Graduation Date

Spring 2022

Document Type



Master of Science degree with a major in Biology

Committee Chair Name

Mark Wilson

Committee Chair Affiliation

HSU Faculty or Staff

Second Committee Member Name

Catalina Cuellar-Gempeler

Second Committee Member Affiliation

HSU Faculty or Staff

Third Committee Member Name

Karen Kiemnec-Tyburczy

Third Committee Member Affiliation

HSU Faculty or Staff

Fourth Committee Member Name

Susan Marshall


Biology, Thermoacidophiles, Alicyclobacillus acidocaldarius, Biogeography

Subject Categories



Many thermoacidophilic bacterial species thrive in hot springs, fumaroles, and geothermally heated soils. These habitats are globally distributed but often small in size and located distantly from similar habitats, and therefore the biogeography of thermoacidophiles may be similar to island biogeography of plants and animals. Little is known about dispersal of thermoacidophiles. In this study I aimed to quantify, isolate, and characterize thermoacidophilic bacteria from non-permissive habitats. I also used genetic approaches to compare these isolates to thermoacidophilic bacteria from permissive habitats to provide insight to their biogeography.

To determine if I could enrich thermoacidophiles from non-permissive habitats, and if so, what taxa could be enriched, ten soil samples were incubated at five different temperatures between 30°C and 70°C in pH 2.5 PTYG medium. Thermoacidophiles were enriched from all ten soil samples, and at all temperatures from most samples. Forty-two isolates were obtained with at least 12 different species identified. Nine potentially novel species were isolated, sharing less than 97% 16S rRNA sequence identity with characterized isolates.

To determine what media would enrich the most thermoacidophiles, Most Probable Number (MPN) trials were carried out with three different media (PTYG, K medium, and Potato Dextrose medium [PD]) at pH 3 and 60°C. Five soil samples from the non-permissive Trinity Alps location produced growth up to at least the 10-2 dilution in all media. The PTYG medium produced the highest concentrations. Seven of the ten isolates from these experiments were novel species based on 16S rRNA gene analysis.

To quantify thermoacidophiles in non-permissive environments, twelve soil samples from each of six different locations were incubated at 60°C, pH 3 in PTYG medium in a MPN trial (one replicate by three ten-fold dilutions). Growth occurred from every location in at least eight of the 12 samples. The concentration of thermoacidophilic endospores was between 13-325/g. From the 72 samples, 50 isolates were collected with the most prevalent being A. pomorum (22) and A. acidocaldarius (8). Seventeen isolates were novel species based on 16S rRNA gene analysis.

To explore biogeographical distributions of A. acidocaldarius in non-permissive environments, I used comparative multilocus sequence typing (MLST) of six different highly conserved genes (cpn60, eftu, emrB, gmp, gyrB, and rpoB). These genes were PCR-amplified and sequenced from 29 A. acidocaldarius isolates from non-permissive locations. I found that isolates from different non-permissive locations had identical or nearly identical sequences. These isolates were compared to isolates from permissive habitats using MLST and individual gene trees which showed isolates from permissive and non-permissive habitats clustering together, with some isolates from these opposing habitats sharing identical or near identical alleles. Our findings support evidence for broad, on-going dispersal of spores to the permissive and non-permissive regions sampled.

Surprisingly, thirty-three isolates failed to meet the 97% criterion for alignment of their 16S rRNA gene in BLASTn. Many of these isolates also did not share 97% identity with each other, suggesting that multiple potentially novel species were cultured from non-permissive habitats. Because so many studies have looked at thermoacidophiles in permissive environments, it seems unlikely that these novel species were not previously detected because of undersampling, and instead it suggests that non-permissive habitats may host a variety of novel thermoacidophiles. The source environments for these novel thermoacidophiles is unclear.

Collectively, these results suggest that massive numbers of thermoacidophilic spores are broadly dispersed in short timeframes across large scales and accumulate at high concentrations in diverse habitats. These spores come from diverse species, many of which have not yet been isolated or characterized.

Citation Style


Included in

Biology Commons



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