A unique method to cultivate previously uncultured but ubiquitous bacteria using extended incubation and low nutrient levels

A unique method to cultivate previously uncultured but ubiquitous bacteria using extended incubation and low nutrient levels

Bacteria

Bacteriology -- Cultures and culture media

Rosbach, Derren Thompson

2006

O'Connell, Sean

http://rightsstatements.org/vocab/CNE/1.0/

application/pdf

manuscripts (documents)

Text

61724

https://southernappalachiandigitalcollections.org/object/61724

Limited to on-campus users

Development of 16S rDNA comparative phylogenetic assessment techniques have revealed many taxonomic groups yet to have members grown in pure culture. This discrepancy has only recently been addressed using innovative cultivation techniques aimed at re-creating environmental conditions in the lab, mimicking niche requirements for unstudied groups, so that isolated species can be obtained. Without pure culture representatives these species cannot be evaluated metabolically or ecologically. Several studies have had success using low nutrient media and extended incubation times to isolate species from groups with few or no cultured representatives. These species are most likely oligotrophic due to their ability to grow under low nutrient conditions by growing slowly and as small colonies. We evaluated a novel approach to culturing soil bacteria from three forested ecosystems in Great Smoky Mountains National Park (GSMNP). Albright Grove is an old growth forest while Purchase Knob and Cataloochee study plots are second growth forests, with the former having been logged and the latter impacted by chestnut blight. The cultivation approach combined an ex tended incubation period (3 months) under extremely oligotrophic conditions created by liquid serial dilution followed with cultivation on low nutrient solid media. Long-term enrichment culturing increased bacterial numbers even under extremely oligotrophic conditions. Twenty- five isolates were obtained and yielded 20 unique 16s rDNA sequences, with the same sequence observed at two different sites three times and at the same site twice. Only one sequence provided a 100% match to organisms databased in RDP II, while the rest fell within 91- 99% sequence similarity to isolates or clones in the database. Five major phyla of bacteria were cultured in this experiment, with three groups of the Proteobacteria dominating at the second growth forests, the Low G+C Gram Positives found to be the most abundant at Albright Grove, and Cytophaga/Flavobacteria/Bacteroides common at all three sites. Community members previously isolated from these sites using different culturing techniques revealed a different composition dominated by Low G+C Gram Positives with fewer Proteobacteria, Cytophaga/Flavobacteria/Bacteroides, and the presence of High G+C Gram Positives which were completely absent from the current study. All identified isolates were determined to be of a ""facultative"" oligotrophic nature due to their ability to grow under extreme oligotrophic conditions but displaying a preference for higher nutrient conditions. The cultivation of novel species is important because it will lead to insight into the biology, physiology, and ecology of the smallest organisms living on this planet, which play some of the largest biogeochemical roles. Future work will focus on creation of conditions conducive to growth of unidentified species by manipulating factors such as pH, unique carbon sources, dilution and incubation parameters

2014-09-16

All rights reserved. For permissions, contact Hunter Library Digital Collections, Western Carolina U, Cullowhee, NC 28723

Great Smoky Mountains National Park (N.C. and Tenn.)

8979 KB(file size)

viii, 59 pages(pages)

Western Carolina University Restricted Electronic Theses and Dissertations