Cultivation and characterization of the bacterial assemblage of epsomic Basque Lake, BC

  • James D. Crisler
  • Fei Chen
  • Benton C. Clark
  • Mark A. SchneegurtEmail author
Original Paper


Athalassohaline waters that are rich in divalent ions are good analogues for the chemical environments of Mars and the ocean worlds. Sulfate salts, along with chlorides, are important in Mars regolith with Ca, Fe, Mg, and Na counterions. Certain lakes in the Pacific Northwest are saturated with MgSO4 as epsomite. Here we report on the microbial community of Basque Lake, BC, a group of playas that is saturated with MgSO4. More than 60 bacterial isolates were obtained from Basque Lake soils by enrichment culture and repetitive streak-plating using media containing 10% (~ 1.7 M) NaCl or 50% (~ 2 M) MgSO4. Most of the isolates (~ 75%) were Gram-positive, motile, and produced endospores. Isolates related to Marinococcus halophilus and Virgibacillus marismortui dominated the collection. Halomonas and Salinivibrio were Gram-negative genera found at Basque Lake. Nearly all of the Basque Lake isolates grew at 50% MgSO4, with 65% growing at 60% MgSO4. Several isolates could grow in saturated (67%) MgSO4 (aw = 0.90). All of the isolates grew at 10% NaCl with 70% growing at 20% salinity (~ 3.5 M NaCl; aw = 0.82). Basque Lake isolates grew better at basic pH than acidic pH, with 80% growing at pH 9 and 30% growing at pH 10. Only 20% of the isolates grew at pH 5. Numerical taxonomy dendrograms based on 44 phenetic characteristics showed a strong correspondence to phylogenetic trees constructed from 16S rRNA gene sequences. Pyrosequencing of 16S rRNA gene sequences from direct DNA extracts of Basque Lake soils recovered predominantly Proteobacteria (60%), Firmicutes (11%), and unclassified bacteria (27%). Microbes capable of growth under the extreme chemical conditions of Mars are a particular concern for forward planetary protection should they contaminate a spacecraft.


Astrobiology Epsomite Extremophiles Halotolerance Mars Salinotolerance 



The authors are thankful for the preliminary and supportive work done by Amer Al Soudi, Bishal Bista, John Dille, Timothy Eberl, Casper Fredsgaard, Brian Kilmer, Tony Mai, Donald Moore, Trista Newville, Kyle Rowe, Namrata Shrestha, and Karen Woltersdorf. We are grateful to Bruce Madu and Jim Britton (British Columbia Ministry of Energy and Mines; BCMEM) for collecting and documenting Basque Lake samples. We thank Fadi Aramouni (Kansas State University) for performing water activity measurements and Stephen Lindemann (Purdue University) for deep sequencing analyses. Preliminary accounts of this work have been presented previously (Crisler et al. 2010a, b, 2018; Kilmer et al. 2012). This work was supported by awards from National Aeronautics and Space Administration (NASA), Research Opportunities in Space and Earth Science (ROSES), Planetary Protection Research (09-PPR09-0004 and 14-PPR14-2-0002). Additional student support was from Kansas Institutional Development Award (IDeA) Networks of Biomedical Research Excellence (KINBRE), National Institute of General Medical Sciences (NIGMS), National Institutes of Health (NIH) (P20 GM103418). The content is solely the responsibility of the authors and does not necessarily represent the official views of BCMEM, KINBRE, NASA, NIGMS or NIH.

Authors’ Contributions

JDC, FC, BCC and MAS conceived and designed the study. JDC, FC and MAS performed research and analyzed data. JDC and MAS wrote the paper.

Compliance with ethical standards

Conflicts of interest

The authors have no conflicts of interest.

Human and animal rights statement

Neither animal nor human subjects were part of this work.

Supplementary material

10482_2019_1244_MOESM1_ESM.png (40 kb)
Figure S1. Phylogenetic tree for Gram-positive bacteria from Basque Lake based on 16S rRNA gene sequences. GenBank accession numbers, sampling site, enrichment temperature, and enrichment medium (10% NaCl or 50% MgSO4) are given for each isolate. (PNG 40 kb)
10482_2019_1244_MOESM2_ESM.png (17 kb)
Figure S2. Phylogenetic tree for Gram-negative bacteria from Basque Lake based on 16S rRNA gene sequences. GenBank accession numbers, sampling site, enrichment temperature, and enrichment medium (10% NaCl or 50% MgSO4) are given for each isolate. (PNG 16 kb)
10482_2019_1244_MOESM3_ESM.docx (129 kb)
Supplementary material 3 (DOCX 128 kb)
10482_2019_1244_MOESM4_ESM.docx (125 kb)
Supplementary material 4 (DOCX 124 kb)
10482_2019_1244_MOESM5_ESM.docx (164 kb)
Supplementary material 5 (DOCX 163 kb)
10482_2019_1244_MOESM6_ESM.docx (168 kb)
Supplementary material 6 (DOCX 168 kb)
10482_2019_1244_MOESM7_ESM.docx (129 kb)
Supplementary material 7 (DOCX 129 kb)
10482_2019_1244_MOESM8_ESM.docx (125 kb)
Supplementary material 8 (DOCX 125 kb)
10482_2019_1244_MOESM9_ESM.docx (60 kb)
Supplementary material 9 (DOCX 59 kb)


  1. Al Soudi AF, Farhat O, Chen F, Clark BC, Schneegurt MA (2017) Bacterial growth tolerance to concentrations of chlorate and perchlorate salts relevant to Mars. Int J Astrobiol 16:229–235CrossRefGoogle Scholar
  2. Altheide T, Chevrier V, Nicholson C, Denson J (2009) Experimental investigation of the stability and evaporation of sulfate and chloride brines on Mars. Earth Planet Sci Lett 282:69–78CrossRefGoogle Scholar
  3. Anderson GC (1958) Some limnological features of a shallow saline meromictic lake. Limnol Oceanog 3:259–270CrossRefGoogle Scholar
  4. Boring J, Kushner DJ, Gibbons NE (1963) Specificity of the salt requirement of Halobacterium cutirubrum. Can J Microbiol 2:143–154CrossRefGoogle Scholar
  5. Caton IR, Schneegurt MA (2012) Culture-independent analysis of the soil bacterial assemblage at the Great Salt Plains of Oklahoma. J Basic Microbiol 52:16–26CrossRefGoogle Scholar
  6. Caton TM, Witte LR, Ngyuen HD, Buchheim JA, Buchheim MA, Schneegurt MA (2004) Halotolerant aerobic heterotrophic bacteria from the Great Salt Plains of Oklahoma. Microb Ecol 48:449–462CrossRefGoogle Scholar
  7. Chen F, Vaishampayan P, La Duc M, Probst A, Schneegurt MA, Clark B (2016) Comparative phylogenetic analysis of the microbial communities in a spacecraft assembly facility and in epsomite lakes. In: Abstracts and program, 116th General Meeting of the American Society for Microbiology, Boston, June 2016Google Scholar
  8. Chevrier VF, Altheide TS (2008) Low temperature aqueous ferric sulfate solutions on the surface of Mars. Geophys Res Lett 35:L22101CrossRefGoogle Scholar
  9. Chevrier VF, Hanley J, Altheide TS (2009) Stability of perchlorate hydrates and their liquid solutions at the Phoenix landing site, Mars. Geophys Res Lett 36:L10202CrossRefGoogle Scholar
  10. Clark BC (1993) Geochemical components in Martian soil. Geochim Cosmochim Acta 57:4575–4581CrossRefGoogle Scholar
  11. Clark BC, Kounaves SP (2016) Evidence for the distribution of perchlorates on Mars. Int J Astrobiol 15:311–318CrossRefGoogle Scholar
  12. Clark BC, van Hart D (1981) The salts of Mars. Icarus 45:370–378CrossRefGoogle Scholar
  13. Clark BC, Morris RV, McLennan SM, Gellert R, Jolliff B, Knoll AH, Squyres SW, Lowenstein TK, Ming DW, Tosca NJ, Yen A, Christensen PR, Gorevan S, Brückner J, Calvin W, Dreibus G, Farrand W, Klingelhoefer G, Waenke H, Zipfel J, Bell JF III, Grotzinger J, McSween HY, Rieder R (2005) Chemistry and mineralogy of outcrops at Meridiani Planum. Earth Planet Sci Lett 240:73–94CrossRefGoogle Scholar
  14. Crisler JD, Kilmer BR, Cunderla B, Madu BE, Schneegurt MA (2010a) Isolation and characterization of microbes from Basque Lake, BC, and Hot Lake, WA, environments with high magnesium sulfate concentrations. In: Abstracts and program, 110th General Meeting of the American Society for Microbiology, San Diego, May 2010Google Scholar
  15. Crisler JD, Kilmer BR, Rowe K, Cunderla B, Madu BE, Schneegurt MA (2010b) Isolation and characterization of microbes from Basque Lake, BC, and Hot Lake, WA, environments with high magnesium sulfate concentrations. In: 142nd Annual Meeting of the Kansas Academy of Science, Hays, April 2010. Trans KS Acad Sci 113:122Google Scholar
  16. Crisler JD, Newville TM, Chen F, Clark BC, Schneegurt MA (2012) Bacterial growth at the high concentrations of magnesium sulfate found in Martian soils. Astrobiology 12:98–106CrossRefGoogle Scholar
  17. Crisler JD, Chen F, Clark BC, Schneegurt MA (2018) Numerical taxonomy and phylogenetic analyses of the microbial community of epsomic Basque Lake, BC. In: Abstracts and program, 118th General Meeting of the American Society for Microbiology, Atlanta, June 2018Google Scholar
  18. Cull SC, Arvidson RE, Catalano JG, Ming DW, Morris RV, Mellon MT, Lemmon M (2010) Concentrated perchlorate at the Mars phoenix landing site: evidence for thin film liquid water on Mars. Geophys Res Lett 37:L22203Google Scholar
  19. Edwards U, Rogall H, Blöcker H, Emde M, Böttger EC (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucl Acids Res 17:7843–7853CrossRefGoogle Scholar
  20. Foster IS, King PL, Hyde BC, Southam G (2010) Characterization of halophiles in natural MgSO4 salts and laboratory enrichment samples: astrobiological implications for Mars. Planet Space Sci 58:599–615CrossRefGoogle Scholar
  21. Fox-Powell MG, Hallsworth JE, Cousins CR, Cockell CS (2016) Ionic strength is a barrier to habitability of Mars. Astrobiology 16:427–442CrossRefGoogle Scholar
  22. Fredsgaard C, Moore DB, Al Soudi AF, Crisler JD, Chen F, Clark BC, Schneegurt MA (2017a) Relationships between sucretolerance and salinotolerance in bacteria from hypersaline environments and their implications for the exploration of Mars and the icy worlds. Int J Astrobiol 16:156–162CrossRefGoogle Scholar
  23. Fredsgaard C, Moore DB, Chen F, Clark BC, Schneegurt MA (2017b) Prevalence of sucretolerant bacteria in common soils and their isolation and characterization. Ant Leeuwen 110:995–1005CrossRefGoogle Scholar
  24. Gendrin A, Mangold N, Bibring JP, Langevin Y (2005) Sulfates in Martian layered terrains: the OMEGA/Mars Express view. Science 307:1587–1591CrossRefGoogle Scholar
  25. Grant WD (2004) Life at low water activity. Philos Trans R Soc Lond B 359:1249–1267CrossRefGoogle Scholar
  26. Hammer UT (1978) The saline lakes of Saskatchewan. III. Chemical characterization. Int Rev Hydrobiol 63:311–335CrossRefGoogle Scholar
  27. Hammer UT (1986) Saline lake ecosystems of the world. Junk, DordrechtGoogle Scholar
  28. Hammer UT, Haynes RC (1978) The saline lakes of Saskatchewan. 2. Locale, hydrology and other physical aspects. Int Rev Ges Hydrobiol Hydrog 63:179–203CrossRefGoogle Scholar
  29. Handy FM (1916) An investigation of the mineral deposits of northern Okanogan County. Department of Geology Bulletin 100, State College of Washington, Pullman, WA, pp 27Google Scholar
  30. Haynes RC, Hammer UT (1978) The saline lakes of Saskatchewan. IV. Primary production by phytoplankton in selected saline ecosystems. Int Rev Hydrobiol 63:337–351CrossRefGoogle Scholar
  31. Hussmann H, Sohl F, Spohn T (2006) Subsurface oceans and deep interiors of medium-sized outer planet satellites and large trans-neptunian objects. Icarus 185:258–273CrossRefGoogle Scholar
  32. Hyde BC, Foster IS, King PL, Southam G, Nushaj D (2007) Limits of detection for life on Mars: an example using IR spectroscopy of sulfate salts and halophiles from lakes in British Columbia, Canada. Lunar Planet Sci Conf 38:2278Google Scholar
  33. Kargel JS, Kaye JZ, Head JW III, Marion GM, Sassen R, Crowley JK, Ballesteros OP, Grant SA, Hogenbloom DL (2000) Europa’s crust and ocean: origin, composition, and the prospects for life. Icarus 148:226–265CrossRefGoogle Scholar
  34. Kilmer BR, Eberl TC, Crisler JD, Cunderla B, Madu BE, Schneegurt MA (2012) Cultivation and molecular analysis of the microbial community in epsomite lakes of the Pacific Northwest. In: Abstracts and program, 112th General Meeting of the American Society for Microbiology, San Francisco, June 2012Google Scholar
  35. Kilmer BR, Eberl TC, Cunderla B, Chen F, Clark BC, Schneegurt MA (2014) Molecular and phenetic characterization of the bacterial assemblage of Hot Lake, WA, an environment with high concentrations of magnesium sulphate, and its relevance to Mars. Int J Astrobiol 13:69–80CrossRefGoogle Scholar
  36. Kim JS, Makama M, Petito J, Park NH, Cohan FM, Dugan RS (2012) Diversity of bacteria and archaea in hypersaline sediment from Death Valley National Park, California. MicrobiologyOpen 1:135–148CrossRefGoogle Scholar
  37. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874CrossRefGoogle Scholar
  38. Laiz L, Recio D, Hermosin B, Saiz-Jimenez C (2000) Microbial communities in salt efflorescences. In: Ciferri O, Tiano P, Mastromei G (eds) Of microbes and art: the role of microbial communities in the degradation and protection of cultural heritage. Kluwer, New York, pp 77–88CrossRefGoogle Scholar
  39. Lanza NL, Meyer GA, Okubo CH, Newson HE, Wiens RC (2010) Evidence for debris flow gully formation initiated by shallow subsurface water on Mars. Icarus 205:103–112CrossRefGoogle Scholar
  40. Last WM, Ginn FM (2005) Saline systems of the Great Plains of western Canada: an overview of the limnogeology and paleolimnology. Saline Syst 1:10CrossRefGoogle Scholar
  41. Last WM, Slezak LA (1988) The salt lakes of western Canada: a paleolimnological overview. Hydrobiologia 158:310–316CrossRefGoogle Scholar
  42. Li X, Yu YH (2015) Biodiversity and screening of halophilic bacteria with hydrolytic and antimicrobial activities from Yuncheng Salt Lake, China. Biologia 70:151–156Google Scholar
  43. Lindemann SR, Moran JJ, Stegen JC, Renslow RS, Hutchison JR, Cole JK, Dohnalkova AC, Tremblay J, Singh K, Malfatti SA, Chen F, Tringe SG, Beyenal H, Fredrickson JK (2013) The epsomitic phototrophic microbial mat of Hot Lake, Washington: community structural responses to seasonal cycling. Front Microbiol 4:323CrossRefGoogle Scholar
  44. Litzner BR, Caton TM, Schneegurt MA (2006) Carbon substrate utilization, antibiotic sensitivity, and numerical taxonomy of bacterial isolates from the Great Salt Plains of Oklahoma. Arch Microbiol 185:286–296CrossRefGoogle Scholar
  45. Mandrioli P, Saiz-Jimenez C (2002) Biodeterioration: macromonitoring and microeffects on cultural heritage and the potential benefits of research to society. In: EC Advanced Study Course Technical Notes, Sessions 7–8, pp 1–5Google Scholar
  46. Marion GM, Fritsen CH, Eicken H, Payne MC (2003) The search for life on Europa: limiting environmental factors, potential habitats, and Earth analogues. Astrobiology 3:785–811CrossRefGoogle Scholar
  47. Markovitz A (1961) Method for the selection of bacteria that synthesize uronic acid-containing polysaccharides. J Bacteriol 82:436–441Google Scholar
  48. Markovitz A, Sylvan S (1962) Effect of sodium sulfate and magnesium sulfate on heteropolysaccharide synthesis in gram-negative soil bacteria. J Bacteriol 83:483–489Google Scholar
  49. Martín-Torres FJ, Zorzano M-P, Valentín-Serrano P, Harri A-M, Genzer M, Kemppinen O, Rivera-Valentin EG, Jun I, Wray J, Madsen MB, Goetz W, McEwen AS, Hardgrove C, Renno N, Chevrier VF, Mischna M, Navarro-González R, Martínez-Frías J, Conrad P, McConnochie T, Cockell C, Berger G, Vasavada AR, Sumner D, Vaniman D (2015) Transient liquid water and water activity at Gale crater on Mars. Nat Geosci 8:357–361CrossRefGoogle Scholar
  50. McEwen AS, Ojha L, Dundas CM, Mattson SS, Byrne S, Wray JJ, Cull SC, Murchie SL, Thomas N, Gulick VC (2011) Seasonal flows on warm Martian slopes. Science 333:740–743CrossRefGoogle Scholar
  51. McKay CP, Stoker CR, Glass BJ, Davé AI, Davila AF, Heldmann JL, Marinova MM, Fairen AG, Quinn RC, Zacny KA, Paulsen G, Smith PH, Parro V, Andersen DT, Hecht MH, Lacelle D, Pollard WH (2013) The Icebreaker Life Mission to Mars: a search for bimolecular evidence of life. Astrobiology 13:334–353CrossRefGoogle Scholar
  52. Möhlmann D, Thomsen K (2011) Properties of cryobrines on Mars. Icarus 212:123–130CrossRefGoogle Scholar
  53. Mohr V, Larsen H (1963) On the structural transformations and lysis of Halobacterium salinarum in hypotonic and isotonic solutions. J Gen Microbiol 31:267–280CrossRefGoogle Scholar
  54. Montoya L, Vizioli C, Rodríguez N, Rastoll MJ, Amils R, Marin I (2013) Microbial community composition of Tirez lagoon (Spain), a highly sulfated athalassohaline environment. Aquat Biosyst 9:19CrossRefGoogle Scholar
  55. Mottl MJ, Glazer BT, Kaiser RI, Meech KJ (2007) Water and astrobiology. Chem Erde 67:253–282CrossRefGoogle Scholar
  56. Mullakhanbhai MF, Larsen H (1975) Halobacterium volcanii spec. nov., a Dead Sea halobacterium with a moderate salt requirement. Arch Microbiol 104:207–214CrossRefGoogle Scholar
  57. Nesbitt HW (2004) Groundwater evolution, authigenic carbonates and sulfates, of the Basque Lake No. 2 basin, Canada. In: Spencer RJ, Chou I-M (eds.) Fluid-mineral interactions: a tribute to H.P. Eugster. Special Publication, Vol 2, Geochemical Society, pp 355–371Google Scholar
  58. Nimmo F, Pappalardo RT (2016) Ocean worlds in the outer solar system. J Geophys Res Plan 121:1378–1399CrossRefGoogle Scholar
  59. Ojha L, Wilhelm MB, Murchie SL, McEwen AS, Wray JJ, Hanley J, Massé M, Chojnacki M (2015) Spectral evidence for hydrated salts in recurring slope lineae on Mars. Nat Geosci 8:828–832CrossRefGoogle Scholar
  60. Onishi H, Fuchi H, Konomi K, Hidaka O, Kamekura M (1980) Isolation and distribution of a variety of halophilic bacteria and their classification by salt-response. Agric Biol Chem 44:1253–1258Google Scholar
  61. Pontefract A, Zhu TF, Walker VK, Hepburn H, Lui C, Zuber MT, Ruvkun G, Carr CE (2017) Microbial diversity in a hypersaline sulfate lake: a terrestrial analog of ancient Mars. Front Microbiol 8:1819CrossRefGoogle Scholar
  62. Postberg F, Schmidt J, Hillier J, Kempf S, Srama R (2011) A salt-water reservoir as the source of a compositionally stratified plume on Enceladus. Nature 474:620–622CrossRefGoogle Scholar
  63. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P et al (2013) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41:D590–D596CrossRefGoogle Scholar
  64. Rivera-Valentin EG, Gough RV, Chevrier VF, Primm KM, Martinez GM, Tolbert M (2018) Constraining the potential liquid water environment at Gale Crater, Mars throughout MSL’s traverse. Lunar Planet Sci Conf 2018:2752Google Scholar
  65. Schloss PD, Westcott SL (2011) Assessing and improving methods used in operational taxonomic unit-based approaches for 16S rRNA gene sequence analysis. Appl Environ Microbiol 77:3219–3226CrossRefGoogle Scholar
  66. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB et al (2009) Introducing MOTHUR: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541CrossRefGoogle Scholar
  67. Schneegurt MA (2012) Media and conditions for the growth of halophilic and halotolerant bacteria and archaea. In: Vreeland RH (ed) Advances in understanding the biology of halophilic microorganisms. Springer, Dordrecht, pp 35–58CrossRefGoogle Scholar
  68. Tang J, Zheng A, Bromfield ESP, Zhu J, Li S, Wang S, Deng Q, Li P (2011) 16S rRNA gene sequence analysis of halophilic and halotolerant bacteria isolated from a hypersaline pond in Sichuan, China. Ann Microbiol 61:375–381CrossRefGoogle Scholar
  69. Tosca NJ, Knoll AH, McLennan SM (2008) Water activity and the challenge for life on early Mars. Science 320:1204–1207CrossRefGoogle Scholar
  70. Vaishampayan P, Schneegurt M, Clark B, Chen F (2013) Comparative phylogenetic analysis of the microbial community in spacecraft assembly facility and in epsomite lakes. In: Abstracts and program, 113th General Meeting of the American Society for Microbiology, Denver, May 2013Google Scholar
  71. Vaniman DT, Bish DL, Chipera SJ, Fialips CI, Carey JW, Feldman WC (2004) Magnesium sulphate salts and the history of water on Mars. Nature 431:663–665CrossRefGoogle Scholar
  72. Vreeland RH, Martin EL (1980) Growth characteristics, effects of temperature, and ion specificity of the halotolerant bacterium Halomonas elongata. Can J Microbiol 26:746–752CrossRefGoogle Scholar
  73. Wänke H, Brückner G, Dreibus G, Rieder R, Ryabchikov I (2001) Chemical composition of rocks and soils at the Pathfinder site. Space Sci Rev 96:317–330CrossRefGoogle Scholar
  74. Wilks J, Chen F, Clark BC, Schneegurt MA (2019) Bacterial growth in saturated and eutectic solutions of magnesium sulphate and potassium chlorate with relevance to Mars and the ocean worlds. Int J Astrobiol. Google Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Department of Biological SciencesWichita State UniversityWichitaUSA
  2. 2.Planetary Protection Group, Jet Propulsion LaboratoryNASAPasadenaUSA
  3. 3.Space Science InstituteBoulderUSA

Personalised recommendations