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.
- SourceAvailable from: Wilfred F M Röling[Show abstract] [Hide abstract]
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; · 3.31 Impact Factor
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ABSTRACT: Nitrate-reducing, Fe(II)-oxidizing bacteria were suggested to couple with enzymatic Fe(II) oxidation to nitrate reduction. Denitrification proceeds via intermediates (NO2 -, NO) that can oxidize Fe(II) abiotically at neutral and particularly at acidic pH. Here, we present a revised Fe(II) quantification protocol preventing artifacts during acidic Fe extraction and evaluate the contribution of abiotic vs. enzymatic Fe(II) oxidation in cultures of the nitrate-reducing, Fe(II) oxidizer Acidovorax sp. BoFeN1. Sulfamic acid used instead of HCl reacts with nitrite and prevents abiotic Fe(II) oxidation during Fe extraction. Abiotic experiments without sulfamic acid showed that acidification of oxic Fe(II) nitrite samples leads to 5.6-fold more Fe(II) oxidation than in anoxic samples because the formed NO becomes rapidly reoxidized by O(2) , therefore leading to abiotic oxidation and underestimation of Fe(II). With our revised protocol using sulfamic acid, we quantified oxidation of approximately 7 mm of Fe(II) by BoFeN1 within 4 days. Without addition of sulfamic acid, the same oxidation was detected within only 2 days. Additionally, abiotic incubation of Fe(II) with nitrite in the presence of goethite as surface catalyst led to similar abiotic Fe(II) oxidation rates as observed in growing BoFeN1 cultures. BoFeN1 growth was observed on acetate with N(2) O as electron acceptor. When adding Fe(II), no Fe(II) oxidation was observed, suggesting that the absence of reactive N intermediates (NO2 -, NO) precludes Fe(II) oxidation. The addition of ferrihydrite [Fe(OH)(3) ] to acetate/nitrate BoFeN1 cultures led to growth stimulation equivalent to previously described effects on growth by adding Fe(II). This suggests that elevated iron concentrations might provide a nutritional effect rather than energy-yielding Fe(II) oxidation. Our findings therefore suggest that although enzymatic Fe(II) oxidation by denitrifiers cannot be fully ruled out, its contribution to the observed Fe(II) oxidation in microbial cultures is probably lower than previously suggested and has to be questioned in general until the enzymatic machinery-mediating Fe(II) oxidation is identified.Geobiology 12/2012; · 3.69 Impact Factor
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ABSTRACT: Microbial reduction of ferric iron is partly dependent on Fe hydroxide particle size: nanosized Fe hydroxides greatly exceed the bioavailability of their counterparts larger than 1 μm. Citrate as a low molecular weight organic acid can likewise stabilize colloidal suspensions against aggregation by electrostatic repulsion but also increase Fe bioavailability by enhancing Fe hydroxide solubility. The aim of this study was to see whether adsorption of citrate onto surfaces of large ferrihydrite aggregates results in the formation of a stable colloidal suspension by electrostatic repulsion and how this effect influences microbial Fe reduction. Furthermore, we wanted to discriminate between citrate-mediated colloid stabilization out of larger aggregates and ferrihydrite dissolution and their influence on microbial Fe hydroxide reduction. Dissolution kinetics of ferrihydrite aggregates induced by different concentrations of citrate and humic acids were compared to microbial reduction kinetics with Geobacter sulfurreducens. Dynamic light scattering results showed the formation of a stable colloidal suspension and colloids with hydrodynamic diameters of 69 (±37) to 165 (± 65) nm for molar citrate:Fe ratios of 0.1 to 0.5 and partial dissolution of ferrihydrite at citrate:Fe ratios ⩾ 0.1. No dissolution or colloid stabilization was detected in the presence of humic acids. Adsorption of citrate, necessary for dissolution, reversed the surface charge and led to electrostatic repulsion between sub-aggregates of ferrihydrite and colloid stabilization when the citrate:Fe ratio was above a critical value (⩽ 0.1). Lower ratios resulted in stronger ferrihydrite aggregation instead of formation of a stable colloidal suspension, owing to neutralization of the positive surface charge. At the same time, microbial ferrihydrite reduction increased from 0.029 to 0.184 mM h−1 indicating that colloids stabilized by citrate addition enhanced microbial Fe reduction. Modelling of abiotic dissolution kinetics revealed that colloid stabilization was most pronounced at citrate:Fe ratios of 0.1 – 0.5, whereas higher ratios led to enhanced dissolution of both colloidal and larger aggregated fractions. Mathematical simulation of the microbial reduction kinetics under consideration of partial dissolution and colloid stabilization showed that the bioaccessibility increases in the order large aggregates < stable colloids < Fe-citrate. These findings indicate that much of the organic acid driven mobilization of Fe oxy(hydr)oxides is most likely due to colloid formation and stabilization rather than solubilisation.Geochimica et Cosmochimica Acta 08/2014; 139:434–446. · 4.25 Impact Factor
Reevaluation of colorimetric iron determination methods commonly used
Juliane Braunschweiga, Julian Boscha, Katja Heisterb, Christine Kuebecka, Rainer U. Meckenstocka,⁎
aInstitute of Groundwater Ecology, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany
bLehrstuhl für Bodenkunde, Technische Universität München, D-85350 Freising-Weihenstephan, Germany
a b s t r a c ta r t i c l e i n f o
Received 21 November 2011
Received in revised form 30 January 2012
Accepted 30 January 2012
Available online 12 February 2012
Microbial iron oxidation
Microbial iron reduction
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 dissolu-
tion and inductively coupled plasma atomic emission spectroscopy (ICP-AES). Iron dissolution prior to the
photometric assays included dissolution in 1 M or 6 M HCl, at 21 or 60 °C, and oxic or anoxic conditions. Re-
sults indicated a good reproducibility of quantitative total iron determinations by the ferrozine and phenan-
throline 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 1 M HCl by 10 to 13%, whereas phenanthroline matched the results determined by ICP-AES with a devia-
tion of 5%. Total iron concentrations after dissolution in 1 M HCl of highly crystalline oxides such as magne-
tite, a mixture of goethite and magnetite, and goethite were underestimated by up to 95% with both methods.
When dissolving these minerals in 6 M 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 re-
liable for the determination of total pyritic iron as well as ferrous iron after incubation in 1 M HCl at 21 °C
in the Fe(II)(aq)sample with a recovery of 98%. Low ferrous iron concentrations of less than 0.5 mM were
overestimated in a Fe(III) background by up to 150% by both methods. Heating of mineral samples in 6 M
HCl increased their solubility and susceptibility for both photometric assays which is a need for total iron de-
termination of highly crystalline minerals. However, heating also rendered a subsequent reliable determina-
tion 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 6 M 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 6 M HCl, centrifu-
gation, 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.
© 2012 Elsevier B.V. All rights reserved.
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
are commonly used to assess total (Fe(tot)) or ferrous iron (Fe(II)) con-
tents due to their simple application in everyday's laboratory routine
(Anastácio et al., 2008). In current geomicrobiological studies, the ferro-
zine (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.,
Journal of Microbiological Methods 89 (2012) 41–48
⁎ Corresponding author. Tel.: +49 89 3187 2561; fax: +49 89 3187 3361.
E-mail addresses: email@example.com
(J. Braunschweig), firstname.lastname@example.org (J. Bosch),
email@example.com (K. Heister), firstname.lastname@example.org
(C. Kuebeck), email@example.com (R.U. Meckenstock).
0167-7012/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
Contents lists available at SciVerse ScienceDirect
Journal of Microbiological Methods
journal homepage: www.elsevier.com/locate/jmicmeth
2011), mineralogy (Osborne et al., 2010), and engineering (Paipa et al.,
2005). For investigations of siliceous mineral systems, the phenanthro-
line 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-phe-
nanthroline and exposure to ambient UV radiation (David et al., 1972;
nation 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)-phe-
nanthroline 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 com-
plex (adsorption at 560 nm) with ferrozine. For ferrozine, a light sen-
sitivity 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 dissolu-
tion and quantification of Fe(II) and Fe oxides in large sample num-
bers 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 min-
erals 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 in-
crease of dissolution temperature and incubation time is also neces-
sary to achieve sufficient dissolution (Kuslu and Bayramoglu, 2002).
Despite their broad application in today's geomicrobiological re-
search, a systematic investigation of the accuracy and applicability
of the two colorimetric Fe assays with respect to samples typically an-
alyzed 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 in-
vestigated, 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 proce-
dures. This might result in a decrease in reproducibility or accuracy of
This study intends to close the scientific gapon 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 goe-
and a microbially-formed mixture of magnetite and goethite. These
minerals representa set of Fe oxides widely applied in recentgeomicro-
biological studies (Cutting et al., 2009; Zhang et al., 2009).
2. Materials and methods
2.1. 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 fresh-
water 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 6.5–6.9. This medium was supplemented
with 0.1 mL L−1trace elements solution SL10 (Widdel et al., 1983),
0.1 mL L−1selenite–tungsten solution, 0.03 mL L−1of 7 vitamin solu-
tion (Widdel and Pfennig, 1981), and 5 μM anthraquinone-2,6-disulfo-
nate (AQDS). As stimulating agent for anaerobic growth, cAMP was
added at 10 μM. Fifty millimolar synthesized ferrihydrite (Lovley and
Phillips, 1986b) was used as electron acceptor. Ten millimolar sodium
acetate or 500 μM toluene was used as electron donor. Microorganisms
were cultivated in 60 mL medium in 100 mL glass serum bottles sealed
er resin XAD-7 (0.3 g) was added to each bottle to decrease the actual
toluene concentration to sub-toxic levels. The bottles were incubated
at 30 °C in the dark. Addition of toluene, inoculation of the media with
bacteria, and sampling were performed carefully with anoxic syringes
through the closed stoppers.
2.2. Fe bearing solutions and minerals
As reference solutions for the evaluation of crystalline Fe oxide dis-
solution, 9 mM Fe(II) and Fe(III) solutions were prepared with FeSO4·
7 H2O (A.C.S. grade, Sigma-Aldrich, Inc., USA) and Fe(NO3)3· 9 H2O
(A.C.S. grade, Sigma-Aldrich) in 1 M HCl, respectively. Additionally,
both solutions were mixed at the ratio 1:40 (v/v) to mimic potentially
reduced or oxidized minerals. Fifty-one millimolar synthesized ferrihy-
drite (Lovley and Phillips, 1986b) was dissolved in 1 M and 6 M HCl
(1:10, v/v). Magnetite and a mixture of goethite and magnetite, formed
ic ferrihydrite, were also dissolved in 1 M and 6 M HCl (1:10, v/v) with
final Fe(tot) concentrations of 31 and 36 mM, respectively. Solid syn-
thetic goethite (Bayferrox 920Z, Lanxess GmbH, Germany) was dis-
solved in 1 M and 6 M HCl with final concentrations of 10 mM,
whereas pyrite (Georg Maisch, Freising, Germany) was dissolved only
in 6 M HCl (1:10, v/v) with a final concentration of 35 mM.
2.3.1. Ferrozine assay
The ferrozine stock solution was prepared by dissolving 1 g of fer-
rozine (purum p.a., Fluka Sigma-Aldrich) in 1 L of a 6.5 M ammonium
acetate solution (97%+, A.C.S., Sigma-Aldrich). A 1.4 M hydroxyl-
amine stock solution (99%, Sigma-Aldrich) was prepared in 1 L of
1 M HCl. Both solutions were stored at 4 °C in the dark.
2.3.2. Phenanthroline assay
A 7 mM phenanthroline stock solution (≥99%, Sigma-Aldrich)
was prepared. When Fe(III) was reduced to Fe(II) for determination
of the total iron content, a 1.2 M sodium acetate solution (99%,
ReagentPlus®, Sigma-Aldrich) was used as buffer. When the sample
contained Fe(III), a 1.3 M ammonium acetate solution was used in-
stead. Furthermore, a 2 M ammonium fluoride solution (99.99+%
trace metals basis, Sigma-Aldrich) was used as masking agent for
If not otherwise stated, all reagents were prepared with Millipore
water (R=18.2 MΩ, 4 ppb TOC, MilliporeElix+Milli-Q Advantage
2.4. Experimental procedures
2.4.1. Influence of acid concentration on iron extraction
Dissolved Fe(II) and Fe(III) (9 to 10 mM Fe(tot)) and solutions
containing goethite (31 mM), a mixture of goethite and magnetite
(36 mM), magnetite (31 mM), and ferrihydrite (51 mM) were diluted
in 1 M or 6 M HCl in an Eppendorf tube (1:9, v/v in 1 mL) and shaken
J. Braunschweig et al. / Journal of Microbiological Methods 89 (2012) 41–48
over night or for 4 days (goethite) at 21 °C. In parallel, all samples di-
luted in 6 M HCl were shaken at 60 °C over night or for 4 days (goe-
thite and pyrite). One sample of each approach was taken and three
aliquots per sample were measured with ferrozine or phenanthroline.
For the measurement with ferrozine, the acidified and shaken sam-
ples were mixed with the ferrozine solution at a ratio of 1:9 (v/v in
0.2 mL) in a microtiter plate (0.3 mL/well, Nunc, Denmark), enabling a
faster measurement procedure for large sample numbers as compared
to the use of cuvettes. Absorbance of three wells per sample was mea-
sured with a Wallac Victor31420 plate reader (PerkinElmer, Inc., USA)
at 560 nm.
For the phenanthroline method, 0.16 mL of ammonium fluoride,
0.2 mL of phenanthroline, and 0.4 mL of ammonium acetate were
filled in 2 mL reaction tubes (Eppendorf, Germany). The acidified
iron samples were added in 10 μL steps to the mixture, until an ap-
propriate orange color developed (>0.2 absorbance units). After 1 h
incubation, three aliquots of 0.2 mL from each sample were filled
into microtiter plates and absorbance was measured at 490 nm. For
both methods, iron concentrations and standard deviations were cal-
culated from the mean value of the absorbance of three aliquots.
For determination of total Fe content with the phenanthroline
method, the acidified samples were diluted and reduced with hy-
droxylamine (1:10, v/v in 1 mL) and shaken for 15 min at 21 °C to re-
duce all Fe(III) to Fe(II). Afterwards, the samples were diluted with
ferrozine and measured as described above. For the phenanthroline
procedure, 0.2 mL of phenanthroline and 0.4 mL of sodium acetate
were mixed in a 2 mL tube and the acidified and reduced samples
were added in 10 μL steps as described above. After 1 h incubation,
the absorbance was measured. Masking of Fe(III) with ammonium
fluoride was not necessary due to the prior reduction with
2.4.2. Anoxic preparation of samples
To investigate the impact of ambient oxygen during the proce-
dure, the same experiments were also conducted under anoxic condi-
tions in a glove box (b3 ppm O2, N2/H2=95/5%, v/v, Coy Laboratory
Products, USA). One sample of each approach was prepared in 6 M
HCl and shaken over night at 60 °C. Three aliquots per sample were
analyzed as described above. All chemicals were filled into anoxic
100 mL glass serum bottles, sealed with butyl rubber stoppers and
flushed with 20/80% CO2/N2for 3 min to exchange the headspace be-
fore introducing into the glove box.
2.4.3. Growth experiments
For evaluation of the performance of the different methods, G.
sulfurreducens was grown in a freshwater medium as described
above. Cells and ferrihydrite were added via anoxic syringes to a
final concentration of 52 mM. The bottles were incubated at 30 °C in
the dark. Immediately after inoculation, the first samples for Fe deter-
mination were taken under anoxic conditions and dissolved in 1 M
and 6 M HCl as described above. Subsequently, the undiluted samples
were transferred out of the glove box, exposed to O2, and dissolved in
oxic 1 M or 6 M HCl, respectively. All samples dissolved in 1 M HCl
and one reaction tube with samples in 6 M HCl were shaken over
night at 21 °C. The remaining reaction tubes with samples diluted in
6 M HCl were shaken over night at 60 °C. The procedure of sampling
remained unchanged over the entire experiment. Three aliquots of
each sample were measured with the ferrozine and phenanthroline
method as described above. Iron concentrations and standard devia-
tions were calculated from the mean value of the absorbance of
Total iron was determined with both methods for all time points,
and mean values and standard deviations (n=11) are calculated in
percent of the concentration measured with ICP-AES. Ferrous and
total iron were measured for all three batches separately. Data of
one batch are shown, whereas the other two batches serve as inde-
2.4.4. Influence of medium composition
Ferric iron solutions were prepared with FeCl2· 4H2O (p.a., Sigma-
Aldrich) in different concentrations (Table 1). Every solution was
mixed with a single medium additive. In order to visualize potential
effects of additives, their added concentrations are increased. One
part of the samples was flushed with N2/CO2(20/80%) (Table 1). All
samples were dissolved in 1 M HCl with subsequent shaking over
night at 21 °C. Ferrous and total iron determinations were performed
as described above under oxic conditions. All approaches were done
twice to obtain independent replicas.
2.4.5. Influence of light on ferrous iron determination with ferrozine
plexes, two aqueous Fe(III) samples with final concentrations of 1.4 or
0.7 mM were incubated for 0, 3, 5, 7, 10, or 30 min under ambient
were calculated from the mean absorbance values.
2.4.6. Total iron verification and characterization of iron oxides
As external control of the quality of both assays and the dissolution
procedures, the total Fe content of all samples was measured with in-
ductively coupled plasma atomic emission spectrometry (ICP-AES).
Minerals were totally dissolved using aqua regia (mixture of HCl:
HNO3=3:1) with heating at 70 °C for 1 h. Total Fe concentrations in
the digestions were determined using a Ciros Vision ICP-AES (Spectro
Analytical Instruments, Germany) in 15 L min−1Ar.
Powder X-ray diffraction (XRD) patterns for crystallographic struc-
USA) aliquots. A Philips vertical goniometer (PW 1050, Philips, The
Netherlands), equipped with a diffracted beam graphite monochroma-
tor,andCoKαX-rayradiationat40 kVand30 mAwereapplied.Powder
specimens were measured from 5 to 80° 2θ with increments of 0.02° 2θ
anda countingtimeof5 s perstep.Theobserved peaks of haliteandsyl-
vite stem from the diluted freshwater medium which became concen-
trated during the freeze-drying procedure.
2.4.7. Statistical analysis
Smirnov test. The Student's t test at 5% significance level (Harris, 2002)
was used to compare the iron concentrations obtained with ferrozine
and phenanthroline, respectively. All statistical analyses were per-
To compare the photometric ferrozine and phenanthroline assays
for Fe determination, we measured the Fe(II) and Fe(tot) contents of
Fe(II)aqand Fe(III)aq, mixed valence solutions, synthetic goethite, fer-
rihydrite, 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 syn-
thesized ferrihydrite as well as the microbially-formed magnetite
and the mixture of goethite and magnetite were confirmed by XRD
analysis (Fig. 1).
3.1. Efficiency of total iron determination
For Fe(II)aq, Fe(III)aq, ferrihydrite, and magnetite, the concentra-
tions of Fe(tot) obtained from the phenanthroline assay after dissolu-
tion in 1 M HCl (at 21 °C) were significantly higher than those from
J. Braunschweig et al. / Journal of Microbiological Methods 89 (2012) 41–48
the ferrozine method. For ferrihydrite and aqueous Fe(II) or Fe(III)
standard solutions, the phenanthroline assay perfectly matched the
concentrations of the external control measurements via ICP-AES
(Fig. 2a), while the ferrozine assay led to systematic underestimation
of Fe(tot) concentrations between 13 and 10%. Due to the low solubil-
ity of goethite, microbially-formed magnetite, and the mixture of goe-
thite and magnetite in 1 M HCl, the concentrations of these crystalline
iron forms were significantly underestimated with both assays by 78
to 92% for goethite and the mixture of goethite and magnetite and 5
to 12% for magnetite (Fig. 2a). In case goethite and the mixture of
goethite and magnetite, the minerals were visibly not dissolved
completely, which led to additional disturbance of the photometric
measurements by turbidity.
The Fe(tot) concentrations after dissolution in 6 M HCl at 21 °C for
4 days for goethite and pyrite were as low as concentrations obtained
after shaking in 1 M HCl. For the other minerals, the phenanthroline
method led to slightly higher values than the ferrozine test and in
most cases to an overestimation of Fe(tot) in comparison to the exter-
nal ICP-AES control (Fig. 2a). For ferrihydrite and the mixture of goe-
thite and magnetite, the ferrozine test precisely matched the ICP-AES
results,whiletheFe(tot)concentrationsof goethite,pyrite, and magne-
difference between both methods is not significant neither for aqueous
Fe solutions nor ferrihydrite.
Elevation of the incubation temperature to 60 °C and an HCl con-
centration of 6 M completely dissolved all tested minerals, including
highly crystalline oxides and sulfides like goethite, magnetite, and py-
rite. Dissolution in 6 M HCl at 60 °C for 1 or 4 days for goethite and
pyrite led to a good agreement of measured Fe(tot) concentrations
vs. the external control for all sample types (Fig. 2a). Here, the ferro-
zine assay almost exactly reflected the ICP-AES results with a devia-
tion of less than 5%. The phenanthroline method often led to an
overestimation by 5 to 10% (Fig. 2a) but the difference between
Fig. 1. X-ray diffraction patterns of iron bearing minerals from anaerobic microbial re-
duction experiments: (a) ferrihydrite, (b) magnetite, and (c) mixture of goethite and
magnetite. Bars show theoretical peaks of ferrihydrite (gray), magnetite (dotted),
and goethite (black). Peaks of NaCl (32.04, 37.14, 53.42, 66.8° 2θ) and KCl (33.12°
2θ) originate from the cultivation medium, peak at 45.02° 2θ from the aluminum sam-
Composition of FeCl2solutions including different medium additives for the investigation of their interferences with ferrozine and phenanthroline.
TE 7 Vits
Vol.% of additive in sample
Total Fe concentration1
1Determined with ICP-AES.
2Determined after acidification of the samples with 1 M HCl and adjustment to 1.0–1.2 with 1 M NaOH.
Fig. 2. Comparison of extraction methods and iron determination from different sam-
ples. a) Total iron related to ICP measurements as external standard. b) Ferrous iron
concentrations under oxic conditions and c) ferrous iron under oxic (ox) and anoxic
(anox) conditions. Values were obtained by the ferrozine and the phenanthroline
(phen) assays. Error bars depict the standard deviations from three parallel measure-
ments of one sample.
J. Braunschweig et al. / Journal of Microbiological Methods 89 (2012) 41–48
both methods was not significant. For pyrite, the measurements with
phenanthroline resulted in 91% of the externally determined concen-
tration, while the ferrozine method led to a severe overestimation of
145% of the actual concentration.
3.2. Ferrous iron determination
When Fe(II) was the only Fe species, the phenanthroline meth-
od exactly matched the concentrations determined by ICP-AES,
whereas ferrozine overestimated by 14% (1.5 mM). No reliable Fe
(II) determination was achieved, when the concentration in the
samples was below 0.5 mM, since deviations of expected to mea-
sured values were larger than 100%. Ferrous iron concentrations
of magnetite and the mixture of goethite and magnetite after disso-
lution in 1 M HCl at 21 °C (Fig. 2b) were higher for the phenanthro-
line assay than for ferrozine. However, the true amounts of Fe(II)
in magnetite and in the mixture of goethite and magnetite are
Samples dissolved in 6 M HCl (at 21 °C) yielded slightly reduced
Fe(II) concentrations as compared to 1 M HCl treatments (Fig. 2b).
Ferrous iron concentrations after 6 M HCl dissolution were somewhat
higher for the phenanthroline assay than for the ferrozine assay.
However, the Fe(II) concentrations determined with ferrozine
matched the ICP-AES concentrations for the samples Fe(II) and Fe
(II)/(III). The Fe(II) concentrations of pyrite were underestimated by
70–90% by the photometrical tests.
Determined Fe(II) concentrations for Fe(II), magnetite, and the
mixture of goethite and magnetite after dissolution in 6 M HCl at
60 °C were 4 to 6-fold lower than after shaking at 21 °C in 1 M or
6 M HCl (Fig. 2b). At very low Fe2+concentrations as in the Fe(II)/
(III) and ferrihydrite samples, Fe(II) was not detectable. For pyrite,
the phenanthroline method overestimated the Fe(II) concentrations
by about 10%, whereas the ferrozine method led to an underestima-
tion of 23%. For pyrite, the dissolution in 6 M HCl at 60 °C and the sub-
sequent measurement using the phenanthroline assay was the only
method almost matching the externally determined concentration
For ferrozine, we could observe a photochemical reduction of Fe
(III)-ferrozine complexes by light (Fig. 3). During a maximal incuba-
tion time of a sample with ferrozine in a microtiter plate of 5 min dur-
ing which all samples were processed, 8 to 15% of the Fe(III) were
However, for ferrous iron determinations the ferrozine and phe-
nanthroline methods did not show systematical differences.
3.3. Oxic vs. anoxic extraction
For the minerals magnetite, ferrihydrite, a mixture of goethite and
magnetite as well as the Fe(II)aqand mixed valence solutions, anoxic
incubation almost doubled the detectable Fe2+concentrations after
shaking in 6 M HCl at 60 °C for 4 days (Fig. 2c). Nevertheless, the
measured concentrations were only 20 to 30% of the Fe2+measured
after shaking in 1 M HCl at 21 °C, whereas the values obtained from
the phenanthroline method were slightly higher than those obtained
3.4. Growth experiments
When Fe(II) and Fe(tot) were analyzed in batch experiments with
G. sulfurreducens and ferrihydrite as electron acceptor, the ferrozine
method matched the externally determined Fe(tot) concentrations
to 100% (Fig. 4a). In two other experiments, deviations of 5 and 8% oc-
curred, respectively. Concentrations obtained with the phenanthro-
line method were 8 to 13% higher than the reference concentrations
determined with ICP-AES. Single measurements of the samples dis-
solved in 1 M HCl showed that both methods overestimated the
ICP-AES concentrations during the first 100 h. Afterwards, ferrozine
tended to underestimate, whereas phenanthroline matched the ICP-
AES results. After dissolution in 6 M HCl at 60 °C, the single values
of every time point determined with ferrozine varied between
±5 mM (10% of total Fe), while phenanthroline overestimated in
general about 10%, occasionally up to 40%.
For oxic treatment at 21 °C, the Fe(II) values after dissolution in
6 M HCl were less than 50% of the measured concentration after dis-
solution in 1 M HCl for both methods (Fig. 4b). After dissolution in
6 M HCl at 60 °C, no Fe(II) was detectable. Anoxic treatment im-
proved all results. Especially after dissolution in 6 M HCl at 21 °C,
the recovery was up to 40% of the Fe(tot) concentration and led to re-
sults close to 1 M HCl samples (Fig. 4c). However, the concentrations
measured after dissolution at 60 °C in 6 M HCl were up to 50% lower
than the results obtained after dissolution at 21 °C.
In almost all treatments, phenanthroline led to higher concentra-
tions than ferrozine, in a range of 1 to 9% of the total Fe concentration
(Fig. 4a) and up to 22% in other batches (related to ICP-AES data).
Only after treatment with 6 M HCl at 21 °C, ferrozine yielded higher
concentrations of 1 and 8% of Fe(tot).
3.5. Influence of medium composition
In most cases, results determined for total Fe (Fig. 5a) showed sig-
nificantly higher values for ferrozine than for phenanthroline, which
contradicts to Figs. 2a and 3a. Addition of trace element solution as
well as the vitamin solution led to underestimation of up to 20% for
For Fe(II) determination, the trend of significant phenanthroline
over- and ferrozine underestimation of the ICP data could be con-
firmed (Fig. 5b). The addition of trace element solution led to an un-
derestimation of 5 and 9% for the ferrozine and phenanthroline
methods, respectively. An opposite effect was observed after mixing
with the vitamin solution, which led to overestimations of >20% for
This study compares the application of the colorimetric phenan-
throline 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 concen-
trations in geomicrobiological studies.
Fig. 3. Effect of photochemical reduction of Fe(III)-ferrozine complexes of pure Fe(III)
stock solutions caused by light irradiation. Error bars depict the standard deviations
from six parallel measurements of one sample.
J. Braunschweig et al. / Journal of Microbiological Methods 89 (2012) 41–48
4.1. Quantification of total iron content
The comparison of photometric Fe quantifications with external
validation by ICP-AES of different Fe oxides proved the general suit-
ability of both the phenanthroline and ferrozine assays for the Fe
(tot) determination of typical geomicrobiological Fe samples. Espe-
cially the easy dissolvable species such as aqueous Fe(II) and Fe(III)
solutions, and amorphous ferrihydrite were reliably determined, in-
dependent of the assay type or dissolution procedure, with phenan-
throline being slightly more accurate.
The main problem for the analysis of crystalline Fe oxides was
their poor solubility. Dissolution procedures turned out to be decisive
for precise Fe quantification. Mean values of several measurements of
the growth experiment showed that the results obtained with the fer-
rozine assay fitted best, regardless of acidic strength or dissolution
temperature. During growth of G. sulfurreducens, ferrihydrite recrys-
tallized to magnetite which is hardly soluble in 1 M HCl. However, re-
sults of the phenanthroline method were frequently higher than
results obtained with ferrozine and the ICP-AES reference concentra-
tions. This effect is already described by Anastácio et al. (2008) and is
attributed to the complete dissolution of minerals by phenanthroline.
Interferences with Co2+, Cu2+, Mn2+, and Zn2+(Ali, 1993;
Evans, 1983; Jyothi et al., 1987; Kompany-Zareh and Massoumi,
1999; Mutaftchiev et al., 1999), which have absorption maxima be-
tween 300 and 530 nm, could be excluded. These cations were pre-
sent in the applied freshwater medium with concentrations of 0.96,
0.01, 0.76, and 0.84 μM, respectively. Besides, Fe(tot) determination
of Fe(II) solutions spiked with trace elements led to underestimated
values for both methods. Taylor et al. (1994) showed that metabolic
products of cells can form strong complexes with Fe(II) and Fe(III)
which are characterized by pH independent thermodynamic stability.
is also able to produce such strong complexation agents which are not
Fig. 5. Total a) and ferrous iron concentrations b) of FeCl2solutions treated with differ-
ent medium additives. N2/CO2: flushed with N2/CO2, Medium: freshwater medium, TE:
trace elements, 7 Vits: vitamin solution.
Fig. 4. Comparison of extraction methods and iron determination from samples of a
growth experiment of G. sulfurreducens on synthetic ferrihydrite. a) Total iron concen-
trations of added iron oxides related to ICP measurements as external standard and b)
ferrous iron concentrations under anoxic and c) oxic conditions. Total iron concentra-
tions and their standard deviations are calculated as mean values from all taken
J. Braunschweig et al. / Journal of Microbiological Methods 89 (2012) 41–48
destroyable by 1 M HCl. Possibly, phenanthroline can dissolve these
complexes in contrast to ferrozine which may explain lower values
obtained with ferrozine compared to phenanthroline in biological sam-
ples in contrast to the abiotic experiments (Fig. 5a). Vitamins, having
similar complexing properties, might explain the underestimation of
the Fe(tot) content with ferrozine and phenanthroline by 5 and 16%, re-
mation of the Fe(tot) content with phenanthroline in biological samples
still remained unclear.
The phenanthroline method proved to be suited best for the deter-
mination of total pyritic iron after dissolution in 6 M HCl at 60 °C.
High concentrations of sulfide seem to affect the ferrozine complex
(Chapin et al., 2002), leading to overestimation in the photometric
measurements. However, a brownish precipitate, as described by
these authors, was not observed in our study.
The best results for Fe(tot) determination of highly crystalline Fe
oxides, such as goethite and magnetite, were obtained with the ferro-
zine assay after dissolution in 6 M HCl at 60 °C (Fig. 6). However, the
increase of acidic strength without increasing temperature did not
improve the results apart from goethite and a mixture of goethite
and magnetite. For easily dissolvable compounds in biotic samples
such as ferrihydrite or Fe(II)aqwhich were incubated in 1 M HCl at
21 °C, the best results are obtained by the phenanthroline assay.
Mean values of several measurements with the ferrozine method
were in excellent accordance with the external validation. Therefore,
and due to the time consuming procedure of the phenanthroline
method, we advocate the ferrozine assay for measurements of large
4.2. Ferrous iron determination
Ferrous iron concentrations were measured with highest accuracy
at 21 °C. Nevertheless, both photometric methods tended to overesti-
mate actual Fe(II) concentrations in biological samples. For ferrozine,
this can be attributed to a photochemical reduction of the Fe(III)-fer-
rozine complex which was also observed by Anastácio et al. (2008).
For the phenanthroline assay, photochemical reduction can be ex-
cluded due to the addition of a masking agent. A similar overestima-
tion of Fe(II) determination was observed for the mixed valence
sample Fe(II)/(III). We assigned this overestimation to a decreasing
reliability of both assays with decreasing concentrations.
Iron determinations of the growth experiment frequently showed
higher concentrations for the phenanthroline method compared to fer-
rozine (Fig. 4b). However, the exact Fe(II) concentrations are not
known. Nevertheless, in the context of overestimation for Fe(tot) with
phenanthroline in biotic samples, we assume an overestimation here
as well. Conspicuously, single measurements of the same sample are
more erratic for ferrozine than for phenanthroline. Anastácio et al.
(2008) explained these fluctuations with the light sensitivity of ferro-
zine and the partly dissolution of highly crystalline Fe oxides. For Fe
(II), interferences with medium additives could be excluded for both
methods. Independently of the additive, values obtained by phenan-
throline are mostly up to 5% higher than ICP data, whereas ferrozine
showed up to 5% lower concentrations. The high concentrations after
addition of the vitamin solution could be attributed to interference
As described for determination of the total iron content, the low
solubility of highly crystalline samples reduced the measurable con-
centration of Fe(II). The detected concentrations of Fe(II) in the mix-
ture of goethite and magnetite at 21 °C were not convincing due to
the low solubility of goethite and magnetite and the resulting high
turbidity of the sample. Ferrous iron incorporated into the crystal
lattice could probably not react with phenanthroline and ferrozine.
The low Fe(II) concentrations determined for incubations at 60 °C
might be due to a fast abiotic oxidation of Fe(II) (Millero et al., 1987;
Sung and Morgan, 1980). Porsch and Kappler (2011) explained this
observation with the formation of Fe(II)–Cl−complexes and undisso-
ciated HCl, leading to Fe–HCl complexes. Both complexes lead to a
faster oxidation of Fe(II). Anoxic sampling improved the Fe(II) recov-
ery, but the underestimation was still significant, indicating that Fe
(II) might oxidize during the 4 days incubation in 6 M HCl. The only
mineral which was not affected by oxidation was pyrite. Schoonen
et al. (2000) attributed this effect to the faster sulfide oxidation, caus-
ing suppression of iron oxidation at pH values below 3 and under il-
lumination with visible light. A concentration close to the reference
ICP-AES concentration of the pyrite sample was measured with phe-
nanthroline. The lower results with ferrozine could also be evoked
by the interference between ferrozine and sulfide.
In summary, for easily dissolvable compounds such as ferrihydrite
and Fe(II)aq, we recommend 1 M HCl for dissolution with an incuba-
tion time of 24 h (Fig. 6). More crystalline minerals such as goethite
or magnetite have to be dissolved in 6 M HCl at 60 °C for accurate
iron measurement. In contrast to the Fe(tot) determination, heating
of the acidified samples is not recommended for subsequent mea-
surements of Fe(II) because Fe(II) will oxidize. An opportunity to
overcome the difficulty of Fe(II) determination in samples with
hardly soluble iron oxides is the partial dissolution of the samples
in 6 M HCl at 21 °C. To avoid the interference of the turbid solutions
with the colored ferrozine or phenanthroline complexes, the acidi-
fied samples should be centrifuged with subsequent determination
of the Fe2+content in the supernatant. Most accurate results for
pyrite were obtained using the time consuming phenanthroline
assay. To avoid possible interferences with different cations, we
recommend using the ferrozine assay which is usually accurate
within ±10% for both Fe(II) and Fe(tot) measurements.
Fe(aq) / Fh
(hardly soluble oxides)
Gt / Magn
1 M, 21˚C
1 M + 6M
6 M, 60˚C
1 M, 21˚C
6 M, 60˚C6 M, 60˚C
Fig. 6. Proposedextractionmethodsandirondeterminationassaysfordifferentsampletypes.+/+depictscombinationsofextractionandanalysismethodsproducingveryaccurateresults.
+/− stands for less accurate but recommended methods whereas combinations labeled with −/− are not recommended to use. The upper part of the figure shows combinations for total
iron (Fe(tot)), the lower for ferrous iron (Fe(II)) analysis.
J. Braunschweig et al. / Journal of Microbiological Methods 89 (2012) 41–48
We thank Bernhard Michalke for ICP-AES measurements and
Thomas Braunschweig for help with the statistic analysis. This study
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