Reevaluation of colorimetric iron determination methods commonly used in geomicrobiology
ABSTRACT The ferrozine and phenanthroline colorimetric assays are commonly applied for the determination of ferrous and total iron concentrations in geomicrobiological studies. However, accuracy of both methods depends on slight changes in their protocols, on the investigated iron species, and on geochemical variations in sample conditions. Therefore, we tested the performance of both methods using Fe(II)((aq)), Fe(III)((aq)), mixed valence solutions, synthetic goethite, ferrihydrite, and pyrite, as well as microbially-formed magnetite and a mixture of goethite and magnetite. The results were compared to concentrations determined with aqua regia dissolution and inductively coupled plasma atomic emission spectroscopy (ICP-AES). Iron dissolution prior to the photometric assays included dissolution in 1M or 6M HCl, at 21 or 60°C, and oxic or anoxic conditions. Results indicated a good reproducibility of quantitative total iron determinations by the ferrozine and phenanthroline assays for easily soluble iron forms such as Fe(II)((aq)), Fe(III)((aq)), mixed valence solutions, and ferrihydrite. The ferrozine test underestimated total iron contents of some of these samples after dissolution in 1M HCl by 10 to 13%, whereas phenanthroline matched the results determined by ICP-AES with a deviation of 5%. Total iron concentrations after dissolution in 1M HCl of highly crystalline oxides such as magnetite, a mixture of goethite and magnetite, and goethite were underestimated by up to 95% with both methods. When dissolving these minerals in 6M HCl at 60°C, the ferrozine method was more reliable for total iron content with an accuracy of ±5%, related to values determined with ICP-AES. Phenanthroline was more reliable for the determination of total pyritic iron as well as ferrous iron after incubation in 1M HCl at 21°C in the Fe(II)((aq)) sample with a recovery of 98%. Low ferrous iron concentrations of less than 0.5mM were overestimated in a Fe(III) background by up to 150% by both methods. Heating of mineral samples in 6M HCl increased their solubility and susceptibility for both photometric assays which is a need for total iron determination of highly crystalline minerals. However, heating also rendered a subsequent reliable determination of ferrous iron impossible due to fast abiotic oxidation. Due to the low solubility of highly crystalline samples, the determination of total iron is solely possible after dissolution in 6M HCl at 60°C which on the other hand makes determination of ferrous iron impossible. The recommended procedure for ferrous iron determination is therefore incubation at 21°C in 6M HCl, centrifugation, and subsequent measurement of ferrous iron in the supernatant. The different procedures were tested during growth of G. sulfurreducens on synthetic ferrihydrite. Here, the phenanthroline test was more accurate compared to the ferrozine test. However, the latter provided easy handling and seemed preferable for larger amounts of samples.
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- "The concentration of volatile fatty acids (VFAs) from the slurry waters was measured by a high-pressure liquid chromatograph (Alliance e26951, Waters, Tokyo, Japan) equipped with an RSpak KC-811 column (Shodex, Tokyo, Japan) and a photodiode array (2998, Waters, Tokyo, Japan). Ferrous iron was determined as described previously (Braunschweig et al., 2012). Briefly, 1 g of the sediment slurry samples was extracted for 5 days using 6 M HCl, and the extracted Fe(II) was determined using the ferrozine method. "
ABSTRACT: Organically enriched sediment has been found in water environments. The tsunami originating from the Great East Japan Earthquake in 2011 deposited large amount of sediment, thus providing evidence about its huge accumulation in coastal marine areas possibly due to human activities such as fish culture and marine product processing of industries. Here, degradation potential of organically enriched sediment deposited on a coastal site at Higashi-Matsushima, Miyagi, Japan was investigated under both sulfate- and iron-reducing conditions. Sediment slurry was prepared by mixing the sediment with artificial seawater. The effects of supplementation with sulfate and lepidocrocite (a crystalline Fe[III] oxide) on the structure and activity of the slurry microorganisms were examined by the combined physicochemical analyses and 16S rRNA deep sequencing. The sediment slurry was incubated for 5 days, during which the concentrations of TOC, sulfate, and ferrous iron remained at constant levels and the TG-DTA patterns did not change. The composition of dominant members of the microbial communities was stable, although the rare microbial populations slightly changed. The result in this study revealed that the organically enriched sediment was resistant to biodegradation under the sulfate- and iron-reducing conditions.Journal of Water and Environment Technology 08/2014; 12(4):357-366. DOI:10.2965/jwet.2014.357
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- "Fe 2+ was measured using the ferrozine assay according to Braunschweig et al. . Acetate and butyrate were measured by HPLC (Shimadzu, Japan) on an Aminex HPX87H column (Bio-Rad) with 0.5 mM H 2 SO 4 as a mobile phase (column temperature: 50 • C, flow rate: 0.5 mL min −1 , UV detection at 220 nm). "
ABSTRACT: For microorganisms that play an important role in bioremediation, the adaptation to swift changes in the availability of various substrates is a key for survival. The iron-reducing bacterium Geobacter metallireducens was hypothesized to repress utilization of less preferred substrates in the presence of high concentrations of easily degradable compounds. In our experiments, acetate and ethanol were preferred over benzoate, but benzoate was co-consumed with toluene and butyrate. To reveal overall physiological changes caused by different single substrates and a mixture of acetate plus benzoate, a nano-liquid chromatography–tandem mass spectrometry-based proteomic approach (nano-LC–MS/MS) was performed using label-free quantification. Significant differential expression during growth on different substrates was observed for 155 out of 1477 proteins. The benzoyl-CoA pathway was found to be subjected to incomplete repression during exponential growth on acetate in the presence of benzoate and on butyrate as a single substrate. Peripheral pathways of toluene, ethanol, and butyrate degradation were highly expressed only during growth on the corresponding substrates. However, low expression of these pathways was detected in all other tested conditions. Therefore, G. metallireducens seems to lack strong carbon catabolite repression under high substrate concentrations, which might be advantageous for survival in habitats rich in fatty acids and aromatic hydrocarbons.Systematic and Applied Microbiology 06/2014; 37(4). DOI:10.1016/j.syapm.2014.02.004 · 3.31 Impact Factor
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ABSTRACT: The apoprotein of Pseudomonas aeruginosa azurin binds iron(II) to give a 1:1 complex, which has been characterized by electronic absorption, Mössbauer, and NMR spectroscopies, as well as X-ray crystallography and quantum-chemical computations. Despite potential competition by water and other coordinating residues, iron(II) binds tightly to the low-coordinate site. The iron(II) complex does not react with chemical redox agents to undergo oxidation or reduction. Spectroscopically calibrated quantum-chemical computations show that the complex has high-spin iron(II) in a pseudotetrahedral coordination environment, which features interactions with side chains of two histidines and a cysteine as well as the C═O of Gly45. In the (5)A(1) ground state, the d(z(2)) orbital is doubly occupied. Mutation of Met121 to Ala leaves the metal site in a similar environment but creates a pocket for reversible binding of small anions to the iron(II) center. Specifically, azide forms a high-spin iron(II) complex and cyanide forms a low-spin iron(II) complex.Journal of the American Chemical Society 11/2012; 134(48). DOI:10.1021/ja308346b · 11.44 Impact Factor