Date Thesis Awarded
5-2024
Access Type
Honors Thesis -- Access Restricted On-Campus Only
Degree Name
Bachelors of Science (BS)
Department
Biology
Advisor
Kurt Williamson
Committee Members
Mark Forsyth
Bjorg Larson
Abstract
Microbial communities, though unseen, are an essential part of all ecosystems. Fixation of atmospheric dinitrogen into ammonium and nitrate by soil bacteria is critical to the nitrogen cycle, which all plants and primary producers depend upon. Bacteriophages, viruses that infect bacteria, are known to impact bacterial activity and could affect these processes, yet their role in soil nitrogen cycling is largely unknown. Current predictions regarding phage impacts on terrestrial nitrogen cycling are largely based upon sequencing analyses and highly speculative. Accordingly, this project aims to directly quantify phage impact on soil nitrogen cycling. To investigate this, we designed a series of microcosm experiments with soil denitrifier Bacillus subtilis and its phage, HA. First, we determined baseline B. subtilis nitrate metabolism through liquid microcosm experiments in minimal medium with nitrate as the sole nitrogen source. In measuring broth nitrate levels with vanadium colorimetry, we found that B. subtilis uses nitrate only in low-oxygen conditions. Addition of phage to B. subtilis cultures in subsequent microcosms resulted in a near 5-fold decrease (p < 0.0001) in broth nitrate concentration over two weeks, suggesting that host nitrate reduction is stimulated by phage infection. Pilot synthetic soil microcosms demonstrated no significant difference in nitrate concentrations between B. subtilis-inoculated replicates and uninoculated controls. However, further investigation indicated that the soil medium used may not have been sufficiently sterilized to prevent microbial growth. In addition to analyzing autoclave sterilization efficiency, we also investigated the infectious particle recovery phage concentration methods for our chosen phage. While we used ultracentrifugation to resuspend phage in phage buffer prior to inoculation of liquid microcosms, we noted that it significantly reduced phage infectivity in comparison to concentration via polyethylene glycol precipitation and ultrafiltration. Another characteristic of phage that we examined was replication activity in different media. Nutrient limitations presented by minimal medium extended phage latent period by 30 minutes and significantly decreased burst size when compared to phage replication in rich media. Determining the latent period of HA is key in deciding the frequency of data collection in future microcosm experiments, as soil is also a low-nutrient medium which may alter the rate of HA progeny production and therefore the potential impact of phage on B. subtilis denitrification.
In the future, we plan to refine our methodology further and continue this approach with doubly-sterilized soil microcosms inoculated with B. subtilis and phage HA, and time series data collection to determine change in nitrate, bacteria, and phage concentrations over two weeks.
Recommended Citation
Assaad, Madeleine R., "Investigating Viral Impact on Nitrogen Cycling Through Phage-host Interactions in Soil" (2024). Undergraduate Honors Theses. William & Mary. Paper 2133.
https://scholarworks.wm.edu/honorstheses/2133
