Abstract
Uncultured microorganisms comprise most of the microbial diversity existing on our planet. Despite advances in environmental sequencing and single-cell genomics, in-depth studies about bacterial metabolism and screening of novel bioproducts can only be assessed by culturing microbes in the laboratory. Here we report uncultured, or recalcitrant, microorganisms from an Antarctic soil sample, using relatively simple methods: oligotrophic media, extended incubation periods, observation under stereo microscopy, and selection of slow-growing bacteria. We managed to isolate several rare microorganisms belonging to infrequently isolated or recently described genera, for example Lapillicoccus, Flavitalea, Quadrisphaera, Motilibacter, and Polymorphobacter. Additionally, we obtained isolates presenting 16S rRNA sequence similarity ranging from 92.08 to 94.46% with any other known cultured species, including two distinct isolates from the class Thermoleophilia, that although common in Antarctic soils (as identified by metagenomics), was never reported to be isolated from such samples. Our data indicates that simple methods are still useful for cultivating recalcitrant microorganisms, even when dealing with samples from extreme environments.
Highlights
Most environmental microorganisms are still classified as uncultivable (Hug et al, 2016), or using the best definition, “yet-to-be cultivable,” considering that all organisms should be able to grow and divide, and capable of growing under the proper conditions (Lewis et al, 2010; Vartoukian et al, 2010; Stewart, 2012)
Samples were collected on December 29, 2014, at Hennequin Point, Admiralty Bay, King George Island, Antarctica (Figure 1) during the XXXIII Brazilian Antarctic Operation (2014/2015)
We did not collect all slow-growing colonies that appeared on the plates, as several similar slow-growing morphotypes were observed after the same incubation period
Summary
Reasons for failing to grow most of the existing microorganisms in the laboratory vary. For example failing to supplement the correct nutrients (Kopke et al, 2005), using the wrong pH, insufficient incubation periods (Janssen et al, 2002; Davis et al, 2011), or the use of extremely rich-media (which favors fast growing cells, that can overgrow slower microorganisms). Some are more complex, such as the need for specific growth signals (Bruns et al, 2002; Nichols et al, 2008), dependence on other microorganisms (D’Onofrio et al, 2010; He et al, 2015), or development as microcolonies (Davis et al, 2011). Many reasons justify the interest in growing “uncultivable” organisms. Features such as determination of growth preferences, consumption or production of environmental metabolites
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