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Culturing uncultured environmental microorganisms.


Research on natural environments, over the last decade, is replete with microbial diversity studies that used culture -independent approaches. The cloning and sequencing of the 16S rRNA genes has been the driving force in the expansion of awareness about the great diversity of previously undiscovered microorganisms. Well-known uncultured groups of microorganisms are numerous, and half of the known phylogenetic divisions of bacteria are not represented in any culture collection. It is no longer assumed that cultures acquired from an environment represent the dominant or physiologically important organisms from that environment. A high throughput culturing (HTC) technique was developed in an attempt to bring into culture some of these widespread and uncultured microorganisms. Over the course of 3 years, 2,484 culturing attempts were screened for microbial growth from sample collections off the coast of Oregon and 576 attempts from groundwater at McClellan Air Force Base (MAFB). However, using the HTC approach up to 14% of the microorganisms counted by direct microscopy were cultured. In contrast, less than 1% of the microorganisms from natural environments that are observed under a microscope can be grown using standard agar plating techniques. This newly developed technique was successful at bringing into culture 11 previously uncultured or undescribed Proteobacteria. Four were isolated from the marine environment including, members of the SAR11 clade (alpha subclass), OM43 (beta-subclass), SAR92 (gamma subclass), and OM60/OM241 (gamma subclass). SARI 1 was transiently cultured in this study but was later successfully brought into culture using these HTC techniques by Mike Rappe. Eight were isolated from a trichloroethene (TCE) and cis-dichloroethene (cis-DCE) contaminated aquifer, including members of the MHP14 clade (alpha subclass), 4-Org1-14 clade (alpha subclass), Herbaspirillum/Oxalobacter clade (beta subclass), HTCC333 (beta subclass), HTCC410 (beta subclass), PM1 clade (beta subclass), Boom-7m-04 clade (beta subclass) and OM43 clade (beta subclass). Culturing microorganisms is an important step towards understanding their physiology and ecology, and in most cases is necessary for the formal systematic description of a new species. For microorganisms of global significance, such as the major uncultured bacterioplankton and soil microbiota, obtaining cultures is a prerequisite for obtaining complete genome sequences and understanding the relevance of these microorganisms to biogeochemical cycles.