The implementation of IsotopAmor involved the analysis of different ACC formation pathways under controlled abiotic and biogenic conditions to investigate the role of amorphous precursors in determining the isotopic composition of the final crystalline CaCO₃ phases.
During the reporting period, the experimental workflows for ACC synthesis, characterization, and transformation were successfully established. A combination of mineralogical, geochemical, isotopic, and microbiological approaches was applied depending on the objectives of each scientific work package.
1) Abiotic approach (WP1: ACC synthesis and crystallization under controlled abiotic conditions)
ACC was synthesized using two methods: slow and rapid mixing methods (respectively using titration and centrifugation) to investigate the isotopic evolution during the ACC formation and transformation into crystalline phases in solution as a function of pH and temperature, and under subaerial conditions at different relative humidities.
The solid phases were characterized using complementary mineralogical and geochemical techniques:
- Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR): molecular bonding and mineral composition, identification of amorphous and crystalline phases.
- X-ray Diffraction (XRD): crystal structure and mineral phases, identification of crystalline polymorphs.
- Scanning Electron Microscopy (SEM): high-resolution surface morphology and microstructural characterization, particle size distribution, qualitative and semi-quantitative elemental composition, and relationships between chemical composition and microstructure.
- X-ray Photoelectron Spectroscopy (XPS): surface chemistry (chemical states, surface functional groups, and atomic ratios).
- Isotope Ratio Mass Spectrometry (IRMS): stable isotope composition, determination of carbon (δ¹³C) and oxygen (δ¹⁸O) isotope ratios in carbonate minerals.
The aqueous phases were characterized with the following techniques:
- Ion Chromatography (IC): ionic composition, quantitative determination of dissolved anions and cations to monitor changes in solution chemistry during amorphous carbonate precipitation and transformation.
- Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES): elemental composition, quantitative determination of major element concentrations and monitoring of elemental variations during carbonate precipitation and crystallization.
- Isotope Ratio Mass Spectrometry (IRMS): stable isotope composition: determination of carbon (δ¹³C) and oxygen (δ¹⁸O) isotope ratios of the amorphous and crystalline carbonate phases.
2) Biotic approach (WP2: ACC formation under biogenic conditions and crystallization pathways)
This WP of the project was focused on the study of intracellular ACC (iACC) formed by cyanobacteria. During the research stay at Paris with Dr Benzerara, cyanobacterial cultures were grown and protocols for iACC production and extraction were developed.
The experiments consist of cyanobacteria cultivation, CellDeg treatment to promote iACC formation. iACC development was monitored by SEM, FTIR, calcium quantification, and pH measurements. Following cell lysis, the presence and preservation of iACC were evaluated using ATR-FTIR and SEM. Additional high-resolution characterization of the extracted iACC is planned using the following techniques:
- Transmission Electron Microscopy (TEM) (to study the internal structure and morphology at the nanoscale): particle size determination, identification of amorphous and crystalline phases.
- Nano Secondary Ion Mass Spectrometry (NanoSIMS) (nanoscale elemental and isotopic composition): high-resolution elemental and isotopic mapping, spatial distribution of stable isotopes, and characterization of chemical heterogeneity.
The aqueous phase associated with cyanobacterial growth and biomineralization was monitored using:
- Calcium quantification by spectrometry: quantitative determination of dissolved Ca²⁺ concentration to monitor carbonate precipitation and dissolution processes.
- pH monitoring: alkalinity evolution, pH variations during carbonate precipitation experiments.
- Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES): elemental composition, quantitative determination of major and minor element concentrations.
- Nutrient analysis: nutrient availability, quantitative determination of dissolved nutrients to monitor microbial activity and carbonate biomineralization processes.
