, Volume 29, Issue 6, pp 1085–1095 | Cite as

A universal assay for the detection of siderophore activity in natural waters



Siderophores, a family of biogenic metal chelating agents, play critical roles in the biogeochemical cycling of Fe and other metals by facilitating their solubilization and uptake in circumneutral to alkaline oxic environments. However, because of their small concentrations (ca. nM) and large number of molecular structures, siderophore detection and quantification in environmental samples requires specialized equipment and expertise, and often requires pre-concentration of samples, which may introduce significant bias. The “universal” CAS assay, which was originally designed for use in bacterial cultures, quantifies the iron chelating function of a pool of siderophores but only at concentrations (>2 µM) well above the concentrations estimated to be present in marine, freshwater, and soil samples. In this manuscript, we present a high sensitivity modification of this universal assay (HS-CAS) suitable for detecting and quantifying siderophore activity in the nM concentration range, allowing for direct quantitation of siderophore reactivity in transparent aqueous samples.


Siderophores Environmental assay Metal uptake Biogeochemistry 



We thank Lauren Saal and Tyler Sowers for assistance in method development and sampling. We thank the North Carolina Agricultural Research Service (02440) for support.

Supplementary material

10534_2016_9979_MOESM1_ESM.docx (17 kb)
Supplementary material 1 (DOCX 17 kb)


  1. Ahmed E, Holmström SJM (2014) The effect of soil horizon and mineral type on the distribution of siderophores in soil. Geochim Cosmochim Acta 131:184–195. doi:10.1016/j.gca.2014.01.031 CrossRefGoogle Scholar
  2. Alexander DB, Zuberer DA (1991) Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria. Biol Fertil Soil 12:39–45CrossRefGoogle Scholar
  3. Almaraz N, Whitaker AH, Andrews MY, Duckworth OW (in review) Assessing biomineral formation by iron-oxidizing bacteria in a circumneutral creekGoogle Scholar
  4. Brausam A, van Eldik R (2012) Advances in the mechanistic understanding of selected reactions of transition metal polyaminecarboxylate complexes. In: van Rudi E, Ivana I-B (eds) Advances in inorganic chemistry, vol 64. Academic Press, Cambridge, pp 141–181. doi:10.1016/B978-0-12-396462-5.00005-2 Google Scholar
  5. Bylund D, Norstrom SH, Essen SA, Lundstrom US (2007) Analysis of low molecular mass organic acids in natural waters by ion exclusion chromatography tandem mass spectrometry. J Chromatogr A 1176:89–93. doi:10.1016/j.chroma.2007.10.064 CrossRefPubMedGoogle Scholar
  6. Callahan JH, Cook KD (1984) Mechanism of surfactant-induced changes in the visible spectrometry of metal–chrome azurol s complexes. Anal Chem 56:1632–1640. doi:10.1021/ac00273a022 CrossRefPubMedGoogle Scholar
  7. Cervini-Silva J, Kearns J, Banfield J (2012) Steady-state dissolution kinetics of mineral ferric phosphate in the presence of desferrioxamine-B and oxalate ligands at pH 4–6 and T = 24 ± 0.6 °C. Chem Geol 320–321:1–8. doi:10.1016/j.chemgeo.2012.05.022 CrossRefGoogle Scholar
  8. Crumbliss AL, Harrington JM (2009) Iron sequestration by small molecules: thermodynamic and kinetic studies of natural siderophores and synthetic model compounds. Adv Inorg Chem 61:179–250CrossRefGoogle Scholar
  9. Csáky TZ, Hassel O, Rosenberg T, Lång S, Turunen E, Tuhkanen A (1948) On the estimation of bound hydroxylamine in biological materials. Acta Chem Scand 2:450–454. doi:10.3891/acta.chem.scand.02-0450 CrossRefGoogle Scholar
  10. Duckworth OW, Holmstrom SJM, Pena J, Sposito G (2009) Biogeochemistry of iron oxidation in a circumneutral freshwater habitat. Chem Geol 260:149–158CrossRefGoogle Scholar
  11. Emerson D (2012) Biogeochemistry and microbiology of microaerobic Fe(II) oxidation. Biochem Soc Trans 40:1211–1216. doi:10.1042/BST20120154 CrossRefPubMedGoogle Scholar
  12. Emerson D, Fleming EJ, McBeth JM (2010) Iron-oxidizing bacteria: an environmental and genomic perspective. Annu Rev Microbiol 64:561–583. doi:10.1146/annurev.micro.112408.134208 CrossRefPubMedGoogle Scholar
  13. Essen SA, Bylund D, Holmstrom SJ, Moberg M, Lundstrom US (2006) Quantification of hydroxamate siderophores in soil solutions of podzolic soil profiles in Sweden. Biometals 19:269–282. doi:10.1007/s10534-005-8418-8 CrossRefPubMedGoogle Scholar
  14. Frausto da Silva JRR, Williams RJP (1991) The biological chemistry of the elements. Clarendon Press, OxfordGoogle Scholar
  15. Gimbert LJ, Worsfold PJ (2007) Environmental applications of liquid-waveguide-capillary cells coupled with spectroscopic detection. TrAC Trends Anal Chem 26:914–930. doi:10.1016/j.trac.2007.08.005 CrossRefGoogle Scholar
  16. Glass J, Orphan V (2012) Trace metal requirements for microbial enzymes involved in the production and consumption of methane and nitrous oxide. Front Microbiol. doi:10.3389/fmicb.2012.00061 Google Scholar
  17. Gledhill M, Buck KN (2012) The organic complexation of iron in the marine environment: a review. Front Microbiol 3:69. doi:10.3389/fmicb.2012.00069 PubMedPubMedCentralGoogle Scholar
  18. Gledhill M, McCormack P, Ussher S, Achterberg EP, Mantoura RFC, Worsfold PJ (2004) Production of siderophore type chelates by mixed bacterioplankton populations in nutrient enriched seawater incubations. Mar Chem 88:75–83. doi:10.1016/j.marchem.2004.03.003 CrossRefGoogle Scholar
  19. Harrington JM, Parker DL, Bargar JR, Jarzecki AA, Tebo BM, Sposito G, Duckworth OW (2012) Structural dependence of Mn complexation by siderophores: donor group dependence on complex stability and reactivity. Geochim Cosmochim Acta 88:106–119. doi:10.1016/j.gca.2012.04.006 CrossRefGoogle Scholar
  20. Harrington JM, Duckworth OW, Haselwandter K (2015) The fate of siderophores: antagonistic environmental interactions in exudate-mediated micronutrient uptake. Biometals 28:461–472. doi:10.1007/s10534-015-9821-4 CrossRefPubMedGoogle Scholar
  21. Haselwandter K, Winkelmann G (1998) Identification and characterization of siderophores of mycorrhizal fungi. Springer Lab Manual Series; Mycorrhiza manual. Springer, BerlinGoogle Scholar
  22. Haselwandter K et al (2006) Basidiochrome—a novel siderophore of the Orchidaceous Mycorrhizal Fungi Ceratobasidium and Rhizoctonia spp. Biometals 19:335–343. doi:10.1007/s10534-006-6986-x CrossRefPubMedGoogle Scholar
  23. Holmström SJM, Lundström US, Finlay RD, Van Hees PAW (2004) Siderophores in forest soil solution. Biogeochemistry 71:247–258CrossRefGoogle Scholar
  24. Hussein KA, Joo JH (2014) Potential of siderophore production by bacteria isolated from heavy metal: polluted and rhizosphere soils. Curr Microbiol 68:717–723. doi:10.1007/s00284-014-0530-y CrossRefPubMedGoogle Scholar
  25. Jarosz M, Malát M (1988) Spectrophotometric study of the formation of ternary complexes of iron(III) with some triphenylmethane dyes and cationic surfactants. Microchem J 37:268–274. doi:10.1016/0026-265x(88)90136-1 CrossRefGoogle Scholar
  26. Kem MP, Zane HK, Springer SD, Gauglitz JM, Butler A (2014) Amphiphilic siderophore production by oil-associating microbes. Metallomics 6:1150–1155. doi:10.1039/c4mt00047a CrossRefPubMedGoogle Scholar
  27. Kloepper JW, Leong J, Teintze M, Schroth MN (1980) Pseudomonas siderophores: a mechanism explaining disease-suppressive soils. Curr Microbiol 4:317–320. doi:10.1007/bf02602840 CrossRefGoogle Scholar
  28. Kraemer SM (2004) Iron oxide dissolution and solubility in the presence of siderophores. Aquat Sci 66:3–18. doi:10.1007/s00027-003-0690-5 CrossRefGoogle Scholar
  29. Kraemer SM, Butler A, Borer P, Cervini-Silva J (2005) Siderophores and the dissolution of iron-bearing minerals in marine systems. Rev Mineral Geochem 59:53–84. doi:10.2138/rmg.2005.59.4 CrossRefGoogle Scholar
  30. Kraemer SM, Crowley DE, Kretzschmar R (2006) Geochemical aspects of phytosiderophore-promoted iron acquisition by plants. Adv Agron 91:1–46. doi:10.1016/s0065-2113(06)91001-3 CrossRefGoogle Scholar
  31. Kraemer SM, Duckworth OW, Harrington JM, Schenkeveld WDC (2014) Metallophores and Trace Metal Biogeochemistry. Aquat Geochem 21:159–195. doi:10.1007/s10498-014-9246-7 CrossRefGoogle Scholar
  32. Kraepiel AM, Bellenger JP, Wichard T, Morel FM (2009) Multiple roles of siderophores in free-living nitrogen-fixing bacteria. Biometals 22:573–581. doi:10.1007/s10534-009-9222-7 CrossRefPubMedGoogle Scholar
  33. Kuhn KM, Maurice PA, Neubauer E, Hofmann T, von der Kammer F (2014) Accessibility of humic-associated Fe to a microbial siderophore: implications for bioavailability. Environ Sci Technol 48:1015–1022. doi:10.1021/es404186v CrossRefPubMedGoogle Scholar
  34. Kustka AB, Jones BM, Hatta M, Field MP, Milligan AJ (2015) The influence of iron and siderophores on eukaryotic phytoplankton growth rates and community composition in the Ross Sea. Mar Chem 173:195–207. doi:10.1016/j.marchem.2014.12.002 CrossRefGoogle Scholar
  35. Langmyhr FJ, Klausen KS (1963) Complex formation of iron (III) with chrome azurol s. Anal Chim Acta 29:149–167. doi:10.1016/s0003-2670(00)88596-7 CrossRefGoogle Scholar
  36. Lemanceau P, Bauer P, Kraemer S, Briat J-F (2009) Iron dynamics in the rhizosphere as a case study for analyzing interactions between soils, plants and microbes. Plant Soil 321:513–535. doi:10.1007/s11104-009-0039-5 CrossRefGoogle Scholar
  37. Machuca A, Navias D, Milagres AMF, Chavez D, Guillen Y (2014) Effects of metal ions (Cd2+, Cu2+, Zn2+) on the growth and chelating-compound production of three ectomycorrhizal fungi. Interciencia 39:221–227Google Scholar
  38. Macrellis HM, Trick CG, Rue EL, Smith G, Bruland KW (2001) Collection and detection of natural iron-binding ligands from seawater. Mar Chem 76:175–187. doi:10.1016/s0304-4203(01)00061-5 CrossRefGoogle Scholar
  39. Madison AS, Tebo BM, Luther GW (2011) Simultaneous determination of soluble manganese(III), manganese(II) and total manganese in natural (pore)waters. Talanta 84:374–381. doi:10.1016/j.talanta.2011.01.025 CrossRefPubMedGoogle Scholar
  40. Mawji E et al (2008) Hydroxamate siderophores: occurrence and importance in the Atlantic Ocean. Environ Sci Technol 42:8675–8680. doi:10.1021/es801884r CrossRefPubMedGoogle Scholar
  41. Moberg M, Holmstrom SJ, Lundstrom US, Markides KE (2003) Novel approach to the determination of structurally similar hydroxamate siderophores by column-switching capillary liquid chromatography coupled to mass spectrometry. J Chromatogr A 1020:91–97. doi:10.1016/s0021-9673(03)01236-6 CrossRefPubMedGoogle Scholar
  42. Mucha P, Rekowski P, Kosakowska A, Kupryszewski G (1999) Separation of siderophores by capillary electrophoresis. J Chromatogr A 830:183–189. doi:10.1016/s0021-9673(98)00907-8 CrossRefGoogle Scholar
  43. Neilands JB (1981) Microbial iron compounds. Annu Rev Biochem 50:715–731. doi:10.1146/ CrossRefPubMedGoogle Scholar
  44. Parker DL, Sposito G, Tebo BM (2004) Manganese (III) binding to a pyoverdine siderophore produced by a manganese (II)-oxidizing bacterium. Geochim Cosmochim Acta 68:4809–4820CrossRefGoogle Scholar
  45. Połedniok J, Szpikowska-Sroka B (2012) Spectrophotometric study of colour reaction of vanadium(IV) with chrome azurol s in the presence of cationic and non-ionic surfactants. J Anal Chem 68:45–49. doi:10.1134/s1061934813010085 CrossRefGoogle Scholar
  46. Powell PE, Cline GR, Reid CPP, Szaniszlo PJ (1980) Occurrence of hydroxamate siderophore iron chelators in soils. Nature 287:833–834. doi:10.1038/287833a0 CrossRefGoogle Scholar
  47. Pytlakowska K, Zerzucha P, Czoik R (2011) Influence of mixed cationic–nonionic surfactant systems on the spectral properties of ci mordant blue 29 and its complexes with iron(III). Anal Sci 27:555–560CrossRefPubMedGoogle Scholar
  48. Renshaw JC, Robson GD, Trinci APJ, Wiebe MG, Livens FR, Collison D, Taylor RJ (2002) Fungal siderophores: structures, functions and applications. Mycol Res 106:1123–1142. doi:10.1017/s0953756202006548 CrossRefGoogle Scholar
  49. Ribas X, Salvado V, Valiente M (1989) The chemistry of iron in biosystems. II: a hydrolytic model of the complex formation between iron(III) and citric acid in aqueous solutions. J Chem Res 1989:2533–2553Google Scholar
  50. Robin A, Vansuyt G, Hinsinger P, Meyer JM, Briat JF, Lemanceau P (2008) Iron dynamics in the rhizosphere: consequences for plant health and nutrition. Adv Agron 99:183–225. doi:10.1016/s0065-2113(08)00404-5 CrossRefGoogle Scholar
  51. Roden EE et al (2012) The microbial ferrous wheel in a neutral pH groundwater seep. Front Microbiol 3:172. doi:10.3389/fmicb.2012.00172 CrossRefPubMedPubMedCentralGoogle Scholar
  52. Schalk IJ, Hannauer M, Braud A (2011) New roles for bacterial siderophores in metal transport and tolerance. Environ Microbiol 13:2844–2854. doi:10.1111/j.1462-2920.2011.02556.x CrossRefPubMedGoogle Scholar
  53. Schenkeveld WD, Oburger E, Gruber B, Schindlegger Y, Hann S, Puschenreiter M, Kraemer SM (2014) Metal mobilization from soils by phytosiderophores—experiment and equilibrium modeling. Plant Soil 383:59–71. doi:10.1007/s11104-014-2128-3 CrossRefPubMedPubMedCentralGoogle Scholar
  54. Schroth MN, Hancock JG (1982) Disease-suppressive soil and root-colonizing bacteria. Science 216:1376–1381. doi:10.1126/science.216.4553.1376 CrossRefPubMedGoogle Scholar
  55. Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47–56CrossRefPubMedGoogle Scholar
  56. Semb A, Langmyhr FJ (1966) Complex formation of copper(II) with chrome azurol s. Anal Chim Acta 35:286–292. doi:10.1016/s0003-2670(01)81678-0 CrossRefGoogle Scholar
  57. Shenker M, Hadar Y, Chen Y (1995) Rapid method for accurate determination of colorless siderophores and synthetic chelates. Soil Sci Soc Am J 59:1612–1618CrossRefGoogle Scholar
  58. Shenker M, Hadar Y, Chen Y (1999) Kinetics of iron complexing and metal exchange in solutions by rhizoferrin, a fungal siderophore. Soil Sci Soc Am J 63:1681–1687CrossRefGoogle Scholar
  59. Siebner-Freibach H, Hadar Y, Chen Y (2004) Interaction of iron chelating agents with clay minerals. Soil Sci Soc Am J 68:470–480CrossRefGoogle Scholar
  60. Sorichetti RJ, Creed IF, Trick CG (2014) The influence of iron, siderophores and refractory DOM on cyanobacterial biomass in oligotrophic lakes. Freshw Biol 59:1423–1436. doi:10.1111/fwb.12355 CrossRefGoogle Scholar
  61. Sowers TD, Harrington JM, Polizzotto ML, Duckworth OW (2016) Sorption of arsenic to biogenic iron (oxyhydr)oxides produced in circumneutral environments. Geochim Cosmochim Acta (in press)Google Scholar
  62. Springer SD, Butler A (2016) Microbial ligand coordination: consideration of biological significance. Coord Chem Rev 306:628–635. doi:10.1016/j.ccr.2015.03.013 CrossRefGoogle Scholar
  63. Stintzi A, Barnes C, Xu L, Raymond KN (2000) Microbial iron transport via a siderophore shuttle: a membrane ion transport paradigm. Proc Natl Acad Sci 97:10691–10696CrossRefPubMedPubMedCentralGoogle Scholar
  64. Upase AB, Zade AB, Kalbende PP (2011) Spectrophotometric microdetermination of thorium(IV) and uranium(VI) with chrome azurol-s in presence of cationic surfactant. J Chem 8:1132–1141Google Scholar
  65. van der Helm D, Winkelmann G (1994) Hydroxamates and polycarboxylates as iron transport agents (siderophores) in fungi. In: Winkelmann G, Winge FR (eds) Metal ions in fungi. M. Dekker, New York, pp 39–98Google Scholar
  66. Velasquez I, Nunn BL, Ibisanmi E, Goodlett DR, Hunter KA, Sander SG (2011) Detection of hydroxamate siderophores in coastal and Sub-Antarctic waters off the South Eastern Coast of New Zealand. Mar Chem 126:97–107. doi:10.1016/j.marchem.2011.04.003 CrossRefGoogle Scholar
  67. Vestin JLK, Norström SH, Bylund D, Lundström US (2008) Soil solution and stream water chemistry in a forested catchment II: influence of organic matter. Geoderma 144:271–278. doi:10.1016/j.geoderma.2007.11.027 CrossRefGoogle Scholar
  68. Wei G-Z (1992) A chemical tool for the detection of siderophores. Lamar University, BeaumontGoogle Scholar
  69. Wichard T, Bellenger JP, Loison A, Kraepiel AM (2008) Catechol siderophores control tungsten uptake and toxicity in the nitrogen-fixing bacterium Azotobacter Vinelandii. Environ Sci Technol 42:2408–2413. doi:10.1021/es702651f CrossRefPubMedGoogle Scholar
  70. Yehuda Z, Shenker M, Romheld V, Marschner H, Hadar Y, Chen Y (1996) The role of ligand exchange in the uptake of iron from microbial siderophores by gramineous plants. Plant Physiol 112:1273–1280CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Crop and Soil SciencesNorth Carolina State UniversityRaleighUSA
  2. 2.Center for Integrated Fungal ResearchNorth Carolina State UniversityRaleighUSA

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