In terms of the number and diversity of living units, the prokaryotic empire is the most represented form of life on Earth, and yet it is still to a significant degree shrouded in darkness. This microbial "dark matter" hides a great deal of potential in terms of phylogenetically or metabolically diverse microorganisms, and thus it is important to acquire them in pure culture. However, do we know what microorganisms really need for their growth, and what the obstacles are to the cultivation of previously unidentified taxa? Here we review common and sometimes unexpected requirements of environmental microorganisms, especially soil-harbored bacteria, needed for their replication and cultivation. These requirements include resuscitation stimuli, physical and chemical factors aiding cultivation, growth factors, and co-cultivation in a laboratory and natural microbial neighborhood.

• Keywords
• VBNC, cultivation techniques, difficult-to-culture microorganisms, dormancy, environmental microbiome, growth factors, improved cultivation, microbial ecology,
• Publication type
• Journal Article MeSH
• Review MeSH

Balneotherapeutic water springs, such as those with thermal, saline, sulfur, or any other characteristics, have recently been the subject of phylogenetic studies with a closer focus on the description and/or isolation of phylogenetically novel or biotechnologically interesting microorganisms. Generally, however, most such microorganisms are rarely obtained in pure culture or are even, for now, unculturable under laboratory conditions. In this culture-dependent study of radioactive water springs of Jáchymov (Joachimstahl), Czech Republic, we investigated a combination of classical cultivation approaches with those imitating sampling source conditions. Using these environmentally relevant cultivation approaches, over 1,000 pure cultures were successfully isolated from 4 radioactive springs. Subsequent dereplication yielded 121 unique taxonomic units spanning 44 genera and 9 taxonomic classes, ~10% of which were identified as hitherto undescribed taxa. Genomes of the latter were sequenced and analyzed, with a special focus on endogenous defense systems to withstand oxidative stress and aid in radiotolerance. Due to their origin from radioactive waters, we determined the resistance of the isolates to oxidative stress. Most of the isolates were more resistant to menadione than the model strain Deinococcus radiodurans DSM 20539T. Moreover, isolates of the Deinococcacecae, Micrococcaceae, Bacillaceae, Moraxellaceae, and Pseudomonadaceae families even exhibited higher resistance in the presence of hydrogen peroxide. In summary, our culturomic analysis shows that subsurface water springs contain diverse bacterial populations, including as-yet-undescribed taxa and strains with promising biotechnological potential. Furthermore, this study suggests that environmentally relevant cultivation techniques increase the efficiency of cultivation, thus enhancing the chance of isolating hitherto uncultured microorganisms. IMPORTANCE The mine Svornost in Jáchymov (Joachimstahl), Czech Republic is a former silver-uranium mine and the world's first and for a long time only radium mine, nowadays the deepest mine devoted to the extraction of water which is saturated with radon and has therapeutic benefits given its chemical properties. This healing water, which is approximately 13 thousand years old, is used under medical supervision for the treatment of patients with neurological and rheumatic disorders. Our culturomic approach using low concentrations of growth substrates or the environmental matrix itself (i.e., water filtrate) in culturing media combined with prolonged cultivation time resulted in the isolation of a broad spectrum of microorganisms from 4 radioactive springs of Jáchymov which are phylogenetically novel and/or bear various adaptive or coping mechanisms to thrive under selective pressure and can thus provide a wide spectrum of capabilities potentially exploitable in diverse scientific, biotechnological, or medical disciplines.

• Keywords
• environmentally relevant cultivation approaches, extremophiles, novel taxa, oxidative stress response, radioactive water springs, subsurface microbiology,
• MeSH
• Bacteria MeSH
• Phylogeny MeSH
• Humans MeSH
• Adolescent MeSH
• Hydrogen Peroxide MeSH
• Radium * MeSH
• Radon * MeSH
• Sulfur MeSH
• Silver MeSH
• Uranium * MeSH
• Vitamin K 3 MeSH
• Water MeSH
• Check Tag
• Humans MeSH
• Adolescent MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hydrogen Peroxide MeSH
• Radium * MeSH
• Radon * MeSH
• Sulfur MeSH
• Silver MeSH
• Uranium * MeSH
• Vitamin K 3 MeSH
• Water MeSH