Date of Award

9-2019

Document Type

Thesis

Degree Name

Master of Science in Biology

Department

Biology

First Reader/Committee Chair

Jeremy Dodsworth

Abstract

‘Aigarchaeota’, a deeply branching lineage in the domain Archaea with no cultivated representatives, includes both thermophilic and hyperthermophilic microorganisms that reside in terrestrial and marine geothermal environments. The ‘Aigarchaeota’ consists of at least nine proposed genus-level groups that have been confirmed via 16S rRNA sequencing, with ‘Aigarchaeota’ Group 1 (AigG1) being the focus of this study. Based on cultivation-independent genomic data available from several AigG1 members in Great Boiling Spring (GBS), NV, and Yellowstone National Park, 22 different types of growth media were designed and tested for their ability to support growth of AigG1. One of these cultures, G1-10, was found to contain AigG1 at ~5% abundance, as well as other novel thermophilic microbial groups including a new species of the genus Pyrobaculum, members of the candidate phyla ‘Calescamentes’ and ‘Fervidibacteria’, and the novel archaeal lineage NAG1 (‘Geoarchaeota’). To attempt to obtain pure cultures of AigG1 and other novel thermophiles, a single-cell sorting system using an optical trap and a microfluidic device was constructed. The system was validated by sorting E. coli cells, which demonstrated that single, viable cells could be reliably obtained. Using this single cell sorting device on the G1-10 culture, a pure culture of a member of the genus Pyrobaculum was obtained, which was shown to represent a distinct species in this phylum by whole genome sequencing and in silico DNA-DNA hybridization. Additionally, a pure culture of the first representative of the candidate phylum ‘Fervidibacteria’ from an enrichment culture derived from G1-10. Additionally, to aid in morphology-based sorting of AigG1 and stable isotope labeling studies, fluorescence in situ hybridization (FISH) based on catalyzed reporter deposition (CARD-FISH) were developed and an AigG1-specific probe was tested. CARD-FISH was successfully used to detect AigG1 in both the G1-10 culture and in natural sediment samples from GBS. Stable isotope labeling incubations were performed with a variety of 13C-labeled substrates (bicarbonate, amino acids, sugars, and short chain fatty acids) on GBS sediments and G1-10 culture samples, and CARD-FISH was used to specifically detect AigG1 in the fixed samples. Nanometer-scale secondary-ion mass spectrometry (nano-SIMS) will then be used to determine whether AigG1 was capable of taking up the different carbon substrates tested. Overall, the results and accomplishments from this project and follow up nano-SIMS analysis will allow a better understanding of the metabolic potential of AigG1 and will aid future efforts to attempt to obtain pure cultures of this novel lineage.

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