, Volume 32, Issue 6, pp 819–828 | Cite as

H-Aquil: a chemically defined cell culture medium for trace metal studies in Vibrios and other marine heterotrophic bacteria

  • Donald E. MartocelloIII
  • François M. M. Morel
  • Darcy L. McRoseEmail author


A variety of trace metals, including prominently iron (Fe) are necessary for marine microorganisms. Chemically defined medium recipes have been used for several decades to study phytoplankton, but similar methods have not been adopted as widely in studies of marine heterotrophic bacteria. Medium recipes for these organisms frequently include tryptone, casamino acids, as well as yeast and animal extracts. These components introduce unknown concentrations of trace elements and organic compounds, complicating metal speciation. Minimal medium recipes utilizing known carbon and nitrogen sources do exist but often have high background trace metal concentrations. Here we present H-Aquil, a version of the phytoplankton medium Aquil adapted for marine heterotrophic bacteria. This medium consists of artificial seawater supplemented with a carbon source, phosphate, amino acids, and vitamins. As in Aquil, trace metals are controlled using the synthetic chelator EDTA. We also address concerns of EDTA toxicity, showing that concentrations up to 100 µM EDTA do not lead to growth defects in the copiotrophic bacterium Vibrio harveyi or the oligotrophic bacterium Candidatus Pelagibacter ubique HTCC1062, a member of the SAR11 clade. H-Aquil is used successfully to culture species of Vibrio, Phaeobacter, and Silicibacter, as well as several environmental isolates. We report a substantial decrease in growth rate between cultures grown with or without added Fe, making the medium suitable for conducting Fe-limitation studies in a variety of marine heterotrophic bacteria.


Vibrio Iron Marine heterotrophic bacteria EDTA Defined medium SAR11 



This work was funded by the National Science Foundation (OCE 1657639 Granted to F.M.M.), the Princeton Environmental Institute (Grand Challenges to F.M.M. and Mary and Randall Hack Water Quality Award to D.L.M.), and the Princeton University Department of Geosciences (Undergraduate research funding support awarded to D.E.M.). We thank the Dyhrman lab (Columbia U.), Giovannoni lab (Oregon State U.), Bassler lab (Princeton U.) and Seyedsayamdost lab (Princeton U.) for providing bacterial cultures. We are especially grateful to K. R. Frischkorn for assistance with Trichodesmium epibionts, J. S. Valastyan for assistance with V. harveyi and S. Noell for assistance with SAR11.

Supplementary material

10534_2019_215_MOESM1_ESM.pdf (682 kb)
Supplementary material 1 (PDF 683 kb)


  1. Baars O, Frischkorn KR, Dyhrman ST (in prep) Siderophore production by Trichodesmium epibionts isolated from the North and South PacificGoogle Scholar
  2. Bassler BL, Wright M, Silverman MR (1994) Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway. Mol Microbiol 13:273–286. CrossRefPubMedGoogle Scholar
  3. Bertani G (1951) Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300PubMedPubMedCentralGoogle Scholar
  4. Brand LE, Sunda WG, Guillard RRL (1986) Reduction of marine phytoplankton reproduction rates by copper and cadmium. J Exp Mar Biol Ecol 96:225–250. CrossRefGoogle Scholar
  5. Brown MRW, Melling J (1969) Role of divalent cations in the action of polymyxin B and EDTA on Pseudomonas aeruginosa. Microbiology 59:263–274. CrossRefGoogle Scholar
  6. Carini P, Steindler L, Beszteri S, Giovannoni SJ (2013) Nutrient requirements for growth of the extreme oligotroph ‘Candidatus Pelagibacter ubique’ HTCC1062 on a defined medium. The ISME Journal 7:592–602. CrossRefPubMedGoogle Scholar
  7. Eagle H (1955) The specific amino acid requirements of a mammalian cell (strain L) in tissue culture. J Biol Chem 214:839–852PubMedGoogle Scholar
  8. Farmer JI, Hickman-Brenner F (2006) The genera Vibrio and Photobacterium. In: Falkow S, Rosenberg E, Schleifer K-H, Stackebrant E (eds) The prokaryotes. A handbook on the biology of bacteria. Volume 6: proteobacteria: gamma subclass. Springer, New York, pp 508–563Google Scholar
  9. Frischkorn KR, Rouco M, Van Mooy BAS, Dyhrman ST (2017) Epibionts dominate metabolic functional potential of Trichodesmium colonies from the oligotrophic ocean. ISME J 11:2090–2101CrossRefGoogle Scholar
  10. Fuhrman JA, Sleeter TD, Carlson CA, Proctor LM (1989) Dominance of bacterial biomass in the Sargasso Sea and its ecological implications. Mar Ecol Prog Ser 57:207–217. CrossRefGoogle Scholar
  11. Granger J, Price NM (1999) The importance of siderophores in iron nutrition of heterotrophic marine bacteria. Limnol Oceanogr 44:541–555CrossRefGoogle Scholar
  12. Greenberg EP, Hastings JW, Ulitzur S (1979) Induction of luciferase synthesis in Beneckea harveyi by other marine bacteria. Arch Microbiol 120:87–91CrossRefGoogle Scholar
  13. Guillard RRL, Ryther JH (1962) Studies of marine planktonic diatoms. I. Cyclotella nana (Hustedt), and Detonula confervacea (Cleve) Gran. Can J Microbiol 8:229–239CrossRefGoogle Scholar
  14. Hancock RE (1984) Alterations in outer membrane permeability. Annu Rev Microbiol 38:237–264CrossRefGoogle Scholar
  15. Hering JG, Morel FMM (1988) Kinetics of trace metal complexation: role of alkaline-earth metals. Environ Sci Technol 22:1469–1478CrossRefGoogle Scholar
  16. Hopkinson BM, Roe KL, Barbeau KA (2008) Heme uptake by Microscilla marina and evidence for heme uptake systems in the genomes of diverse marine bacteria. Appl Environ Microbiol 74:6263–6270. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hutner SH, Provasoli L, Schatz A, Haskins CP (1950) Some approaches to the study of the role of metals in the metabolism of microorganisms. Proc Am Philos Soc 94:152–170. CrossRefGoogle Scholar
  18. King EO, Ward MK, Raney DE (1954) Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med 44:301–307PubMedGoogle Scholar
  19. Laboratories CSH (2010) M9 minimal mediuma (standard). Cold Spring Harbor Protoc. CrossRefGoogle Scholar
  20. Laboratories CSH (2010b) Terric broth (TB) medium. Cold Spring Harbor Protoc.
  21. Leive L (1965) A nonspecific increase in permeability in Escherichia coli produced by EDTA. Proc Natl Acad Sci USA 53:745–750. CrossRefPubMedGoogle Scholar
  22. Lin B et al (2010) Comparative genomic analyses identify the Vibrio harveyi genome sequenced strains BAA-1116 and HY01 as Vibrio campbellii. Environ Microbiol Rep 2:81–89. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Madigan MT, Martinko JM, Dunlap PV, Clark DP (2009) Nutrition, culture and metabolism of microorganisms. Brock biology of microorganisms. Pearson Education Inc., San Francisco, pp 107–140Google Scholar
  24. Martin JH, Fitzwater SE, Gordon RM (1990) Iron deficiency limits phytoplankton growth in Antarctic waters. Glob Biogeochem Cycles 4:5–12. CrossRefGoogle Scholar
  25. Morel FMM, Rueter JG, Anderson DM, Guillard RRL (1979) Aquil: a chemically defined phytoplankton culture medium for trace metal studies. J Phycol 15:135–141. CrossRefGoogle Scholar
  26. Neidhardt FC, Bloch PL, Smith DF (1974) Culture medium for enterobacteria. J Bacteriol 119:736–747PubMedPubMedCentralGoogle Scholar
  27. Nicas TI, Hancock REW (1983) Alteration of susceptibility to EDTA, polymyxin B and gentamicin In Pseudomonas aeruginosa by divalent cation regulation of outer membrane protein H1. Microbiology 129:509–517. CrossRefGoogle Scholar
  28. Nikaido H (2003) Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev 67:593–656. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Nikaido H, Vaara M (1985) Molecular basis of bacterial outer membrane permeability. Microbiol Rev 49:1PubMedPubMedCentralGoogle Scholar
  30. Price NM, Harrison GI, Hering JG, Hudson RJ, Nirel PMV, Palenik B, Morel FMM (1989) Preparation and chemistry of the artificial algal culture medium Aquil. Biol Oceanogr 6:443–461Google Scholar
  31. Provasoli L, McLaughlin J, Droop MR (1956) The development of artifical media for marine algae. Archiv für Mikrobiologie 25:392–428CrossRefGoogle Scholar
  32. Rappé MS, Connon SA, Vergin KL, Giovannoni SJ (2002) Cultivation of the ubiquitous SAR11 marine bacterioplankton clade. Nature 418:630. CrossRefPubMedGoogle Scholar
  33. Roe KL, Barbeau K, Mann EL, Haygood MG (2011) Acquisition of iron by Trichodesmium and associated bacteria in culture. Environ Microbiol 14:1681–1695. CrossRefPubMedGoogle Scholar
  34. Roe KL, Hogle SL, Barbeau KA (2013) Utilization of heme as an iron source by marine Alphaproteobacteria in the Roseobacter clade. Appl Environ Microbiol 79:5753–5762. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Sawabe T et al (2013) Updating the Vibrio clades defined by multilocus sequence phylogeny: proposal of eight new clades, and the description of Vibrio tritonius sp. nov. Front Microbiol. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Schneck E et al (2010) Quantitative determination of ion distributions in bacterial lipopolysaccharide membranes by grazing-incidence X-ray fluorescence. Proc Natl Acad Sci USA 107:9147–9151. CrossRefPubMedGoogle Scholar
  37. Schwarzenbach G, Ackerman H (1948) Komplexone XII. Die homologen der Äthyendiamin-tetraessigsäure und ihre Erdalkalikomplexe. Helv Chim Acta 31:1029–1048CrossRefGoogle Scholar
  38. Sunda WG, Price NM, Morel FMM (2005) Trace metal ion buffers and their use in culture studies. In: Andersen RA (ed) Algal culturing techniques. Elsevier Academic Press, Burlington, pp 35–63Google Scholar
  39. Thompson CC, Thompson FL, Vicente ACP, Swings J (2007) Phylogenetic analysis of vibrios and related species by means of atpA gene sequences. Int J Syst Evol Microbiol 57:2480–2484. CrossRefPubMedGoogle Scholar
  40. Westall J, Zachary J, Morel FMM (1976) MINEQL: A computer program for the calculation of chemical equilibrium composition in aqueous systems. MIT, Tech. Note 18Google Scholar
  41. Zobell CE (1941) Studies of marine bacteria. I. The cultural requirements of heterotrophic aerobes. J Mar Res 4:42–75Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of GeosciencesPrinceton UniversityPrincetonUSA
  2. 2.Department of Marine Chemistry and GeochemistryWoods Hole Oceanographic InstitutionWoods HoleUSA
  3. 3.Department of Earth, Atmospheric, and Planetary SciencesMassachusetts Institute of TechnologyCambridgeUSA
  4. 4.Divisions of Geological and Planetary Sciences and Biology and Biological EngineeringCalifornia Institute of TechnologyPasadenaUSA

Personalised recommendations