Isolation and Investigation of Natural Rare Earth Metal Chelating Agents From Calothrix brevissima - A Step Towards Unraveling the Mechanisms of Metal Biosorption

Abstract

In this study water soluble compounds that form complexes with Rare Earth Elements (REE) and other metals were isolated from Calothrix brevissima biomass with chromatographic methods for the first time. Molecular characterization showed that the isolated compounds are most likely polysaccharides comprised of arabinose, xylose, mannose, galactose and glucose. FT-IR analysis revealed functional groups involved in the binding mechanism of Tb are likely sulfate- and to a lesser extend hydroxyl-groups. The binding specificity of the isolated compounds was investigated with different metal solutions. Here, ions of the alkali and alkaline earth metals Na, K, Mg and Ca showed no competition for Tb-binding even at 10-fold excess concentration. Ions of the elements Co and Pb on the other hand replaced Tb at higher concentrations. Addition of the isolated compounds significantly reduced the precipitation of Eu at pH-values between 6.7 and 9.5, indicating that the interaction between the isolated chelators and Rare Earth Metals is stable even at high pH-values.

Keywords: biosorption; calothrix; complexation; cyanobacteria; mechanism; rare earth elements.

FIGURE 1

Schematic overview of proposed mechanisms for biosorption of metal-ions.

FIGURE 2

Schematic workflow for the extraction of biomass derived chelators.

FIGURE 3

Schematic workflow for the molecular characterization of isolated metal chelators.

FIGURE 4

Anion exchange chromatography of the C. brevissima biomass extract; The UV-signal in mAU is plotted against the elution volume from the wavelengths 230 nm (black), 270 nm (red), 360 nm (blue) and 400 nm (pink). The conductivity is displayed in green. 2.5 ml fractions were collected (numbered 1-15). Each fraction was tested for interaction with Tb via luminescence spectrometry. The ∆RFU-values (before and after addition of Tb) measured at 545 nm (200–5000, if not noted otherwise) are plotted against the excitation wavelength (230–402 nm). Also, the H₂O-background measurement is displayed. Fractions 9-12 were pooled.

FIGURE 5

Size exclusion chromatography of the concentrate of C. brevissima; The UV-signal in mAU is plotted against the elution volume for the wavelengths 215 nm (black), 255 nm (red), 280 nm (blue) and 495 nm (pink). The conductivity is displayed in green. 2.5 ml fractions were collected (numbered 1-7). Each fraction was measured via luminescence spectrometry. The ∆RFU-values (before and after addition of Tb) measured at 545 nm (200–5000, if not noted otherwise) are plotted against the excitation wavelength (230–402 nm). Also, the H₂O-background measurement is displayed. Fractions one and two were pooled for further analysis.

FIGURE 6

GC-MS sugar analysis of the isolated fractions.

FIGURE 7

Change in europium-concentration at different pH-values; without the addition of REE-interacting chelators europium precipitated at high pH values leading to a decrease in Eu-concentration. In presence of REE-interacting chelators however a significant proportion of europium remained in solution. (error bars indicate ±σ, n = 3).

FIGURE 8

IR-spectra of the isolated (<10 kDa) compounds; before (black) and after adding 1 µmol of Tb³⁺ (orange).

FIGURE 9

Competition of different metals for complex formation with the isolated chelators (<10 kDa), tracked by analyzing the emission intensity of terbium; Tb concentration in all samples is 0.2 mM. Interfering metals were added in concentrations between 0.1 and 2.0 mM, excitation wavelength was set to 230 nm.