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

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

Mar del Plata

Congreso; VIII Congreso de la Sociedad Argentina de Microbiología General; 2012

SAMIGE

Cadmium, nickel and lead are relevant metals from a toxicological point of view. People are exposed to them primarily through food and water. Biosorption, the passive and energy-independent sorption of molecules onto the surface of an adsorbent such as inactivated microbial biomass, has shown to be an efficient tool to remove metals. In this sense, lactic acid bacteria have been already used as biosorbents (Halttunen et al., 2007, 2008). The S-layer is a macromolecular paracrystalline bidimensional array of proteins that represents the most outer structure covering the cell envelope of several species of microorganisms, including Lactobacillus kefir. S-layer subunits are generally assembled in lattices with oblique, square or hexagonal symmetry and it has been suggested that these cavities may act as appropriate surfaces for biosorption. For this reason, the aim of this work was to get an insight on the molecular mechanisms involved in the interaction L. kefir-metal ions and S-layers-metal ions. 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 [from Cd(NO3)2] or Ni+2 [from Ni(NO3)2.6H2O] ranging from 0 to 0.9 mM. The suspensions were further incubated for 1 h at 30oC (pH 5.5) and then centrifuged. The pellets were kept to register the Raman spectra and the supernatants were used for the analytical determinations of Pb+2, Cd+2 and Ni+2. The S-layers from both L.kefir were extracted and purified as described Mobili, P. et al, 2009. The pure S-layers were suspended in 1 ml milli Q water containing 0.3 mM 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. The Raman spectra differences observed show modifications of both symmetric and asymmetric carboxylate frequency bands indicate that bacteria⁄metal interaction occurs through carboxylate 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 separation between the symmetric and asymmetric carboxylates vibrations in the spectra of the S-layers in 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 (Gerbino, E. et al, 2011, 2012). L. kefir CIDCA 8348 and JCM 5818 bind heavy metals in an efficient way. This analysis allowed obtaining a deeper insight on the molecular interactions involved when heavy metals are attached to the bacterial surface.