Quantitative characterization of biotic iron oxides by analytical electron microscopy

Abstract

Hydrous ferric oxides (HFO) play a tremendous role in the environment, and as a result, they have often been analyzed for their bulk chemical and mineralogical composition, but rarely at the particle level. This study used a combination of transmission electron microscopy (TEM), parallel electron energy loss spectroscopy (TEM-PEELS), and selected area electron diffraction (TEMSAED), to determine the morphology, stoichiometry, and mineralogy of synthetic biotic HFO and assess the effect of specimen preparation (i.e., dehydration) on their overall chemical composition. A mixture of synthetic lepidocrocite (γ-FeOOH) and goethite (α-FeOOH) was used as reference material for all TEM analyses. HFO were formed by the oxidation of Fe 2+ (pH = 8) in the presence of Bacillus subtilis (bacteria without extracellular polymers) and Bacillus licheniformis cells (with extracellular polymers). Biotic Fe-oxides occurred as small particles (5-10 nm diameter) on the bacterial cell walls and associated exopolymers and were identified as 2-line ferrihydrite by TEMSAED. Quantitative TEM-PEELS results showed that (1) freeze-dried abiotic samples had an Fe/O atomic ratio of 0.54 ± 0.03, whereas the Fe/O ratio of the biotic ferrihydrite formed in the presence of B. licheniformis and B. subtilis was 0.48 ± 0.06 and 0.54 ± 0.08, respectively; (2) air-dried biotic ferrihydrite possessed an Fe/O ratio close to 0.30, whereas the ratio of the abiotic air-dried HFO samples remained unchanged (i.e., 0.51 ± 0.04). These results indicate that quantitative TEM-PEELS can be used for the chemical characterization of Fe-oxides, since the measured Fe/O atomic ratios of the reference HFO are in agreement with their known stoichiometry (i.e., 0.50 for FeOOH). Our results also suggest that ferrihydrite dehydrates during freeze-drying and that specimen preparation is very important when assessing chemical composition