Tuesday, April 20, 2010
11-38

Analysis of cellulolytic bacterial diversity within the intestinal tract of herbivores

Sandra Bediako and J. Michael Henson. Biological Sciences, Clemson University, 111 Biosystems Research Complex, Clemson, SC 29632

Amplification and analysis of 16S rDNA was employed to study the diversity of cellulolytic bacteria within the Holstein rumen and the intestinal tract of the Florida manatee Trichechus manatus latirostris. Colony isolation techniques were used to attain pure colonies for characterization. An inoculum of three day old Holstein rumen fluid was enriched for cellulolytic bacteria in an anaerobic medium. After several transfers, a colony was isolated from an anaerobic agar plate containing microcrystalline cellulose, Avicel. The bacterium was grown on two different types of media, PYG-Tween broth and TVC broth, for FAME analysis resulting in the production of two different fatty acid profiles. The 16S rRNA products were sequenced and BLASTed on the NCBI nucleotide data base. The bacterium was identified as Lactobacillus rhamnosus with a 97% maximum identity. DNA was isolated from several manatee fecal samples that were collected by USGS collaborators in Gainesville Florida. The fecal samples were taken from both male and female manatees of various sizes. Two sets of primers, one specific for Bacteria 16S rDNA and another for Archaea 16S rDNA, were used to evaluate the diversity of each domain within the manatee intestinal tract. The PCR amplified products were cloned and sequenced. In order to attain single colonies, enrichment for cellulolytic bacteria was performed using anaerobic broth cultures containing Whatman 1 filter paper, switchgrass, delignified switchgrass, Carboxymethyl cellulose, or cellobiose.

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