Biosorption of Metal ions to Lactobacillus kefir and Lactobacillus kefir S layers. A Vibrational Spectroscopy study

E. GERBINO; P. MOBILI; E.E. TYMCZYSZYN; C. ARAUJO-ANDRADE; C. FRAUSTO-REYES; RUI FAUSTO; A. GÓMEZ-ZAVAGLIA

Mar del Plata

Congreso; VIII Congreso Argentino de Microbiología General; 2012

SAMIGE

Cadmium, nickel and lead are relevant metals from a toxicological point of view. Peopleare exposed to them primarily through food and water. Biosorption, the passive and energyindependentsorption of molecules onto the surface of an adsorbent such as inactivatedmicrobial biomass, has shown to be an efficient tool to remove metals. In this sense, lacticacid bacteria have been already used as biosorbents (Halttunen, 2007).The S-layer is a macromolecular paracrystalline bidimensional array of proteins thatrepresents the most outer structure covering the cell envelope of several species ofmicroorganisms, including Lactobacillus kefir. S-layer subunits are generally assembled in latticeswith oblique, square or hexagonal symmetry and it has been suggested that these cavities may act asappropriate surfaces for biosorption. For this reason, the aim of this work was to get an insight onthe molecular mechanisms involved in the interaction L. kefir-metal ions and S-layers-metalions.L. kefir CIDCA 8348 and L. kefir JCM 5818 were cultured in de MRS broth [De Man, J.O.et al, 1960] for 48 h at 30 oC (stationary phase). One millilitre of cultures was harvested,washed and resuspended into 1 ml milli Q water containing Pb+2 [from Pb(NO3)2], Cd+2 [fromCd(NO3)2] or Ni+2 [from Ni(NO3)2.6H2O] ranging from 0 to 0.9 mM. The suspensions werefurther incubated for 1 h at 30oC (pH 5.5) and then centrifuged. The pellets were kept toregister the Raman spectra and the supernatants were used for the analytical determinations ofPb+2, Cd+2 and Ni+2. The S-layers from both L.kefir were extracted and purified as describedMobili, P. et al, 2009. The pure S-layers were suspended in 1 ml milli Q water containing 0.3mM Pb(NO3)2, Cd(NO3)2 or Ni(NO3)2.6H2O and used for the FTIR determinations.The efficiency in the removal following the pattern: Pb+2 > Cd+2 > Ni+2 in both strains. TheRaman spectra differences observed show modifications of both symmetric and asymmetriccarboxylate frequency bands indicate that bacteria⁄metal interaction occurs throughcarboxylate groups. The metal/S-layer protein interaction, analyzed using FTIR spectroscopy,occurs mainly through the carboxylate groups of the side chains of Asp and Glut residues,with some contribution of the NH groups of the peptide backbone. The frequency separationbetween the symmetric and asymmetric carboxylates vibrations in the spectra of the S-layersin presence of the metal ions was found to be ca. 190 cm-1 for S-layer CIDCA 8348 and ca.170 cm-1 for JCM 5818, denoting an unidentate coordination in both cases.L. kefir CIDCA 8348 and JCM 5818 bind heavy metals in an efficient way. This analysisallowed obtaining a deeper insight on the molecular interactions involved when heavy metalsare attached to the bacterial surface.