Masters Theses

Date of Award


Degree Type


Degree Name

Master of Science



Major Professor

Annette S. Engel

Committee Members

Karen G. Lloyd, Anna Szynkiewicz


Subaerial biofilms (SABs) grow at the interface between the atmosphere and rock surfaces in terrestrial and subterranean environments around the world. Multi-colored SABs colonizing relatively dry and nutrient-limited cave surfaces are known to contain microbes putatively involved in chemolithoautotrophic processes using inorganic carbon like carbon dioxide (CO2) or methane (CH4). However, the importance of CO2 and CH4 to SAB biomass production has not been quantified, the environmental conditions influencing biomass production and diversity have not been thoroughly evaluated, and stable carbon and nitrogen isotope compositions have yet to be determined from epigenic cave SABs. The purpose of this study was two-fold: (i) to quantify the proportion of biomass in cave SABs that could be derived from chemolithoautotrophic processes using stable carbon and nitrogen isotope analysis, and (ii) to characterize and quantify taxonomic groups capable of chemolithoautotrophy using molecular techniques. Bulk stable isotope analysis of biomass carbon had δ13C [delta 13-C] values between -35 and -46‰ [per mil], which were more negative than would be predicted if SAB biomass was due to heterotrophic assimilation of organic carbon having δ13C values of -21 to -25‰. Using isotopic compositions of end-member compounds, two-member mixing models indicated that 31‒100% of total biomass carbon could be produced via CO2-derived carbon, and 32‒66% of total biomass carbon could be due to CH4-derived carbon incorporation. Nitrogen isotope analyses confirmed the presence of nitrifying and nitrogen-fixing microbes, which was supported by 16S rRNA gene sequence analyses that retrieved high relative abundances of putative chemolithoautotrophs belonging to the families Nitrosococcaceae, Nitrosomonadaceae, and Nitrospirales, as well as Pseudonocardiaceae. Pseudonocardiaceae are in the Actinobacteria phylum and can potentially fix CO2. Overall microbial community composition significantly correlated with moisture content, and the relative abundances of Pseudonocardiaceae increased as moisture content decreased. Consequently, dry cave-wall habitats may select for Pseudonocardiaceae, as primary colonizers, that make the habitat conducive for other microbial groups like nitrogen cycling chemolithoautotrophs and eventually heterotrophs. Chemolithoautotrophy and nitrogen cycling in cave SABs likely has implications for terrestrial cave food webs, and their contributions to providing organic carbon and nitrogen sources to nutrient-limited cave ecosystems will require additional research.

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