Reevaluation of colorimetric iron determination methods commonly used in geomicrobiology
Highlights
► Comparison of ferrozine and phenanthroline methods in geomicrobiology. ► Main focus on extraction methods of Fe minerals with different solubility. ► Heating of samples leads to fast abiotic oxidation of ferrous Fe. ► Sulfides strongly interfere with ferrozine, usage of phenanthroline is recommended. ► Cell extracts might complex Fe which is better available for phenanthroline.
Introduction
Iron is an important redox active element and iron-oxidizing and iron-reducing microorganisms catalyze a substantial part of the iron-related reactions in the environment (Weber et al., 2006). Since the isolation of Geobacter metallireducens (Lovley and Phillips, 1986a) and Shewanella putrefaciens MR-1 (Myers and Nealson, 1990), the field of iron geomicrobiology is thriving. However, a robust quantification of iron (Fe) and its oxidation states is essential to explore the multitude of microbial Fe reduction and oxidation processes. Colorimetric methods are commonly used to assess total (Fe(tot)) or ferrous iron (Fe(II)) contents due to their simple application in everyday's laboratory routine (Anastácio et al., 2008). In current geomicrobiological studies, the ferrozine (Stookey, 1970) and phenanthroline assays (Fortune and Mellon, 1938) are most frequently applied. Besides, the sulfosalicylic acid assay (Pal and Lahiri, 1974) is often used in e.g. chemistry (Kozak et al., 2011), mineralogy (Osborne et al., 2010), and engineering (Paipa et al., 2005). For investigations of siliceous mineral systems, the phenanthroline method is widely used because 1,10-phenanthroline dissolves all Fe from mineral matrices. The 1,10-phenanthroline solution forms an orange complex (adsorption at 508 nm) with Fe(II). However, due to the photochemical reduction of Fe(III) in presence of excess 1,10-phenanthroline and exposure to ambient UV radiation (David et al., 1972, Wehry and Ward, 1971), a masking of Fe(III) is necessary for Fe determination in samples containing both Fe(II) and Fe(III). Common masking agents are phosphate (Pollock and Miguel, 1967), pyrophosphate (Mizuno, 1972), and fluoride (Tamura et al., 1973). A disadvantage of the phenanthroline method is the degradation of DNA by Co(II)-phenanthroline complexes (Downey et al., 1980) which might expose health effects to the operator.
For measurements in non-siliceous, microbiological, and aquatic systems, the ferrozine method is extensively used. Ferrous iron can be extracted with ammonium oxalate or HCl and forms a violet complex (adsorption at 560 nm) with ferrozine. For ferrozine, a light sensitivity similar to phenanthroline is hardly documented but some authors conducted the measurements in the dark (Macur et al., 1991, Majestic et al., 2006). For determination of Fe(tot) with both assays, Fe(III) has to be reduced to Fe(II) before measurement.
In field and laboratory geomicrobiology, a fast and simple dissolution and quantification of Fe(II) and Fe oxides in large sample numbers is mandatory. Therefore, the time-consuming procedures of oxalate and dithionite extractions are often not feasible; instead, 1 M HCl is commonly used for extraction of Fe(II) and Fe(III). For highly crystalline minerals like e.g. goethite, hematite, or magnetite, stronger acids such as 3 M to 6 M HCl are applied because these minerals do not dissolve in 1 M HCl (Benner et al., 2002, Fredrickson et al., 1998, Heron et al., 1994). For some minerals like e.g. pyrite, an increase of dissolution temperature and incubation time is also necessary to achieve sufficient dissolution (Kuslu and Bayramoglu, 2002).
Despite their broad application in today's geomicrobiological research, a systematic investigation of the accuracy and applicability of the two colorimetric Fe assays with respect to samples typically analyzed in iron geomicrobiology is lacking in the literature. Anastácio et al. (2008) evaluated the ferrozine test for analyzing soil and clay samples, taking the phenanthroline test as reference. However, especially the influence of HCl as extraction reagent has not been investigated, although the incompatibility with strong acids is known for both tests. Furthermore, Fe(II) can oxidize in HCl extractions (Posner, 1953) which is probably temperature dependent and will influence the measurements (Porsch and Kappler, 2011). In our own research, we frequently encountered a high susceptibility of both methods to slight changes in boundary conditions of the procedures. This might result in a decrease in reproducibility or accuracy of the measurements.
This study intends to close the scientific gap on interfering processes during the formation of the ferrozine and phenanthroline complexes. Specifically, the impact of variations in dissolution temperature, time, acid strength, and sample type is investigated. A broad set of samples was investigated including solutions of Fe(II) and Fe(III) as well as goethite, pyrite, synthesized ferrihydrite, and microbially-formed magnetite and a microbially-formed mixture of magnetite and goethite. These minerals represent a set of Fe oxides widely applied in recent geomicrobiological studies (Cutting et al., 2009, Zhang et al., 2009).
Section snippets
Cultivation of microorganisms
Geobacter sulfurreducens DSMZ 12127 (Caccavo et al., 1994) was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany). This strain, as well as a yet undefined, iron-reducing enrichment culture (for the production of a goethite–magnetite mixture), was cultivated in two-fold diluted, modified freshwater medium modified after Widdel and Bak (1992) and Widdel and Hansen (1992) with marble pearls as pH-buffer system (Conrad et al., 2000). The pH was adjusted to
Results
To compare the photometric ferrozine and phenanthroline assays for Fe determination, we measured the Fe(II) and Fe(tot) contents of Fe(II)aq and Fe(III)aq, mixed valence solutions, synthetic goethite, ferrihydrite, and pyrite, as well as microbially-formed magnetite, and a microbially-formed mixture of goethite and magnetite. The accuracy of each test for Fe(tot) was obtained by comparison with the aqua regia dissolved samples measured with ICP-AES. The identity of synthesized ferrihydrite as
Discussion
This study compares the application of the colorimetric phenanthroline and ferrozine assays for the determination of Fe(II) and Fe(tot) in microbiological samples. The experiments were focused on different acidic strengths, incubation temperature, and incubation time. Based on these data, we can now provide a robust dataset for choosing the appropriate method for the determination of Fe concentrations in geomicrobiological studies.
Acknowledgments
We thank Bernhard Michalke for ICP-AES measurements and Thomas Braunschweig for help with the statistic analysis. This study was funded by the research group FOR 580 of the German Research Foundation (DFG) “Electron Transfer Processes in Anoxic Aquifers”, and the EU-project AQUAREHAB (FP7—Grant Agreement Nr. 226565).
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