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Applied and Environmental Microbiology, September 1999, p. 4252-4254, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Role of Humic-Bound Iron as an Electron Transfer
Agent in Dissimilatory Fe(III) Reduction
Derek R.
Lovley* and
Elizabeth L.
Blunt-Harris
Department of Microbiology, University of
Massachusetts, Amherst, Massachusetts 01337
Received 9 March 1999/Accepted 15 June 1999
 |
ABSTRACT |
The dissimilatory Fe(III) reducer Geobacter
metallireducens reduced Fe(III) bound in humic substances, but
the concentrations of Fe(III) in a wide range of highly purified humic
substances were too low to account for a significant portion of the
electron-accepting capacities of the humic substances. Furthermore,
once reduced, the iron in humic substances could not transfer electrons
to Fe(III) oxide. These results suggest that other electron-accepting
moieties in humic substances, such as quinones, are the important
electron-accepting and shuttling agents under Fe(III)-reducing conditions.
| |
TEXT |
Factors controlling the rate and
extent of dissimilatory Fe(III) reduction are of interest because
Fe(III) reduction has an impact on the geochemistry of a variety of
sedimentary environments (4, 5). The presence of humic
substances can significantly stimulate the oxidation of organic
compounds coupled to Fe(III) reduction in aquifer sediments and in
cultures (1, 6, 7, 12). When first noted in sediments, this
phenomenon was presumed to be the result of humic substances
solubilizing Fe(III) from insoluble Fe(III) oxides (12)
because previous studies performed with the same sediments had
demonstrated that solubilization of Fe(III) with synthetic chelators
enhanced Fe(III) reduction (10-12). However, humic
substances were found to solubilize too little Fe(III) to account for
their stimulatory effect (6).
Subsequent studies suggested that humic substances stimulate Fe(III)
reduction because humic substances can act as an electron shuttle
between Fe(III)-reducing microorganisms and insoluble Fe(III) oxides
(6, 7, 14). These studies also suggested that the important
electron-shuttling groups in humic substances are quinone moieties. In
this model, Fe(III)-reducing microorganisms transfer electrons to
quinone moieties in humic substances, and the hydroquinone groups that
are generated from quinone reduction can abiotically transfer electrons
to Fe(III) oxides. This reoxidizes the humic substances into forms
which can undergo another cycle of reduction and oxidation. Thus, in
the presence of Fe(III) oxides, a small amount of soluble humic
substances can become a major electron acceptor for organic matter
oxidation because each humic substance molecule may be reused as an
electron acceptor multiple times. This electron shuttling between
Fe(III)-reducing microorganisms and Fe(III) oxides via humic substances
accelerates the rate of Fe(III) reduction because (i) the Fe(III)
reducers can access soluble humic substances more readily than they can
establish direct contact with insoluble Fe(III) oxides, and (ii)
microbially reduced humic substances can access insoluble Fe(III)
oxides more readily than Fe(III)-reducing microorganisms can (7,
11).
Several lines of evidence support the concept that quinones are the
important electron transfer moieties in electron shuttling between
Fe(III)-reducing microorganisms and Fe(III) oxides. Studies in which
semiquinones were quantified by electron spin resonance revealed that
there was a direct correlation between the quinone contents of various
humic substances and the electron-accepting capacities of the humic
substances (14). It has also been demonstrated that
microbial reduction of humic substances results in an increase in
quinone radicals in direct proportion to the electron-accepting capacity of the humic substances, as would be expected if electrons were being transferred primarily to quinone moieties (14).
Microorganisms that have the ability to transfer electrons to humic
substances invariably have the ability to transfer electrons to
extracellular quinones, whereas organisms that do not reduce
extracellular quinones do not reduce humic substances (3, 6,
7). Low concentrations of extracellular quinones, such as the
humic substance analog anthraquinone-2,6-disulfonate, can stimulate
Fe(III) oxide reduction (6, 7) and enhanced anaerobic
benzene oxidation in the petroleum-contaminated aquifer sediments in
which addition of humic substances stimulated anaerobic benzene
oxidation (1).
It has recently been suggested that iron bound in humic substances
might also be involved in the humic substance redox processes associated with microbial Fe(III) reduction (2). This
suggestion was based on the finding that commercially available Aldrich
humic substances had an iron content that was comparable to the
electron-accepting capacity of the humic substances estimated
indirectly. However, it was not determined whether iron in the humic
substances could in fact serve as an electron acceptor and/or an
electron shuttle to Fe(III) oxide.
Fe(III) is a minor electron-accepting group in true humic
substances.
To determine whether humic substances contain
microbially reducible Fe(III), studies were initially conducted with
highly purified humic substances obtained from the International Humic Substances Society. Such humic substances are preferred over Aldrich humic substances for determining the properties of humic substances because the Aldrich humic substances may not adequately represent the
chemical nature of true humic substances found in soils and sediments
(13). As previously described (6), various
purified humic substances (final concentration, 2 g/liter) were
dissolved in 30 mM anaerobic bicarbonate buffer that contained 10 mM
acetate under N2-CO2 (80:20). A washed
suspension of Geobacter metallireducens cells was added to
the humic substance solution, and the preparation was incubated at
30°C for 2 h. The G. metallireducens was then removed
by anaerobically passing the preparation through a filter (pore
diameter, 0.2 µm). An aliquot of the filtrate was acidified with HCl
(final concentration, 0.5 N), an aliquot of the HCl extract was added
to HEPES buffer containing the Fe(II) reagent ferrozine, and the
absorbance at 562 nm (A562) was quantified in order to measure the Fe(II) content (9). In order to account for the absorbance due to the presence of humic substances in the filtrate, the
A562 of a humic substance solution filtrate without
ferrozine added was also determined. Furthermore, since adding the cell suspension added ca. 20 µM Fe(II), the amount of Fe(II) added with
the cells was determined by adding the cell suspension to bicarbonate
buffer without humic substances and then determining the Fe(II) content
as described above. The amount of Fe(II) produced from microbial
reduction of Fe(III) in the humic substances was calculated from the
A562 of the various preparations as follows: (A562 from the ferrozine analysis of the microbially
reduced humic substance filtrate) 
(A562 attributed
to humic substances) [A562 due to Fe(II) added
with the cell suspension].
The electron-accepting capacity of moieties in the humic substances
other than Fe(III) was determined as previously described (6). Fe(III) citrate (final concentration, 10 mM) was added to a filtrate of microbially reduced humic substances, and after 15 min
an aliquot was acid extracted and analyzed with ferrozine as described
above. As in previous studies (6, 14), the amount of Fe(II)
produced from the reduction of Fe(III) citrate was considered to
represent the electron-accepting capacity of non-Fe(III) moieties in
the humic substances and was calculated as follows: [A562
from the ferrozine analysis of the filtrates of microbially reduced humic substances amended with Fe(III) citrate] [A562
from the ferrozine analysis of filtrates of microbially reduced humic
substances not amended with Fe(III) citrate]. The latter
absorbance value included the absorbance due to any Fe(II) produced as
a result of microbial reduction of Fe(III) in humic substances, the
absorbance due to Fe(II) introduced with the cell suspension, and the
absorbance due to the humic substances.
Soil humic acids, which have been used most frequently for studies of
the electron transfer capability of humic substances
(
6,
7),
contained no detectable microbially reducible Fe(III),
but as
previously demonstrated (
6,
14), these humic substances
did
have a significant electron-accepting capacity due to other
moieties in
them (Table
1). The non-Fe(III)
electron-accepting
capacity of these humic substances and the other
humic substances
evaluated was similar to the capacity observed
previously (
6),
but in this study the results are presented
on a per-gram basis.
Peat humic acids also contained no detectable
microbially reducible
Fe(III) but could accept electrons from
G. metallireducens. The
other highly purified humic substances
obtained from the International
Humic Substances Society that were
evaluated did contain small
amounts of microbially reducible Fe(III)
(Table
1). However,
the electron-accepting capacity of the Fe(III) in
these humic
substances was ca. 10% or less of the electron-accepting
capacity
that was due to moieties other than Fe(III). These results
demonstrate
Fe(III) plays a very minor role as an electron-accepting
group
in a wide range of well-characterized humic substances.
Even though Aldrich humic substances are not considered appropriate
analogues of true humic substances in the environment
(
13),
it was of interest to determine whether, as previously
suggested
(
2), these humic substances contained microbially
reducible
Fe(III). The concentration of microbially reducible
Fe(III) in the
Aldrich humic substances was more than fourfold
higher than the
concentration of microbially reducible Fe(III)
in any of the other
humic substances evaluated, but even in these
highly impure humic
substances the electron-accepting capacity
due to Fe(III) bound in the
humic substances was less than the
electron-accepting capacity due to
other moieties in the humic
substances (Table
1).
In order to determine if Fe(III) or other electron-accepting moieties
in Aldrich humic substances were reduced preferentially,
electron
transfer was monitored over time (Fig.
1). Electrons
were initially transferred
primarily to Fe(III), and only after
the Fe(III) was reduced were the
other electron-accepting moieties
significantly reduced (Fig.
1). This
could have resulted from
an initial transfer solely to Fe(III), but it
is also likely that
any quinone groups that initially accepted
electrons reduced the
Fe(III) in the humic substances, just as they
reduced Fe(III)
oxides. Only after the Fe(III) was completely reduced
would the
quinone moieties remain in a reduced state.
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FIG. 1.
Electron equivalents transferred to Fe(III) and sum of
electron equivalents transferred to Fe(III) and other
electron-accepting groups in Aldrich humic acids by a cell suspension
of G. metallireducens over time. The results are means based
on duplicate incubations.
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Lack of electron shuttling via iron.
Despite the fact that
Fe(III) accounted for little, if any, of the electron-accepting
capacity in highly pure humic substances, the finding that G. metallireducens initially reduced the Fe(III) in humic substances
suggested that even small amounts of iron in humic substances might be
important as an electron transfer agent in Fe(III)-reducing
environments if, once reduced, the iron in humic substances could
transfer electrons to Fe(III) oxide and thus reoxidize the Fe(II) in
the humic substances back to Fe(III). Through such a recycling
mechanism, even small amounts of iron could participate in significant
electron transfer in sediments. Such regeneration mechanisms are
necessary for any electron-accepting moiety in humic substances to play
a significant role as an electron acceptor in sediments because, on a
per-gram basis, the total electron-accepting capacity of humic
substances is low compared to the electron-accepting capacities of
other potential electron acceptors for anaerobic respiration.
Therefore, the potential of iron in humic substances to function as an
electron shuttle between Fe(III) reducers and Fe(III) oxides was evaluated.
The electron shuttling studies were conducted with Aldrich humic
substances because they were the only humic substances available
that
contained high enough concentrations of Fe(III) to accurately
monitor.
Furthermore, it was data obtained with Aldrich humic
substances which
was the basis for the previous suggestion (
2)
that iron may
be important in humic substances. As described above,
the humic
substances (final concentration, 2 g/liter) were dissolved
in anaerobic
bicarbonate buffer containing acetate. The concentration
of Fe(II) was
determined with ferrozine as described above, and
the total iron
concentration was determined after Fe(III) was
reduced with
hydroxylamine, as previously described (
9). The
difference
between the total iron and Fe(II) concentrations represents
the
concentration of Fe(III) (
9). The Aldrich humic substances
were stored aerobically before they were added to the anaerobic
buffer,
and thus it is not surprising that most of the iron in
the initial
humic substance solution was recovered as Fe(III)
(Table
2). As expected from the studies
described above, reduction
of the humic substances with
G. metallireducens reduced all of
the Fe(III) to Fe(II) (Table
2).
If the iron in the humic substances could shuttle the electrons
received from
G. metallireducens to Fe(III) oxide, then
exposure
of microbially reduced humic substances to Fe(III) oxide
should
have oxidized the Fe(II) in the humic substances to Fe(III). To
evaluate this, an anaerobic slurry of poorly crystalline Fe(III)
oxide
(final concentration, 10 mmol per liter), prepared as previously
described (
8), was added to a filtrate of microbially
reduced
humic substances. After 1 h, the Fe(III) oxide was removed
by
filtration, and the Fe(II) and Fe(III) concentrations in the humic
substance solution were determined. No Fe(III) was detected in
the
humic substance solution after exposure to Fe(III) oxide,
indicating
that there was no electron transfer between the Fe(II)
in the humic
substances and the Fe(III) oxide (Table
2). As expected
from the study
described above (Table
1), there was an increase
in the soluble Fe(II)
content after exposure of the microbially
reduced humic substances to
Fe(III) oxide which could be attributed
to other reduced moieties, such
as hydroquinones, that transferred
electrons to the Fe(III)
oxide.
Conclusions.
Our results demonstrate that the
electron-accepting capacities of a wide range of highly purified humic
substances were much higher than the electron-accepting capacities of
the microbially reducible Fe(III), which showed that Fe(III) in humic
substances can at best account for a small amount of the initial
electron transfer to humic substances in environments in which Fe(III) reduction is the terminal electron-accepting process. Furthermore, unlike quinone moieties, the iron in humic substances cannot function as an electron shuttle between Fe(III)-reducing microorganisms and
Fe(III) oxides. The ability to cycle between oxidized and reduced forms
is essential for moieties in humic substances to be quantitatively
significant as electron acceptors in Fe(III)-reducing environments.
Thus, the role of iron bound in humic substances as an electron
transfer agent in Fe(III)-reducing sediments is likely to be minimal.
| |
ACKNOWLEDGMENTS |
This research was supported by grant N0014-96-1-0382 from the
Office of Naval Research and grant DE FG02-97ER62475 from the Department of Energy.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University of Massachusetts, Amherst, MA 01337. Phone: (413) 545-9651. Fax: (413) 545-1578. E-mail:
dlovley{at}microbio.umass.edu.
| |
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Applied and Environmental Microbiology, September 1999, p. 4252-4254, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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