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Applied and Environmental Microbiology, October 2001, p. 4448-4453, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4448-4453.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Growth of Pseudomonas
mendocina on Fe(III) (Hydr)Oxides
L. E.
Hersman,1,*
J. H.
Forsythe,1
L. O.
Ticknor,2 and
P.
A.
Maurice
Bioscience Division1
and Decision Applications Division,2 Los
Alamos National Laboratory, Los Alamos, New Mexico 87545, and
Department of Civil Engineering and Geological Sciences,
University of Notre Dame, Notre Dame, Indiana 465562
Received 20 February 2001/Accepted 8 July 2001
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ABSTRACT |
Although iron (Fe) is an essential element for almost all
living organisms, little is known regarding its
acquisition from the insoluble Fe(III) (hydr)oxides in
aerobic environments. In this study a strict aerobe,
Pseudomonas mendocina, was grown in batch culture
with hematite, goethite, or ferrihydrite as a source of Fe.
P. mendocina obtained Fe from these minerals in
the following order: goethite > hematite > ferrihydrite. Furthermore, Fe release from each of the minerals
appears to have occurred in excess, as evidenced by the growth of
P. mendocina in the medium above that of the insoluble
Fe(III) (hydr)oxide aggregates, and this release was independent of
the mineral's surface area. These results demonstrate that an aerobic
microorganism was able to obtain Fe for growth from several insoluble
Fe minerals and did so with various growth rates.
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INTRODUCTION |
Used in all heme enzymes (proteins
with an Fe cofactor), including cytochromes and hydroperoxidases, a
constituent of ribonucleotide reductase and essential for the activity
of nitrogenases, Fe is required by all microorganisms except those
lactobacilli lacking heme and using a cobalt type of ribonucleotide
reductase (1, 3). Fe's biological importance is a result
of its electronic structure, which is capable of reversible changes in
oxidation state over a wide range of oxidation-reduction potentials:
300 mV in a-type cytochromes to 
490 mV in some Fe-sulfur
proteins (7, 8, 17, 28). Owing to this redox versatility,
Fe occupies a vital role in biological systems (31).
However, Fe is very insoluble at circumneutral pH and in oxic
environments. Although abundant at the Earth's surface, the most
common forms of Fe, the Fe(III) (hydr)oxides (which include oxides,
oxyhydroxides, and hydrated oxides) have solubility products ranging
from 10
39 to 1044, limiting the
Fe3+ aqueous equilibrium concentration to ca.
1017 M (20) in the absence of complexing ligands.
Most microorganisms require micromolar concentrations of Fe to support
growth (18); as a consequence, Fe deprivation of many
species will occur when the culture medium contains <0.1 µM
available Fe. Given this constraint, microorganisms are faced with
overcoming an approximately 10 orders of magnitude discrepancy between
the available Fe (
1017 M) and their metabolic
requirement (107 M) in aerobic environments. As
emphasized by Schwertmann (23), the availability of Fe in
aerobic soils must be governed by mineral dissolution rates.
For more than 50 years microbiologists have been aware of extracellular
ligands, most notably siderophores, which are believed to be the
primary means by which microorganisms acquire Fe (17). In
fact, it has been assumed that because of the high formation constants
of the siderophore-Fe(III) complexes (as high as 1052)
Fe(III) (hydr)oxides would undergo spontaneous dissolution in the presence of a siderophore ligand. However, with the
exception of a few studies (e.g., references 9, 12, and
30), there is little quantitative discussion in the literature
regarding siderophore-mediated dissolution of Fe(III) (hydr)oxides
and virtually no discussion of microbial acquisition of Fe from these
solid phases in aerobic environments. To better understand the
acquisition of Fe, we investigated the removal of Fe from three Fe(III)
(hydr)oxides (geothite, hematite, and ferrihydrite) by a strict
aerobe. We chose these minerals because they are found commonly in
soils and represent major sources of Fe in natural environments. We chose an aerobic microorganism because, in contrast to
facultative anaerobic environments, wherein dissimilatory iron-reducing
bacteria (DIRB) are often abundant, very little is known about
microbial interactions with Fe(III) (hydr)oxides in oxic
environments, even though such environments are of widespread
abundance and importance.
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MATERIALS AND METHODS |
Fe(III) (hydr)oxides.
The minerals used in these
experiments were synthesized according to methods described by
Schwertmann and Cornell (24). BET (Brunauer, Emmett, and
Teller) surface areas (i.e., surface areas determined from gas
adsorption) were measured with an ASAP-2000 volumetric sorption
analyzer from Micrometritics, Inc. (Norcross, Ga.) using N2
adsorption at 195.5°C (4). The resulting specific surface areas were designated As. Method 4 of
Schwertmann and Cornell (24), i.e., "Preparation by
Transformation of Ferrihydrite," was used for the hematite
(
-Fe2O3) synthesis and yielded two batches
consisting of small, roughly hexagonal platelets, with As
values of 15 and 32 m2 g1. Goethite
(-FeOOH) was prepared from the Fe(III) system described in section
5.2.1 of that study (24). Different batches produced needle-like crystals ranging in length from 0.5 to 1 µm for
greater-As (37 m2 g1) to 1 to
3 µm for lower As (27 m2 g1) size particles. Amorphous ferrihydrite was
prepared by precipitation with alkali, as described in section 8.3 (24). This method yielded particles < 20 nm in
diameter with an As = 284 m2 g1. Particle size and shape estimates were confirmed by
atomic force microscopy (AFM) performed at Kent State University using
a Multi Mode NanoScope III AFM (Digital Instruments, Inc., Santa
Barbara, Calif.).
The BET method measured all surface area accessible to gas adsorption.
Each of these minerals, however, formed aggregates during preparation.
Because of this aggregation, not all of the BET surface area may have
been available to the bacterial cells, and exterior aggregate surface
area may have been a better indication of the proportion of total
surface area which was accessible to microorganisms for attachment
(i.e., the "effective" surface area, which we designate
Aeff). Therefore, we estimated Aeff for each Fe(III) (hydr)oxide by using a phase-contrast microscope, in
the absence of bacteria in the succinate medium. This size distribution is presented in Fig. 1. Aeff
values for all the Fe(III) (hydr)oxides were found to differ
substantially from the BET As values (Table 1). In order to determine
Aeff, aggregates were assumed to be spherical for the sake
of approximate calculations. However, this assumption necessarily
neglects surface irregularities. Furthermore, due to limits on
resolution of optical microscopy this method may have undercounted
small aggregates and single particles, both of which have high specific
surface areas. Hence, the Aeff values listed in Table 1 are
likely to be minimum estimates.
Medium, growth conditions, and microorganism.
Fe
deficient medium (FeDM) was prepared by adding to 1.0 liter of
distilled, deionized water the following analytical-grade ingredients
(Fluka Chemie, Buchs, Switzerland): 0.5 g of
K2HPO4, 1.0 g of NH4Cl,
0.2 g of MgSO4 · 7H2O, 0.05 g
of CaCl2, 5.0 g of succinic acid disodium salt
anhydrous (C4H4Na2O4),
and 0.125 ml of trace elements (0.005 g of MnSO4 · H2O, 0.0065 g of CoSO4 · 7H2O, 0.0023 g of CuSO4, 0.0033 g of
ZnSO4, and 0.0024 g of MoO3 per 100 ml of
distilled, deionized water). The pH of the medium was approximately
7.2. No attempt was made to deferrate the medium as suggested by Schwyn
and Neilands (25) because it has been our experience that
such attempts lead to variations in Fe concentrations due to Fe
contamination within the 8-hydroxyquinoline (the agent used to
deferrate growth medium) or chloroform (used to remove
8-hydroxyquinoline from the growth medium) or both. Rather, we used the
analytical-grade ingredients listed above, acid-washed polycarbonate
glassware, and ultrapure distilled and/or deionized water (18 M
cm1). Most importantly, we compared growth on each of the
Fe(III) (hydr)oxides to that of a no-added-Fe control. If
substantial growth occurred in the control flasks (e.g., >0.6
absorbance units), the entire experiment was terminated and then restarted.
Polycarbonate Erlenmeyer flasks (250 ml) containing 30 ml of FeDM were
used for all subsequent experiments. To these flasks
were added various
Fe sources: FeEDTA, goethite, hematite, ferrihydrite,
or no-added-Fe
control. For the FeEDTA growth studies, FeEDTA
was added to yield 0, 0.048, 0.24, 1.2, 6.0, or 30 µM concentrations
of Fe. For the Fe
acquisition experiments, minerals were added
so that the
A
eff was 29 m
2 liter
1 or the
minerals were added in various concentrations (goethite,
0.0, 0.06, 0.22, 1.2, 5.8, or 29 m
2 liter
1; hematite,
0.0, 0.04, 0.19, 0.98, 4.8, or 24 m
2 liter
1;
ferrihydrite, 0.0, 0.06, 0.23, 1.1, 5.7, or 28 m
2 liter
1). We determined in previous experiments that
P. mendocina could
acquire Fe as easily from FeEDTA as it
could from either FeNTA
(NTA = nitrilotriacetic acid) or iron
citrate and that neither
EDTA nor NTA served as a carbon or an electron
donor (
15). Inoculated
controls (no-added-Fe) contained
neither minerals nor FeEDTA.
The flasks were then sterilized by
autoclaving.
The bacterium used for this study was isolated as part of the Yucca
Mountain Project (the proposed site of the Nation's high-level
nuclear
repository) from sediment in a surface holding pond of
a drilling
operation at the Nevada Test Site. It was then identified
as
P. mendocina based on total rRNA sequence analysis at the 0.78%
confidence level (MIDI, Inc.). Also, this organism tested positive
for
catalase, oxidase, and nitrate reduction (assimilatory) but
negative
for fermentation and dissimilatory nitrate, Fe, and sulfate
reduction.
Therefore, this organism is an obligate aerobe, one
not capable of
using nitrate or Fe as a terminal electron acceptor,
and Fe acquisition
is for metabolic needs
only.
Each flask was inoculated with 285 µl of early-log-growth cells, at a
0.2 absorbance at 600 nm. The flasks were incubated
in the absence
of light [to minimize photo-induced Fe(III) reduction]
at
22°C, while agitated at 50 rpm. The cell concentration was
determined
by absorbance (600 nm) and was related to cell numbers
by separate
comparisons of absorbance to CFU. We chose absorbance
of the broth to
determine cell numbers because it assessed the
entire microbial
population with the flasks. For example, total
protein analysis
(
14) revealed no significant difference (
t-
test) between the number of microorganisms growing in the broth
and the
total population (microorganisms in the broth plus those
attached to
particles). Thus, absorbance of the broth accounted
for both the
free-swimming and attached cells, making it unnecessary
to make
separate measurements. Furthermore, the Fe(III) (hydr)oxides
did
not interfere with the absorbance readings because they agglomerated,
settled to the bottom of the flasks, and remained there during
the
course of the experiments (
11). Absorbance readings were
taken twice daily for 4
days.
Fe concentrations.
After maximum microbial growth had
occurred, the concentration of Fe in the FeDM containing either
goethite, hematite, or ferrihydrite as an Fe source was determined by
using three different methods: (i) from the cell numbers (absorbance),
(ii) by spectrophotometric analysis, and (iii) by graphite furnace
atomic absorption spectroscopy (GFAAS). From the number of cells
(absorbance at 600 nm) present in the liquid medium it was possible to
deduce the amount of Fe in the growth medium (i.e., the amount of Fe
removed from the mineral surface) because under our experimental
conditions cell growth was strictly controlled by, and thus could be
used to determine, Fe concentration (10, 20). By comparing
the absorbance of cells grown in FeDM with either the absorbance of
cells grown in FeEDTA (Fig. 2) or
Fe(III) (hydr)oxides (Fig. 3 and
4), one could determine the amount of Fe
removed from the Fe(III) (hydr)oxides by the microorganisms.
Second, Fe was determined spectrophotometrically by the reduction of Fe
with aspartic acid, followed by complexation with bathophenanthroline
and measuring the absorbance at 562 nm. Finally, Fe was measured using
graphite furnace on a Perkin-Elmer atomic absorption spectrometer
equipped with Zeeman background correction. The instrument was
calibrated from 5 to 100 mg of Fe liter1 by diluting a
1,000-mg liter1 AA/ICP Calibration Standard (Aldrich).
The pH of the Fe standards were lowered to pH ~2 by using ultrapure
HCl. Samples of >100 mg of Fe liter1 were diluted 1:10
with Milli-Q water, and the pH was readjusted to ca. 2. The precision
of analysis, as measured by relative standard deviation, was <5% for
all concentrations above 10 mg liter1.
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FIG. 2.
Microbial growth in response to various concentrations
of FeEDTA: 30 µM, y = 0.051, x 0.176, R2 = 0.81; 6.0 µM, y = 0.035x 0.009, R2 = 0.98; 1.2 µM; y = 0.026x 0; R2 = 0.99; 0.24 µM,
y = 0.012x + 0.100, R2 = 0.93;
0.05 µM, y = 0.005x 0, R2 = 0.99; and control, y = 0.002x 0, R2 = 0.99.
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FIG. 3.
Microbial growth on two different hematites and two
different goethites (Ca. 29 m2 liter1
Aeff each) and a no-Fe-added control: goethite 27, y = 0.031x 0.047, R2 = 0.97;
goethite 37, y = 0.031x 0.071, R2 = 0.98; hematite 15, y = 0.027x 0.100, R2 = 0.98; hematite 32, y = 0.027x 0.100, R2 = 0.98; and control,
y = 0.004x 0.011, R2 = 0.97.
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FIG. 4.
Microbial growth on different sources of Fe (30 µM
FeEDTA; minerals approximately 29 m2 11,
Aeff) and a no-Fe-added control: FeEDTA, y = 0.051x 0.176, R2 = 0.81; goethite,
y = 0.034x 0.087, R2 = 0.97;
hematite, y = 0.027x 0.102, R2 = 0.97; ferrihydrite, y = 0.023x + 0.003, R2 = 0.99; and control, y = 0.004x + 0.008, R2 = 0.97.
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Medium-promoted dissolution and nutrient sorption.
Two
different control experiments were performed to assess (i) the effects
of the FeDM and the autoclaving procedure on the dissolution of Fe(III)
(hydr)oxides and (ii) the effects of the Fe(III) (hydr)oxides
on the ingredients of FeDM. First, to determine if either the medium
itself or the autoclave procedure was promoting Fe dissolution, sterile
media (autoclave) containing each of the Fe(III) (hydr)oxides were
set aside for 2 weeks, passed through 0.2-µm (pore-size) filters (to
remove the minerals), inoculated with P. mendocina, and
incubated as described above. Growth in this conditioned medium
relative to the no-added-Fe control would indicate increased Fe
concentrations in the medium due to dissolution promoted by succinate
or the autoclave process.
A second analysis was performed to determine whether the three Fe(III)
(hydr)oxides sorbed ingredients of the FeDM differently.
This was
important to determine because such selective adsorption
could affect
growth on a particular Fe(III) (hydr)oxide different
from another
Fe(III) (hydr)oxide, making it difficult to compare
the relative
growth on each of the Fe(III) (hydr)oxides. The media
were
conditioned as described above, the Fe(III) (hydr)oxides
were
removed by filtration (0.2 µm), and the supernatants were
supplemented with FeEDTA (30 µm) and inoculated. Growth was
compared
to that of the control (unconditioned, FeDM plus 30 µM
FeEDTA).
All experiments described in the Materials and Methods section were
performed in
triplicate.
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RESULTS |
Cell growth and Fe concentration.
As the concentration of
FeEDTA decreased, so did growth rate (Fig. 2). This confirmed previous
observations (10, 18) that within this particular
experimental approach it was possible to control growth by controlling
the concentration of Fe.
When the Fe(III) (hydr)oxides were the sources of Fe, we observed
microbial growth in the medium (as well as on the surface
of the
mineral) that exceeded that of the no-added-Fe control
(a very limited
amount of growth did occur in the control medium,
as seen in Fig.
4).
Apparently, microorganisms attached to the
Fe(III) (hydr)oxide
agglomerates at the bottom of the flasks were
removing enough Fe to
support not only their growth but also the
growth of nonattached cells
in the medium above the minerals.
In fact, all three methods of
measuring Fe concentration in the
medium determined that ample Fe was
present in the FeDM to support
microbial growth. The most conservative
values came from estimates
based on microbial growth (Table
2), in which estimated Fe concentrations
ranged from 0.7 to 0.9 µM. GFAAS analysis yielded intermediate
values. For example, hematite 15 and 32 both had 1 to 2 µM Fe,
as
opposed to geothite 27 and 37 FeDM, which contained 4 to 5
µM Fe.
Finally, analysis by Fe(III) reduction to Fe(II), followed
by
complexation with bathophenanthroline, resulted in higher but
more
varied estimates of liberated Fe (2.6 versus 10.9 µM Fe were
present
in the FeDM for ferrihydrite and goethite 37, respectively).
Together,
these results demonstrate that
P. mendocina removed
Fe from
the Fe(III) (hydr)oxides, resulting in increased Fe concentrations
in the FeDM. As presented in Fig.
2, 1.0 µM Fe present in the
FeDM is
more than enough Fe to support the growth of this microorganism.
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TABLE 2.
Comparison of results from different methods for
determining Fe concentration (micromolar) in the medium
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When Fe was provided as FeEDTA, goethite, hematite, ferrihydrite, or
no-added-Fe control, different rates of microbial growth
were observed
(Table
3 and Fig.
4). The two treatments
whose
95% confidence bounds were the closest were ferrihydrite and
hematite
(the upper bound of ferrihydrite is 0.0246 and the lower bound
for hematite is 0.0282; Table
3). The difference between the
slopes for
these two treatments is 0.007. The small sample standard
deviation for
the difference of these two slopes is 0.000859 (see,
for example, the
study by McClave and Dietrich [
16]) and the
P
value for the
t- test of the difference between the two
slopes
being zero is <0.0001 (using 17 degrees of freedom). Thus,
there
is strong evidence that the two treatments (hematite and
ferrihydrite)
were different. Furthermore, because the two closest
rates differed
significantly at the 0.0001 level, the rates of all the
treatments
would differ at a
P value of this size or less,
thereby providing
strong evidence that all of the treatments differed
significantly
from one another. This analysis confirms that the order
of growth
on the various sources of Fe was as follows: FeEDTA > goethite
> hematite > ferrihydrite > no-added-Fe
control.
Surface area.
As can be seen in Fig.
5, increases in the amount of goethite,
hematite, and ferrihydrite resulted in increased growth rates for this
microorganism. Clearly, the microorganism responded to the increase in
Fe in the form of Fe(III) (hydr)oxides. While it appears that
increased surface area resulted in increased growth, it does not appear
that surface area alone controlled growth. Although different
As values were used [the amount of Fe(III) (hydr)oxides added to the flasks was determined by
Aeff], identical growth rates were obtained for two
goethites (Fig. 3). Identical growth rates also were obtained for two
hematites having different As values. These results suggest
that Aeff and not As controlled the
microbial growth on Fe(III) (hydr)oxides. Furthermore, the growth of P. mendocina was affected differently by the
different Fe(III) (hydr)oxides with the same Aeff (Fig.
3 and 4). Surprisingly, growth rates were greater on the more ordered
goethite than on the poorly ordered and higher-As
ferrihydrite.
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FIG. 5.
Microbial growth in response to various quantities of
available surface area of Fe(III) (hydr)oxides.
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Medium-promoted dissolution and nutrient sorption.
Growth greater than in the controls (FeDM) was not observed in
the conditioned media [FeDM exposed to Fe(III)
(hydr)oxides, filtered and then inoculated], demonstrating
that neither the medium nor the autoclaving procedure were responsible
for Fe dissolution.
Additionally, no difference in the growth of the bacterium was observed
between that of the FeDM exposed to goethite, hematite
or ferrihydrite
[Fe(III) (hydr)oxides were then removed by filtration,
and each
filtrate was supplemented with FeEDTA] and that of the
control (FeDM).
This result confirms that none of the three Fe(III)
(hydr)oxides
selectively sorbed nutrients from the
FeDM.
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DISCUSSION |
Dissolution of Fe.
Measurements of Fe by GFAAS, Fe(III)
reduced to Fe(II) measured with bathophenanthroline, and estimates of
Fe based on microbial growth all indicate that ample Fe was liberated
into FeDM during microbial growth in the presence of Fe(III)
(hydr)oxides. The variability in these measurements may be because
each measured different portions of the Fe held in the aqueous phase.
Estimating Fe based on optical density reflects microbial response to a
particular Fe concentration but does not reveal the total Fe
concentration, only the amount used by the cell. GFAAS analysis
measures the entire concentration (in solution, bound to the cell
walls, and inside the cells). Measurement of Fe(III) reduction measures
Fe(III) accessible to the reductant. In spite of their variability,
together these measurements demonstrate that the P. mendocina removed Fe from the Fe(III) (hydr)oxides.
Furthermore, enough Fe (in a micromolar concentration) was liberated to
support the luxuriant growth of both attached and free-swimming
P. mendocina.
Solubility.
As stated above, P. mendocina was able
to obtain metabolic Fe from Fe(III) (hydr)oxides in the following
order: goethite > hematite > ferrihydrite. These results
are in contrast to typical abiotic dissolution rates (27)
and are in contrast to reports by a number of groups (see, for example,
reference 26), particularly for studies of Fe(III)
reduction by facultative anaerobes such as DIRB. Although DIRB studies
are conducted under reducing conditions, such results are germane to
understanding the relationship between solubility-crystalline order and
dissolution. In one study, Phillips et al. (19) reported
that microorganisms readily reduced poorly ordered Fe(III) oxide (e.g.,
ferrihydrite), extractable by oxalate, and did not reduce
the more ordered Fe(III) forms such as goethite (FeOOH) and
hematite (Fe2O3), which oxalate did not extract.
Simplistically, one might anticipate that the relative
sequence of Fe acquisition would correlate to the relative
solubility
of the three Fe(III) (hydr)oxides. At circumneutral pH,
ferrihydrite
is the most soluble of the three, followed by hematite,
which
is slightly more soluble than goethite (
5). However,
P. mendocina obtained Fe from these minerals in contrast to
their relative
solubilities. Thus, solubility cannot be used to explain
our results,
and it appears that microbial removal of Fe from these
minerals
did not follow conventional wisdom because neither solubility
nor crystalline order controlled
acquisition.
Surface area.
While our results show clearly that increases in
mineral concentration for each of the three Fe(III) (hydr)oxides
resulted in increased growth rate (Fig. 5), our analysis also revealed that the BET surface area (As) did not control Fe
acquisition (Fig. 3). The As values were different for the
different goethites as well as for the different hematites, yet the
growth curves for the two goethites were identical, as were the growth
curves for the two hematites. Differences in As for either
Fe(III) (hydr)oxide had no effect on microbial acquisition of Fe
from either goethite or hematite. Thus, we believe that factors other
than As controlled Fe acquisition in our experiments. This
observation is consistent with reports in the literature (e.g.,
reference 2 and 20). For example, Arnold et al.
(2) suggested that the tendency of hematite and goethite
to aggregate in neutral pH range makes standard measures (such as
nitrogen adsorption, i.e., BET) of particulate surface area
inappropriate. They suggest that "such measurements would
overestimate the surface which is available for microbial contact."
These authors go on to caution that their observed rate in reductive
dissolution is clearly dependent on hematite morphology and cannot be
extended to minerals with a significantly different particle size distribution.
In a recent study, Roden and Zachara (
20) reported that
cell growth correlated with surface area. Although their preparation
of
Fe(III) (hydr)oxide (
6) was different from ours
(
24),
they did use transmission electron microscopy to
determine the
size range of individual particles and to assess the
degree of
particle aggregation (similar to our efforts to determine
A
eff).
Thus, these authors were not correlating dissolution
to BET surface
area but rather to an approximated surface area, in
agreement
to what we report
here.
Al substitution.
We have reported that correlation of the
dissolution of goethite to surface area can be misleading
(15). In that study, normalizing for Aeff
revealed that dissolution was not a function of surface area but rather
correlated positively with increased Al substitution. This result was
counterintuitive because the stability of Fe(III)
(hydr)oxides has been shown to increase with increasing Al
substitution (29, 32); thus, the bacterium should have grown more easily on the less ordered (i.e., less Al substituted) goethite. It may be that particle (and aggregate) morphology was more
important than surface area or thermodynamic stability; goethite needles become shorter and less multidomainic as their Al content increases (15, 24). These results, when combined with the discussion of surface area (above) strongly suggest that neither thermodynamic stability nor BET surface area control microbially mediated Fe(III) (hydr)oxide dissolution.
Hydroxyl coordination.
Recently, Cornell and Schwertmann
(5) discussed the occurrence and importance of singly,
doubly, triply, and geminal coordinated hydroxyl groups, which are in
effect the functional groups of Fe(III) (hydr)oxides, i.e., the
chemically reactive entities at the mineral surface in an aqueous
environment. Adsorption reactions are considered to involve only singly
coordinated groups; the doubly and triply coordinated groups appear to
be nonreactive (13, 21). In general, singly coordinated
hydroxyl groups are believed to be more common on the faces of goethite
than on hematite (5). Unfortunately, little is known about
the relative distributions of hydroxyl groups on ferrihydrite (with
respect to goethite and hematite) because the bulk structure of
ferrihydrite is poorly understood and ferrihydrite surface structure is
likely so dynamic that it may transform during titration analysis.
Although the relative occurrence of surface reactive entities
(goethite > hematite) is consistent with our observed Fe
acquisition from Fe(III) (hydr)oxides, uncertainties with respect
to ferrihydrite make this particular comparison premature at this time.
Certainly, we will be vigilant with respect to developments concerning
hydroxyl group distribution on the surface of ferrihydrite, since this
explanation appears to hold promise.
Transients.
Perhaps the most intriguing influence
comes from Samson and Eggleston (see reference 22
and references therein), who reported that the effect of transients are
responsible for initially rapid Fe dissolution rates. Transients are
nonstructural Fe sorbed to the mineral surface that are difficult to
quantify and are ephemeral, in that once removed by cleaning they may
reform. It is possible that some fraction of the Fe acquired by
P. mendocina from Fe(III) (hydr)oxides was nonstructural
and transient. Thus, the dissolution reported here may be sensitive to
both differences in mineral surface characteristics (hydroxyl groups)
and the presence surface transients. Unfortunately, it is nearly
impossible to determine the relative contribution of transients to the
total budget of acquired Fe. Nevertheless, significant and repeatable
differences were observed in the acquisition of Fe from the different
Fe treatments, suggesting that from the microorganism's point of view,
significant differences in the available Fe at or on the surfaces of
those treatments did exist. If transient Fe is being utilized by the cells, its use was influenced by the Fe(III) (hydr)oxide on which it is found. As with developments in the hydroxyl coordination literature, we will anxiously await further developments in the studies
of transient Fe.
In conclusion, our results demonstrate that
P. mendocina
obtained Fe for growth with increasing difficulty from goethite,
followed by hematite, and finally ferrihydrite, independent of
the BET
surface area. Furthermore, several different lines of
evidence suggest
that Fe acquisition activity occurred at rates
high enough to support
the growth of not only those microorganisms
attached to the mineral
surface but also those growing unattached.
As stated earlier, at
circumneutral pH and oxic conditions, the
solubility of Fe is
approximately 10
17 M. In effect, this microorganism was
increasing the solubility
of Fe by 10 to 11 orders of magnitude, for
each of the Fe(III)
(hydr)oxides examined. We provide here the
first description of
significantly different growth rates of a strict
aerobe on three
environmentally relevant, insoluble Fe(III)
(hydr)oxides. Hopefully,
this work will serve to stimulate
further research into the Fe
metabolism and dissolution process by
microorganisms in Fe-deficient
environments.
| |
ACKNOWLEDGMENTS |
This work was supported by the Department of Energy/Basic Energy Sciences.
We thank Mietek Jaroniec in the Department of Chemistry at Kent State
University, Kent, Ohio, for performing BET surface area measurements
and Garrison Sposito, University of California at Berkeley, for careful
editing of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Bioscience
Division, Los Alamos National Laboratory, P.O. Box 1663, Mail Stop
M888, Los Alamos, NM 87545. Phone: (505) 667 2779. Fax: (505)
665-3024. E-mail: hersman{at}lanl.gov.
| |
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Applied and Environmental Microbiology, October 2001, p. 4448-4453, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4448-4453.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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