BACKGROUND: The extreme conditions of thermal springs constitute a unique aquatic habitat characterized by low nutrient contents and the absence of human impacts on the microbial community composition. Thus, these springs may host phylogenetically novel microorganisms with potential use in biotechnology. With this hypothesis in mind, we examined the microbial composition of four thermal springs of the world-renowned spa town of Karlovy Vary (Carlsbad), Czechia, which differ in their temperature and chemical composition. RESULTS: Microbial profiling using 16S rRNA gene sequencing revealed the presence of phylogenetically novel taxa at various taxonomic levels, spanning from genera to phyla. Many sequences belonged to novel classes within the phyla Hydrothermae, Altiarchaeota, Verrucomicrobia, and TA06. Cultivation-based methods employing oligotrophic media resulted in the isolation of 44 unique bacterial isolates. These include strains that withstand concentrations of up to 12% NaClw/v in cultivation media or survive a temperature of 100 °C, as well as hitherto uncultured bacterial species belonging to the genera Thermomonas, Paenibacillus, and Cellulomonas. These isolates harbored stress response genes that allow them to thrive in the extreme environment of thermal springs. CONCLUSIONS: Our study is the first to analyze the overall microbial community composition of the renowned Karlovy Vary thermal springs. We provide insight into yet another level of uniqueness of these springs. In addition to their unique health benefits and cultural significance, we demonstrate that these springs harbor phylogenetically distinct microorganisms with unusual life strategies. Our findings open up avenues for future research with the promise of a deeper understanding of the metabolic potential of these microorganisms.

Department of Biochemistry and Microbiology Faculty of Food and Biochemical Technology University of Chemistry and Technology Prague Technicka 3 166 28 Prague 6 Czech Republic

Institute of Balneology and Spa Sciences Karlovy Vary Czech Republic

Military Health Institute Ministry of Defence of the Czech Republic Prague Czech Republic

Zobrazit více v PubMed

Alfaro C, Wallace M. Origin and classification of springs and historical review with current applications. Environ Geol. 1994;24(2):112–124. doi: 10.1007/BF00767884. DOI

Des Marais DJ, Walter MR. Terrestrial hot spring systems: introduction. Astrobiology. 2019;19(12):1419–1432. doi: 10.1089/ast.2018.1976. PubMed DOI PMC

Djokic T, Van Kranendonk MJ, Campbell KA, Walter MR, Ward CR. Earliest signs of life on land preserved in ca. 3.5 Ga hot spring deposits. Nat Commun. 2017;8(1):15263. doi: 10.1038/ncomms15263. PubMed DOI PMC

Dalmaso GZ, Ferreira D, Vermelho AB. Marine extremophiles: a source of hydrolases for biotechnological applications. Mar Drugs. 2015;13(4):1925–1965. doi: 10.3390/md13041925. PubMed DOI PMC

Lewis K, Epstein S, D'Onofrio A, Ling LL. Uncultured microorganisms as a source of secondary metabolites. J Antibiot. 2010;63(8):468–476. doi: 10.1038/ja.2010.87. PubMed DOI

Brock TD, Freeze H. Thermus aquaticus gen. n. and sp. N., a nonsporulating extreme thermophile. J Bacteriol. 1969;98(1):289–297. doi: 10.1128/jb.98.1.289-297.1969. PubMed DOI PMC

Raddadi N, Cherif A, Daffonchio D, Neifar M, Fava F. Biotechnological applications of extremophiles, extremozymes and extremolytes. Appl Microbiol Biotechnol. 2015;99(19):7907–7913. doi: 10.1007/s00253-015-6874-9. PubMed DOI

Staley JT, Konopka A. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol. 1985;39(1):321–346. doi: 10.1146/annurev.mi.39.100185.001541. PubMed DOI

Stewart EJ. Growing unculturable bacteria. J Bacteriol. 2012;194(16):4151–4160. doi: 10.1128/JB.00345-12. PubMed DOI PMC

Gutleben J, Chaib De Mares M, van Elsas JD, Smidt H, Overmann J, Sipkema D. The multi-omics promise in context: from sequence to microbial isolate. Crit Rev Microbiol. 2018;44(2):212–229. doi: 10.1080/1040841X.2017.1332003. PubMed DOI

Fox GE, Pechman KR, Woese CR. Comparative cataloging of 16S ribosomal ribonucleic acid: molecular approach to procaryotic systematics. Int J Syst Evol. 1977;27(1):44–57. doi: 10.1099/00207713-27-1-44. DOI

Clarridge JE. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin Microbiol Rev. 2004;17(4):840–862. doi: 10.1128/CMR.17.4.840-862.2004. PubMed DOI PMC

Nygaard AB, Tunsjø HS, Meisal R, Charnock C. A preliminary study on the potential of Nanopore MinION and Illumina MiSeq 16S rRNA gene sequencing to characterize building-dust microbiomes. Sci Rep. 2020;10(1):3209. doi: 10.1038/s41598-020-59771-0. PubMed DOI PMC

Strunecký O, Kopejtka K, Goecke F, Tomasch J, Lukavský J, Neori A, Kahl S, Pieper DH, Pilarski P, Kaftan D, et al. High diversity of thermophilic cyanobacteria in Rupite hot spring identified by microscopy, cultivation, single-cell PCR and amplicon sequencing. Extremophiles. 2019;23(1):35–48. doi: 10.1007/s00792-018-1058-z. PubMed DOI

Najar IN, Sherpa MT, Das S, Das S, Thakur N. Microbial ecology of two hot springs of Sikkim: predominate population and geochemistry. Sci Total Environ. 2018;637–638:730–745. doi: 10.1016/j.scitotenv.2018.05.037. PubMed DOI

Hobel CFV, Marteinsson VT, Hreggvidsson GÓ, Kristjánsson JK. Investigation of the microbial ecology of intertidal hot springs by using diversity analysis of 16S rRNA and chitinase genes. Appl Environ Microbiol. 2005;71(5):2771–2776. doi: 10.1128/AEM.71.5.2771-2776.2005. PubMed DOI PMC

Paces T, Smejkal V. Magmatic and fossil components of mineral waters in the Eager-river continental rift. In: Wanty R, Seal R, editors. Water–rock interaction. II. Rotterdam: AA Balkema Publishers; 2004. pp. 167–172.

Ward L, Taylor MW, Power JF, Scott BJ, McDonald IR, Stott MB. Microbial community dynamics in Inferno Crater Lake, a thermally fluctuating geothermal spring. ISME J. 2017;11(5):1158–1167. doi: 10.1038/ismej.2016.193. PubMed DOI PMC

Kato S, Itoh T, Yuki M, Nagamori M, Ohnishi M, Uematsu K, Suzuki K, Takashina T, Ohkuma M. Isolation and characterization of a thermophilic sulfur- and iron-reducing thaumarchaeote from a terrestrial acidic hot spring. ISME J. 2019;13(10):2465–2474. doi: 10.1038/s41396-019-0447-3. PubMed DOI PMC

Amin A, Ahmed I, Salam N, Kim B-Y, Singh D, Zhi X-Y, Xiao M, Li W-J. Diversity and distribution of thermophilic bacteria in hot springs of Pakistan. Microb Ecol. 2017;74(1):116–127. doi: 10.1007/s00248-017-0930-1. PubMed DOI

Knapik K, Becerra M, González-Siso M-I. Microbial diversity analysis and screening for novel xylanase enzymes from the sediment of the Lobios Hot Spring in Spain. Sci Rep. 2019;9:11195. doi: 10.1038/s41598-019-47637-z. PubMed DOI PMC

Bourrain M, Suzuki MT, Calvez A, West NJ, Lions J, Lebaron P. In-depth prospection of Avène Thermal Spring Water reveals an uncommon and stable microbial community. J Eur Acad Dermatol Venereol. 2020;34(S5):8–14. doi: 10.1111/jdv.16599. PubMed DOI

Inskeep W, Jay Z, Tringe S, Herrgard M, Rusch D. The YNP metagenome project: environmental parameters responsible for microbial distribution in the Yellowstone geothermal ecosystem. Front Microbiol. 2013;4:67. PubMed PMC

Power JF, Carere CR, Lee CK, Wakerley GLJ, Evans DW, Button M, White D, Climo MD, Hinze AM, Morgan XC, et al. Microbial biogeography of 925 geothermal springs in New Zealand. Nat Commun. 2018;9(1):2876. doi: 10.1038/s41467-018-05020-y. PubMed DOI PMC

Vrba J. Thermal mineral water springs in Karlovy Vary. Environ Geol. 1996;27(2):120–125. doi: 10.1007/BF01061684. DOI

Vrba J. Origin and occurrence of carbon dioxide and gaseous mineral waters, Variscian platform, central Europe. Econ Geol. 1964;59(5):874–882. doi: 10.2113/gsecongeo.59.5.874. DOI

Vylita T, Žák K, Cílek V, Hercman H, Mikšíková L. Evolution of hot-spring travertine accumulation in Karlovy Vary/Carlsbad (Czech Republic) and its significance for the evolution of Teplá Valley and Ohře/Eger Rift. Zeitschrift für Geomorphol. 2007;51(4):427–442. doi: 10.1127/0372-8854/2007/0051-0427. DOI

Pěčková M. Properties of a hyperthermophilic bacterium (Thermus sp.) isolated from a Carlsbad spring. Folia Microbiol. 1991;36(6):515–521. doi: 10.1007/BF02884029. DOI

Kolouchová I, Timkina E, Maťátková O, Kyselová L, Řezanka T. Analysis of bacteriohopanoids from thermophilic bacteria by liquid chromatography-mass spectrometry. Microorganisms. 2021;9(10):2062. doi: 10.3390/microorganisms9102062. PubMed DOI PMC

Gharwalová L, Palyzová A, Marešová H, Kolouchová I, Kyselová L, Řezanka T. Identification of homologous polyprenols from thermophilic bacteria. Microorganisms. 2021;9(6):1168. doi: 10.3390/microorganisms9061168. PubMed DOI PMC

Fraraccio S, Strejcek M, Dolinova I, Macek T, Uhlik O. Secondary compound hypothesis revisited: selected plant secondary metabolites promote bacterial degradation of cis-1,2-dichloroethylene (cDCE) Sci Rep. 2017;7(1):8406. doi: 10.1038/s41598-017-07760-1. PubMed DOI PMC

Parada AE, Needham DM, Fuhrman JA. Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environ Microbiol. 2016;18(5):1403–1414. doi: 10.1111/1462-2920.13023. PubMed DOI

Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, Glöckner FO. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 2013;41(1):e1–e1. doi: 10.1093/nar/gks808. PubMed DOI PMC

R Core Team . R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2021.

Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13(7):581–583. doi: 10.1038/nmeth.3869. PubMed DOI PMC

Yilmaz P, Parfrey LW, Yarza P, Gerken J, Pruesse E, Quast C, Schweer T, Peplies J, Ludwig W, Glöckner FO. The SILVA and “All-species Living Tree Project (LTP)” taxonomic frameworks. Nucleic Acids Res. 2014;42(D1):D643–D648. doi: 10.1093/nar/gkt1209. PubMed DOI PMC

McMurdie PJ, Holmes S. Phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One. 2013;8:e61217. doi: 10.1371/journal.pone.0061217. PubMed DOI PMC

Davis NM, Proctor DM, Holmes SP, Relman DA, Callahan BJ. Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data. Microbiome. 2018;6(1):226. doi: 10.1186/s40168-018-0605-2. PubMed DOI PMC

Hsieh TC, Ma KH, Chao A. iNEXT: an R package for rarefaction and extrapolation of species diversity (Hill numbers) Methods Ecol Evol. 2016;7(12):1451–1456. doi: 10.1111/2041-210X.12613. DOI

Lynch MDJ, Neufeld JD. SSUnique: detecting sequence novelty in microbiome surveys. mSystems. 2016;1(6):e00133-00116. doi: 10.1128/mSystems.00133-16. PubMed DOI PMC

Pruesse E, Peplies J, Glöckner FO. SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics. 2012;28(14):1823–1829. doi: 10.1093/bioinformatics/bts252. PubMed DOI PMC

Yu G. Using ggtree to visualize data on tree-like structures. Curr Protoc Bioinform. 2020;69(1):e96. doi: 10.1002/cpbi.96. PubMed DOI

Hugenholtz P. Exploring prokaryotic diversity in the genomic era. Genome Biol. 2002;3(2):REVIEWS0003. doi: 10.1186/gb-2002-3-2-reviews0003. PubMed DOI PMC

Strejcek M, Smrhova T, Junkova P, Uhlik O. Whole-cell MALDI-TOF MS versus 16S rRNA gene analysis for identification and dereplication of recurrent bacterial isolates. Front microbiol. 2018;9:1294. doi: 10.3389/fmicb.2018.01294. PubMed DOI PMC

Thomas P, Sekhar AC, Upreti R, Mujawar MM, Pasha SS. Optimization of single plate-serial dilution spotting (SP-SDS) with sample anchoring as an assured method for bacterial and yeast cfu enumeration and single colony isolation from diverse samples. Biotechnol Rep. 2015;8:45–55. doi: 10.1016/j.btre.2015.08.003. PubMed DOI PMC

Wickham H, Navarro D, Pedersen TL. Ggplot2: elegant graphics for data analysis. New York: Springer; 2016.

Wright ES. RNAconTest: comparing tools for noncoding RNA multiple sequence alignment based on structural consistency. RNA. 2020;26(5):531–540. doi: 10.1261/rna.073015.119. PubMed DOI PMC

Paradis E, Schliep K. Ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics. 2019;35(3):526–528. doi: 10.1093/bioinformatics/bty633. PubMed DOI

Lopez-Echartea E, Suman J, Smrhova T, Ridl J, Pajer P, Strejcek M, Uhlik O. Genomic analysis of dibenzofuran-degrading Pseudomonas veronii strain Pvy reveals its biodegradative versatility. G3 Genes. 2020;11(2):jkaa030. PubMed PMC

Wick RR, Judd LM, Holt KE. Performance of neural network basecalling tools for Oxford Nanopore sequencing. Genome Biol. 2019;20(1):129. doi: 10.1186/s13059-019-1727-y. PubMed DOI PMC

Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017;27(5):722–736. doi: 10.1101/gr.215087.116. PubMed DOI PMC

Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 2015;25(7):1043–1055. doi: 10.1101/gr.186072.114. PubMed DOI PMC

Arkin AP, Cottingham RW, Henry CS, Harris NL, Stevens RL, Maslov S, Dehal P, Ware D, Perez F, Canon S. KBase: the United States department of energy systems biology knowledgebase. Nat Biotechnol. 2018;36(7):566–569. doi: 10.1038/nbt.4163. PubMed DOI PMC

Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068–2069. doi: 10.1093/bioinformatics/btu153. PubMed DOI

Davis JJ, Wattam AR, Aziz RK, Brettin T, Butler R, Butler RM, Chlenski P, Conrad N, Dickerman A, Dietrich EM, et al. The PATRIC Bioinformatics Resource Center: expanding data and analysis capabilities. Nucleic Acids Res. 2019;48(D1):D606–D612. PubMed PMC

Graham E, Heidelberg J, Tully B. Potential for primary productivity in a globally-distributed bacterial phototroph. ISME J. 2018;12(7):1861–1866. doi: 10.1038/s41396-018-0091-3. PubMed DOI PMC

Yoon S-H, Ha S-M, Lim J, Kwon S, Chun J. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek. 2017;110(10):1281–1286. doi: 10.1007/s10482-017-0844-4. PubMed DOI

Kim D, Park S, Chun J. Introducing EzAAI: a pipeline for high throughput calculations of prokaryotic average amino acid identity. J Microbiol. 2021;59(5):476–480. doi: 10.1007/s12275-021-1154-0. PubMed DOI

Na S-I, Kim YO, Yoon S-H, Sung-min H, Baek I, Chun J. UBCG: up-to-date bacterial core gene set and pipeline for phylogenomic tree reconstruction. J Microbiol. 2018;56(4):281–285. doi: 10.1007/s12275-018-8014-6. PubMed DOI

Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547. doi: 10.1093/molbev/msy096. PubMed DOI PMC

Chaudhari NM, Gupta VK, Dutta C. BPGA-an ultra-fast pan-genome analysis pipeline. Sci Rep. 2016;6(1):1–10. doi: 10.1038/srep24373. PubMed DOI PMC

Aramaki T, Blanc-Mathieu R, Endo H, Ohkubo K, Kanehisa M, Goto S, Ogata H. KofamKOALA: KEGG ortholog assignment based on profile HMM and adaptive score threshold. Bioinformatics. 2020;36(7):2251–2252. doi: 10.1093/bioinformatics/btz859. PubMed DOI PMC

Parks DH, Tyson GW, Hugenholtz P, Beiko RG. STAMP: statistical analysis of taxonomic and functional profiles. Bioinformatics. 2014;30(21):3123–3124. doi: 10.1093/bioinformatics/btu494. PubMed DOI PMC

Setlow P. Observations on research with spores of Bacillales and Clostridiales species. J Appl Microbiol. 2019;126(2):348–358. doi: 10.1111/jam.14067. PubMed DOI PMC

Millero FJ, Feistel R, Wright DG, McDougall TJ. The composition of Standard Seawater and the definition of the Reference-Composition Salinity Scale. Deep-Sea Res I Oceanogr Res Pap. 2008;55(1):50–72. doi: 10.1016/j.dsr.2007.10.001. DOI

Larsen H. Halophilic and halotolerant microorganisms-an overview and historical perspective. FEMS Microbiol Rev. 1986;39:3–7. doi: 10.1111/j.1574-6968.1986.tb01835.x. DOI

Chun J, Oren A, Ventosa A, Christensen H, Arahal DR, Da Costa MS, Rooney AP, Yi H, Xu X-W, De Meyer S, et al. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol. 2018;68(1):461–466. doi: 10.1099/ijsem.0.002516. PubMed DOI

Sharma N, Kumar J, Abedin MM, Sahoo D, Pandey A, Rai AK, Singh SP. Metagenomics revealing molecular profiling of community structure and metabolic pathways in natural hot springs of the Sikkim Himalaya. BMC Microbiol. 2020;20(1):246. doi: 10.1186/s12866-020-01923-3. PubMed DOI PMC

Chen Y, Wu L, Boden R, Hillebrand A, Kumaresan D, Moussard H, Baciu M, Lu Y, Colin MJ. Life without light: microbial diversity and evidence of sulfur- and ammonium-based chemolithotrophy in Movile Cave. ISME J. 2009;3(9):1093–1104. doi: 10.1038/ismej.2009.57. PubMed DOI

Rojas-Gätjens D, Arce-Rodríguez A, Puente-Sánchez F, Avendaño R, Libby E, Conejo-Barboza G, Mora-Amador R, Rojas K, Pieper DH, Chavarría M. Rapid shift in microbial community structure in a neutral hydrothermal hot spring from Costa Rica. bioRxiv. 2020.

DeCastro M-E, Doane MP, Dinsdale EA, Rodríguez-Belmonte E, González-Siso M-I. Exploring the taxonomical and functional profile of As Burgas hot spring focusing on thermostable β-galactosidases. Sci Rep. 2021;11(1):101. doi: 10.1038/s41598-020-80489-6. PubMed DOI PMC

Krauze P, Kämpf H, Horn F, Liu Q, Voropaev A, Wagner D, Alawi M. Microbiological and geochemical survey of CO2-dominated mofette and mineral waters of the Cheb Basin, Czech Republic. Front Microbiol. 2017;8:2446. doi: 10.3389/fmicb.2017.02446. PubMed DOI PMC

Deja-Sikora E, Gołębiewski M, Kalwasińska A, Krawiec A, Kosobucki P, Walczak M. Comamonadaceae OTU as a remnant of an ancient microbial community in sulfidic waters. Microb Ecol. 2019;78(1):85–101. doi: 10.1007/s00248-018-1270-5. PubMed DOI PMC

Han Y, Perner M. The globally widespread genus Sulfurimonas: versatile energy metabolisms and adaptations to redox clines. Front Microbiol. 2015;6:989. PubMed PMC

Emerson D, Moyer C. Isolation and characterization of novel iron-oxidizing bacteria that grow at circumneutral pH. Appl Environ Microbiol. 1997;63(12):4784–4792. doi: 10.1128/aem.63.12.4784-4792.1997. PubMed DOI PMC

Bornemann TLV, Adam PS, Turzynski V, Schreiber U, Figueroa-Gonzalez PA, Rahlff J, Köster D, Schmidt TC, Schunk R, Krauthausen B, et al. Genetic diversity in terrestrial subsurface ecosystems impacted by geological degassing. Nat Commun. 2022;13(1):284. doi: 10.1038/s41467-021-27783-7. PubMed DOI PMC

Sun Y, Liu Y, Pan J, Wang F, Li M. Perspectives on cultivation strategies of Archaea. Microb Ecol. 2020;79(3):770–784. doi: 10.1007/s00248-019-01422-7. PubMed DOI

Oliver JD. The viable but nonculturable state for bacteria: status update. Microbe. 2016;11(4):159–164.

Davis KER, Joseph SJ, Janssen PH. Effects of growth medium, inoculum size, and incubation time on culturability and isolation of soil bacteria. Appl Environ Microbiol. 2005;71(2):826–834. doi: 10.1128/AEM.71.2.826-834.2005. PubMed DOI PMC

Bender KE, Glover K, Archey A, Barton HA. The impact of sample processing and media chemistry on the culturable diversity of bacteria isolated from a cave. Int J Speleol. 2020;49(3):3. doi: 10.5038/1827-806X.49.3.2337. DOI

Rahul K, Sharma RC. Microbial diversity and physico-chemical attributes of two hot water springs in the Garhwal Himalaya, India. J Microbiol Biotechnol Food Sci. 2019;8(6):1249–1253. doi: 10.15414/jmbfs.2019.8.6.1249-1253. DOI

Burns DG, Camakaris HM, Janssen PH, Dyall-Smith ML. Combined use of cultivation-dependent and cultivation-independent methods indicates that members of most Haloarchaeal groups in an Australian crystallizer pond are cultivable. Appl Environ Microbiol. 2004;70(9):5258–5265. doi: 10.1128/AEM.70.9.5258-5265.2004. PubMed DOI PMC

Egan M, Dempsey E, Ryan CA, Ross RP, Stanton C. The sporobiota of the human gut. Gut Microbes. 2021;13(1):1–17. doi: 10.1080/19490976.2020.1863134. PubMed DOI PMC

Kurm V, Van Der Putten WH, Hol WHG. Cultivation-success of rare soil bacteria is not influenced by incubation time and growth medium. PLoS ONE. 2019;14(1):e0210073. doi: 10.1371/journal.pone.0210073. PubMed DOI PMC

Kim S, Park MS, Song J, Kang I, Cho J-C. High-throughput cultivation based on dilution-to-extinction with catalase supplementation and a case study of cultivating acI bacteria from Lake Soyang. J Microbiol. 2020;58(11):893–905. doi: 10.1007/s12275-020-0452-2. PubMed DOI

Nichols D, Cahoon N, Trakhtenberg EM, Pham L, Mehta A, Belanger A, Kanigan T, Lewis K, Epstein SS. Use of Ichip for high-throughput in situ cultivation of “uncultivable” microbial species. Appl Environ Microbiol. 2010;76(8):2445. doi: 10.1128/AEM.01754-09. PubMed DOI PMC

Kaeberlein T, Lewis K, Epstein SS. Isolating "uncultivable" microorganisms in pure culture in a simulated natural environment. Science. 2002;296(5570):1127–1129. doi: 10.1126/science.1070633. PubMed DOI

Jung D, Seo E-Y, Owen JS, Aoi Y, Yong S, Lavrentyeva EV, Ahn TS. Application of the filter plate microbial trap (FPMT), for cultivating thermophilic bacteria from thermal springs in Barguzin area, eastern Baikal, Russia. Biosci Biotechnol Biochem. 2018;82(9):1624–1632. doi: 10.1080/09168451.2018.1482194. PubMed DOI

Coker JA. Extremophiles and biotechnology: current uses and prospects. F1000Research. 2016;5:396. doi: 10.12688/f1000research.7432.1. PubMed DOI PMC

Tindall KR, Kunkel TA. Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry. 1988;27(16):6008–6013. doi: 10.1021/bi00416a027. PubMed DOI

Li X, Zhang S, Zhang Q, Gan L, Jiang G, Tian Y, Shi B. Characterization and application of a novel halotolerant protease with no collagenase activity for cleaner dehairing of goatskin. Process Biochem. 2022;113:203–215. doi: 10.1016/j.procbio.2022.01.006. DOI

Moridshahi R, Bahreini M, Sharifmoghaddam M, Asoodeh A. Biochemical characterization of an alkaline surfactant-stable keratinase from a new keratinase producer, Bacillus zhangzhouensis. Extremophiles. 2020;24(5):693–704. doi: 10.1007/s00792-020-01187-9. PubMed DOI

Soy S, Nigam VK, Sharma SR. Cellulolytic, amylolytic and xylanolytic potential of thermophilic isolates of Surajkund hot spring. J Biosci. 2019;44(5):124. doi: 10.1007/s12038-019-9938-7. PubMed DOI

Panosyan H, Margaryan A, Birkeland N-K. Geothermal springs in Armenia and Nagorno-Karabakh: potential sources of hydrolase-producing thermophilic bacilli. Extremophiles. 2020;24(4):519–536. doi: 10.1007/s00792-020-01173-1. PubMed DOI

Timkina E, Drábová L, Palyzová A, Řezanka T, Maťátková O, Kolouchová I. Kocuria strains from unique radon spring water from Jachymov Spa. Ferment. 2022;8(1):35. doi: 10.3390/fermentation8010035. DOI

Jiao JY, Liu L, Hua ZS, Fang BZ, Zhou EM, Salam N, Hedlund BP, Li WJ. Microbial dark matter coming to light: challenges and opportunities. Natl Sci Rev. 2021;8(3):nwaa280. doi: 10.1093/nsr/nwaa280. PubMed DOI PMC

Fasesan D, Dawkins K, Ramirez R, Rasheed-Jada H, Onilude A, Nash O, Esiobu N. Analysis of a tropical warm spring microbiota using 16S rRNA metabarcoding. Adv Microbiol. 2020;10:145–165. doi: 10.4236/aim.2020.104012. DOI

Lloyd KG, Steen AD, Ladau J, Yin J, Crosby L, Neufeld JD. Phylogenetically novel uncultured microbial cells dominate earth microbiomes. mSystems. 2018;3(5):e00055-00018. doi: 10.1128/mSystems.00055-18. PubMed DOI PMC

Inskeep W, Jay Z, Herrgard M, Kozubal M, Rusch D, Tringe S, Macur R, deM R, Jennings R, Boyd E, Spear J, et al. Phylogenetic and functional analysis of metagenome sequence from high-temperature archaeal habitats demonstrate linkages between metabolic potential and geochemistry. Front Microbiol. 2013;4:95. PubMed PMC

Jungbluth SP, Amend JP, Rappé MS. Metagenome sequencing and 98 microbial genomes from Juan de Fuca Ridge flank subsurface fluids. Sci Data. 2017;4(1):170037. doi: 10.1038/sdata.2017.37. PubMed DOI PMC

López-López O, Cerdán ME, González-Siso MI. Hot spring metagenomics. Life. 2013;3(2):308–320. doi: 10.3390/life3020308. PubMed DOI PMC

Marín-Paredes R, Tapia-Torres Y, Martínez-Romero E, Quesada M, Servín-Garcidueñas LE, Newton ILG. Metagenome assembly and metagenome-assembled genome of “Candidatus Aramenus sulfurataquae” from thermal sediments from the Los Azufres volcanic complex. Microbiol Resour Announc. 2021;10(39):e00379-00321. doi: 10.1128/MRA.00379-21. PubMed DOI PMC

Probst AJ, Weinmaier T, Raymann K, Perras A, Emerson JB, Rattei T, Wanner G, Klingl A, Berg IA, Yoshinaga M, et al. Biology of a widespread uncultivated archaeon that contributes to carbon fixation in the subsurface. Nat Commun. 2014;5(1):5497. doi: 10.1038/ncomms6497. PubMed DOI

Bird JT, Baker BJ, Probst AJ, Podar M, Lloyd KG. Culture independent genomic comparisons reveal environmental adaptations for Altiarchaeales. Front Microbiol. 2016;7:1221. doi: 10.3389/fmicb.2016.01221. PubMed DOI PMC

Dombrowski N, Lee J-H, Williams TA, Offre P, Spang A. Genomic diversity, lifestyles and evolutionary origins of DPANN archaea. FEMS Microbiol Lett. 2019;366(2):fnz008. doi: 10.1093/femsle/fnz008. PubMed DOI PMC

Bai Y, Wang J, Zhan Z, Guan L, Jin L, Zheng G, Huang Z. The variation of microbial communities in a depth profile of peat in the Gahai lake wetland natural conservation area. Geomicrobiol J. 2018;35(6):484–490. doi: 10.1080/01490451.2017.1392651. DOI

Schneider D, Arp G, Reimer A, Reitner J, Daniel R. Phylogenetic analysis of a microbialite-forming microbial mat from a hypersaline lake of the Kiritimati Atoll, Central Pacific. PLoS ONE. 2013;8(6):e66662. doi: 10.1371/journal.pone.0066662. PubMed DOI PMC

Chiang E, Schmidt ML, Berry MA, Biddanda BA, Burtner A, Johengen TH, Palladino D, Denef VJ. Verrucomicrobia are prevalent in north-temperate freshwater lakes and display class-level preferences between lake habitats. PLoS ONE. 2018;13(3):e0195112. doi: 10.1371/journal.pone.0195112. PubMed DOI PMC

Erikstad H-A, Ceballos RM, Smestad NB, Birkeland N-K. Global biogeographic distribution patterns of thermoacidophilic Verrucomicrobia methanotrophs suggest allopatric evolution. Front Microbiol. 2019;10:1129. doi: 10.3389/fmicb.2019.01129. PubMed DOI PMC

Magnabosco C, Tekere M, Lau MCY, Linage B, Kuloyo O, Erasmus M, Cason E, van Heerden E, Borgonie G, Kieft TL, et al. Comparisons of the composition and biogeographic distribution of the bacterial communities occupying South African thermal springs with those inhabiting deep subsurface fracture water. Front Microbiol. 2014;5:679. doi: 10.3389/fmicb.2014.00679. PubMed DOI PMC

He S, Stevens SLR, Chan L-K, Bertilsson S, del Rio TG, Tringe SG, Malmstrom RR, McMahon KD, Hallam SJ. Ecophysiology of freshwater Verrucomicrobia inferred from metagenome-assembled genomes. mSphere. 2017;2(5):e00277-00217. doi: 10.1128/mSphere.00277-17. PubMed DOI PMC

Batani G, Bayer K, Böge J, Hentschel U, Thomas T. Fluorescence in situ hybridization (FISH) and cell sorting of living bacteria. Sci Rep. 2019;9(1):18618. doi: 10.1038/s41598-019-55049-2. PubMed DOI PMC

Kumar VS, Maranas CD. GrowMatch: an automated method for reconciling in silico/in vivo growth predictions. PLoS Comput Biol. 2009;5(3):e1000308. doi: 10.1371/journal.pcbi.1000308. PubMed DOI PMC

Lee I, Kim YO, Park S-C, Chun J. OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol. 2016;66(2):1100–1103. doi: 10.1099/ijsem.0.000760. PubMed DOI

Guardabassi L, Perichon B, Heijenoort JV, Blanot D, Courvalin P. Glycopeptide resistance vanA operons in Paenibacillus strains isolated from soil. Antimicrob Agents Chemother. 2005;49(10):4227–4233. doi: 10.1128/AAC.49.10.4227-4233.2005. PubMed DOI PMC

D’Costa VM, King CE, Kalan L, Morar M, Sung WWL, Schwarz C, Froese D, Zazula G, Calmels F, Debruyne R, et al. Antibiotic resistance is ancient. Nature. 2011;477(7365):457–461. doi: 10.1038/nature10388. PubMed DOI

Waglechner N, Wright GD. Antibiotic resistance: it’s bad, but why isn’t it worse? BMC Biol. 2017;15(1):84. doi: 10.1186/s12915-017-0423-1. PubMed DOI PMC

Alves MP, Rainey FA, Nobre MF, da Costa MS. Thermomonas hydrothermalis sp. Nov., a new slightly thermophilic γ-proteobacterium isolated from a hot spring in Central Portugal. Syst Appl Microbiol. 2003;26(1):70–75. doi: 10.1078/072320203322337335. PubMed DOI

Al-Daghistani HI, Mohammad BT, Kurniawan TA, Singh D, Rabadi AD, Xue W, Avtar R, Othman MHD, Shirazian S. Characterization and applications of Thermomonas hydrothermalis isolated from Jordan's hot springs for biotechnological and medical purposes. Process Biochem. 2021;104:171–181. doi: 10.1016/j.procbio.2021.03.010. DOI

Abdollahi P, Ghane M, Babaeekhou L. Isolation and characterization of thermophilic bacteria from Gavmesh Goli hot spring in Sabalan geothermal field, Iran: Thermomonas hydrothermalis and Bacillus altitudinis isolates as a potential source of thermostable protease. Geomicrobiol J. 2021;38(1):87–95. doi: 10.1080/01490451.2020.1812774. DOI

Jessen JE, Sveinsson T, Scully S, Orlygsson J. Ethanol production by a Paenibacillus species isolated from an Icelandic hot spring: production yields from complex biomass. Icel Agric Sci. 2015;28:15–24. doi: 10.16886/IAS.2015.02. DOI

Kumar M, Yadav AN, Tiwari R, Prasanna R, Saxena AK. Evaluating the diversity of culturable thermotolerant bacteria from four hot springs of India. J Biodivers Biopros Dev. 2014;1(3):1000127.

Mead DA, Lucas S, Copeland A, Lapidus A, Cheng J-F, Bruce DC, Goodwin LA, Pitluck S, Chertkov O, Zhang X, et al. Complete genome sequence of Paenibacillus strain Y4.12MC10, a novel Paenibacillus lautus strain isolated from Obsidian hot spring in Yellowstone National Park. Stand Genom Sci. 2012;6(3):366–385. PubMed PMC

Bouraoui H, Rebib H, Aissa MB, Touzel JP, O’donohue M, Manai M. Paenibacillus marinum sp. Nov., a thermophilic xylanolytic bacterium isolated from a marine hot spring in Tunisia. J Basic Microbiol. 2013;53(11):877–883. doi: 10.1002/jobm.201200275. PubMed DOI

Fujinami S, Takeda-Yano K, Onodera T, Satoh K, Sano M, Takahashi Y, Narumi I, Ito M. Draft genome sequence of calcium-dependent Paenibacillus sp. strain TCA20, isolated from a hot spring containing a high concentration of calcium ions. Genome Announc. 2014;2(5):e00866-00814. doi: 10.1128/genomeA.00866-14. PubMed DOI PMC

Characterization of Archaea membrane lipids in radioactive springs using shotgun lipidomics

Compost, plants and endophytes versus metal contamination: choice of a restoration strategy steers the microbiome in polymetallic mine waste

Reaching unreachables: Obstacles and successes of microbial cultivation and their reasons