Examining Complex Microbial Communities Using the Terminal Restriction Fragment Length Polymorphism Method and Dedicated TRFLP Analysis Software

The purpose of this research was to develop a robust methodology that could be used to determine the composition of complex microbial communities found in a variety of environments. In an effort to profile these communities, a 16S rDNA directed, sequencing independent method, Terminal Restriction Fragment Length Polymorphism (TRFLP) was used to generate fragment profiles from genomic DNA isolated from each sample. A custom designed software package, In Silico©, was then used to match these terminal restriction fragments in the sample to patterns of 16S rDNA fragments in a custom database of reference patterns. Identifying these patterns allowed for inferences to be made about the structure of the microbial populations in the environments sampled. TRFLP was chosen as the method of identification since it is a high throughput, cost effective method for community profiling. A combination of this method and the In Silico© software was used to examine microbial communities associated with open air swine waste lagoon systems, the large intestine of the Trichechus manatus latirostrus, Florida manatee, and the microbial populations found in rumen fed fermentors. The community analysis methodology we developed and employed was able to not only detect large and diverse microbial populations within each sample, but was also able to show differences between the detected microbial populations under the various experimental regimes used in the studies presented.

Author dc:creator

• Devine, Anthony Andrew

Advisors dc:contributor.advisor

• Dr. Michael Sikes, Committee Member
• Dr. Amy M. Grunden, Committee Chair
• Dr. James W. Brown, Committee Member
• Dr. Vivek Fellner, Committee Member

• Microbial Community Analysis
• 16S rDNA
• Swine Lagoons
• TRFLP

Statement dc:rights

• I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dis sertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.

Dc Identifier Other

etd-04162009-113944

OAI record oai:identifier

https://repository.lib.ncsu.edu/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Arepository.lib.ncsu.edu%3A1840.16%2F6256