 and blue light with the intensity of 3 µmol photons m-2 s-1 on the growth rates [h-1] of the enrichment culture AOA-AC5 84 16. Primers used to quantify amoA and 16S rRNA gene and RNA abundance in laboratory cultures of AOA and AOB 98 17. Influence of starvation time [days] on growth rate [h-1] of the enrichment cultures AOB-G5-7 and AOA-AC1 117 18. Influence of starvation time [days] on lag phase [h] of the enrichment cultures AOB-G5-7 and AOA-AC1 118 19. Influence of starvation time [days] on amoA copy number [copies/ng RNA] of the enrichment cultures AOB-G5-7 and AOA-AC1 121 20. Influence of starvation time [days] on 16S rRNA copy number [*106 copies/ng RNA] of the enrichment cultures AOB-G5-7 and AOA-AC1 122 iv LIST OF FIGURES Figure Page 1. Simplified schematic diagram of the nitrogen cycle 2 2. Neighbor-joining tree of cultivated AOB species based on 16S rRNA gene sequence 9 3. Neighbor-joining tree of cultivated AOA species and environmental strains based on amoA nucleotide sequence 17 4. Principal Component Analysis of the community composition in the enrichment cultures and the original sediment samples 40 5. Canonical Correspondence Analysis of the relationship between the distribution of the AOB species, the sediment samples and the environmental factors 43 - - 6. Determination of the duration of lag phase based on the NO2 /NO3 - - concentration and log (NO2 /NO3 concentration) in an enrichment culture over time 56 7. Neighbor-joining phylogenetic tree of the AOA enrichment cultures based on amoA gene sequences 61 + 8. Influence of NH4 concentration on the growth rates of the enrichment cultures AOA-AC2; AOA-AC5, AOA-DW, and AOB-G5-7 66 + 9. Influence of NH4 concentration on the lag phase before onset of logarithmic growth in the enrichment cultures AOA-AC2; AOA-AC5, AOA-DW, and AOB-G5-7 69 10. Influence of the calculated O2 concentration in the headspace of the bottle on the growth rate of the enrichment cultures AOA-AC2; AOA-AC5, AOA-DW, and AOB-G5-7 72 11. Influence of the pH of the medium on the growth rates of the enrichment cultures AOA-AC2, AOA-AC5, AOA-DW, and AOB-G5-7 75 12. Influence of white, red, and blue light with the intensity of 30 µmol photons m-2 s-1 and blue light with the intensity of 3 µmol photons m-2 s-1 on the growth rates of the enrichment cultures AOB-G5-7, AOA-DW, AOA-AC2, and AOA-AC5 79 v 13. Chemical and microbial composition of a chemostat initially inoculated with AOB-G5-7 in ammonia-limited, high oxygen conditions 100 14. Chemical and microbial composition of a chemostat initially inoculated with AOA-AC1 in ammonia-limited, high oxygen conditions 102 15. Expression of amoA by AOB-G5-7 (A) and AOA-AC1 (B) during the chemostat growth and competition under ammonia-limited, high oxygen conditions 105 16. Chemical and microbial composition of a chemostat initially inoculated with AOB-G5-7 in ammonia-limited, low oxygen conditions 108 17. Chemical and microbial composition of a chemostat initially inoculated with AOA-AC1 in ammonia-limited, low oxygen conditions 111 18. Expression of amoA by AOB-G5-7 (A) and AOA-AC1 (B) during the chemostat growth and competition under ammonia-limited, low oxygen conditions 113 19. Influence of starvation on the growth rate and lag phase of AOB-G5-7 and AOA-AC-1 recovery cultures 115 20. amoA mRNA and 16S rRNA abundance [copies/ng RNA] during starvation of the AOA enrichment cultures AOA-AC1 (A) and the AOB enrichment culture AOB-G5-7 (B) 119 21. Conceptual model of the niche of AOA and AOB in a eutrophic lake 135 22. Conceptual model of the niches of AOA and AOB in an oligotrophic lake 138 vi ACKNOWLEDGEMENTS A wise person once wrote on a bumper sticker, “graduate school is like hitting yourself on the head with a hammer: it feels good when you stop.” While that might be a bit of an exaggeration, grad school is certainly full of ups and downs, but at the end, I’d like to think my experience was a positive one, where the ups outnumbered and overcame the downs.