Date of Award

5-2010

Degree Type

Thesis

Degree Name

Master of Science

Major

Environmental and Soil Sciences

Major Professor

Michael Essington

Committee Members

Mark Radosevich, Christopher Schadt

Abstract

The aim of this research project was to test new collection and cultivation techniques that may increase the range of cultivable diversity of soil bacteria. Fortified BioSep beads were employed in situ to capture soil bacteria, and the success of the beads was analyzed using Phylochip microarray analysis. In the cultivation phase, three different media substrates and increased incubation period were evaluated for the ability to select novel or rare bacteria. Over 700 agricultural soil bacterial isolates were classified, including a rare Gemmatimonadetes sp., a rare Verrucomicrobia sp., several Acidobacteria sp., and many novel isolates. Land management, media, and incubation period each resulted in lineage specific preferences. The yeast fortified BioSep bead cultivation collection was significantly different from the bulk soil or acyl homoserine lactone (AHL) fortified bead cultivation collections, and there were lineage specific differences in all three collection types.

Phylochip analysis showed a significant difference between bulk soil and all BioSep bead (water, yeast, or AHL fortified) communities based on microarray analysis of 16S rDNA. The yeast fortified BioSep bead community was richer in operational taxonomic units (OTU) than all others. The number of phyla determined by the Phylochip analysis was much higher than that seen in the overall cultivation collection.

Prophage induction assays of 21 isolates were performed, using mitomycin C (mitC) and a mixture of six AHLs, to examine soil lysogenic phage-host interactions. The fraction induced by mitC was 29%, and 10% were induced by AHL. There was no correlation between induction and land management or host growth rate.

This research showed that increases in cultivable diversity can be attained by the use of BioSep beads in the collection process, varying media substrates, and by extending incubation of inoculate cultures. Phylochip analysis, however, revealed that even with altered cultivation methods, there is still a wealth of soil bacterial diversity that remains to be cultivated from this site. We also found that AHLs impact the interactions between soil bacterial hosts and prophage.

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