Date of Award

5-2026

Document Type

Thesis

Degree Name

Master of Science in Biology

Department

Biology

First Reader/Committee Chair

Dodsworth, Jeremy

Abstract

Numerous microbes on Earth have yet to be grown in pure cultures and studied in the laboratory. Cultivation-independent genomic methods, such as metagenomics, can help us predict their metabolic capabilities, but cultivation remains valuable for testing these predictions. These uncultivated microbes are very diverse, especially within hot springs. Examples of these uncultivated microbes include the genus Calditenuis (formerly Aigarchaeota Group 1) and the order Gearchaeales; they are often abundant in terrestrial hot springs such as Great Boiling Springs (GBS) in Nevada. Previous work allowed for the establishment of laboratory enrichment cultures inoculated from sediments of GBS containing Calditenuis and Gearchaeales, under suboxic conditions (~10% oxygen headspace) at 80 °C using casamino acids as a carbon and energy source, with a medium based on GBS spring water.

To learn more about these two novel taxa in GBS, this project had two major aims: (1) find cultivation conditions to enrich for Gearchaeales in mixed culture; and (2) attempt to isolate Gearchaeales or Calditenuis by single-cell sorting, plating and dilution-to-extinction.

Although isolation attempts were not successful, different cultivation conditions were identified to optimize growth of Gearchaeales in mixed culture. The growth period was confirmed to be three weeks, with optimal growth temperature of 80–85 °C. It grows best in suboxic (low-oxygen) conditions, and does not grow in the absence of oxygen. Its growth can be supported by defined carbon sources including sodium propionate and branched-chain amino acids (BCAAs), specifically leucine.  Gearchaeales also grows well in synthetic GBS medium with the addition of tungsten. Additionally, it was found that at 85 °C using propionate as a growth substrate, Calditenuis was also still present in the culture.

Under these conditions, isolation procedures were attempted. Plating on solid medium did not yield single colonies or significant growth, and single-cell sorting was unsuccessful in isolating either of these microbes, possibly due to pluronic toxicity. Dilution-to-extinction worked and successfully enriched Gearchaeales and Calditenuis, although Gearchaeales did not survive further transfers. CARD-FISH was only successful with Calditenuis and suggested that it constituted a large proportion of cells in the enrichments. However, metagenome sequencing revealed that other microbes, Pyrobaculum and Desulfurococcales, were present in the enrichments that were not originally detected due to introns in their 16S rRNA genes or mismatches to primers targeting the 16S rRNA gene. The resulting culture can be used for further attempts to isolate Calditenuis.

Download

  Contact Author

Share

COinS