FEMS Microbiology Ecology 40 (2002) 73^81
www.fems-microbiology.org
Microbial reduction of Fe(III) and turnover of acetate in
Hawaiian soils
Kirsten Ku«sel
a
b
a;
, Christine Wagner a , Tanja Trinkwalter a , Anita S. Go«Mner a ,
Rupert Ba«umler b , Harold L. Drake a
Department of Ecological Microbiology, BITOEK, University of Bayreuth, 95440 Bayreuth, Germany
Department of Soil Science, Technical University of Munich, 85350 Freising-Weihenstephan, Germany
Received 20 November 2001; received in revised form 21 January 2002; accepted 22 January 2002
First published online 26 March 2002
Abstract
Soils contain anoxic microzones, and acetate is an intermediate during the turnover of soil organic carbon. Due to negligible
methanogenic activities in well-drained soils, acetate accumulates under experimentally imposed short-term anoxic conditions. In contrast
to forest, agricultural, and prairie soils, grassland soils from Hawaii rapidly consumed rather than formed acetate when incubated under
anoxic conditions. Thus, alternative electron acceptors that might be linked to the anaerobic oxidation of soil organic carbon in Hawaiian
soils were assessed. Under anoxic conditions, high amounts of Fe(II) were formed by Hawaiian soils as soon as soils were depleted of
nitrate. Rates of Fe(II) formation for different soils ranged from 0.01 to 0.31 Wmol (g dry weight soil)31 h31 , but were not positively
correlated to increasing amounts of poorly crystallized iron oxides. In general, sulfate-reducing and methanogenic activities were
negligible. Supplemental acetate was rapidly oxidized to CO2 via the sequential reduction of nitrate and Fe(III) in grassland soil (obtained
near Kaena State Park). Supplemental H2 stimulated the formation of Fe(II), but H2 -utilizing acetogens appeared to also be involved in
the consumption of H2 . Approximately 270 Wmol Fe(III) (g dry weight soil)31 was available for Fe(III)-reducing bacteria, and acetate
became a stable end product when Fe(III) was depleted in long-term incubations. Most-probable-number estimates of H2 - and acetateutilizing Fe(III) reducers and of H2 -utilizing acetogens were similar. These results indicate that (i) the microbial reduction of Fe(III) is an
important electron-accepting process for the anaerobic oxidation of organic matter in Fe(III)-rich Hawaiian soils of volcanic origin, and
(ii) acetate, formed by the combined activity of fermentative and acetogenic bacteria, is an important trophic link in anoxic microsites of
these soils. < 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Acetate ; Acetogenesis; Fe(III) reduction; Volcanic soil
1. Introduction
Anoxic conditions in soils give rise to anaerobic microbial processes. O2 -driven respiration and the di¡usion of
atmospheric O2 a¡ect the concentration of O2 in soils [1].
The di¡usivity of O2 decreases with increasing water content and decreasing porosity of the soil [2,3]. In aerated
soils, anaerobic processes are stimulated by rainfalls [4,5].
Long-term £ooding of soils initiates sequential reductive
processes, and methanogenesis becomes the dominant terminal electron-accepting process [6,7]. Soil structure also
in£uences the concentration of O2 in aerated soils, and
* Corresponding author. Tel. : +49 (921) 555-642/641;
Fax : +49 (921) 555-799.
E-mail address : [email protected] (K. Ku«sel).
anoxic microzones in soil mineral aggregates can be as
large as 20 mm in radius [8,9]. Anoxic microzones are
also present in the litter layer of forest soils, even though
the interparticle litter pores can be O2 -saturated [10].
Thus, the microbial turnover of organic carbon in aerated
soils is not restricted to aerobic processes.
Low-molecular mass organic acids indicative of anaerobic processes [11] are present in the litter and mineral
horizons of well-drained soils [12^15]. When subjected to
anoxic conditions, litter and mineral soil spontaneously
form acetate, formate, alcohols, H2 , and CO2 , demonstrating that a subcommunity of the microbiota can respond
rapidly to anaerobiosis [16^18]. Soil solutions collected
with small ceramic suction plates from irrigated soil columns display a high spatial and temporal variation of lowmolecular mass organic acids, with maximum concentrations of acetate approximating 3.5 mM [19]. Organic acids
0168-6496 / 02 / $22.00 < 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 0 1 6 8 - 6 4 9 6 ( 0 2 ) 0 0 2 1 8 - 0
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K. Ku«sel et al. / FEMS Microbiology Ecology 40 (2002) 73^81
1 h. Bottles were incubated horizontally in the dark at
30‡C with an initial overpressure of 30 kPa argon at
room temperature. Substrates were added as sterile anoxic
stock solutions or as sterile gas. The concentrations of
supplemental acetate and formate approximated 5 and
20 mM, respectively. The concentrations of H2 ^CO2 supplemented as a gas mixture to soil microcosms approximated 12^20 mmol H2 (l soil suspension)31 and 6^10
mmol CO2 (l soil suspension)31 , respectively.
formed under anoxic conditions in soils are rapidly oxidized to CO2 in the presence of O2 or nitrate, and, thus,
are very reactive in the soil solution [16,18,19]. However,
under anoxic conditions, acetate accumulates, and acetoclastic methanogenesis appears to be a negligible in situ
process [7,16,18]. The formation of acetate in aerated soils
is dependent on the combined activity of facultative and
obligate anaerobes, and the number of cultured acetateproducing anaerobes approximates that of cultured anaerobes [20].
In contrast to the results described above with forest,
agricultural, and prairie soils, preliminary studies with a
non-methane emitting grassland soil from Hawaii demonstrated that certain soils of volcanic origin consumed acetate under anoxic conditions via unknown endogenous
electron acceptors (Wagner and Drake, unpublished
data). The main objective of the present study was to
determine what electron acceptors and associated microbial processes might be linked to the anaerobic oxidation
of organic carbon in Hawaiian soils.
2.2. Enumeration of soil microbiota and cultivation media
Numbers of cultured cells were determined by the mostprobable-number (MPN) technique with three replicates
incubated at 30‡C for 4 months; MPN values were calculated from standard MPN tables and were within 95%
certainty [21]. The medium used for culturing Fe(III)-reducing bacteria contained (in g l31 ): NaHCO3 2.5, NH4 Cl
1.5, CaCl2 W2H2 O 0.1, KCl 0.1, NaH2 PO4 0.6, yeast extract
0.05, trace metal solution [22] 5 ml l31 , vitamin B solution
[22] 5 ml l31 . The ¢nal concentration of synthesized poorly
crystalline Fe(III)-oxyhydroxide [23] approximated 40
mM. The gas phase was N2 ^CO2 (80:20), and the ¢nal
pH approximated 6.8. Either H2 (15 mmol (l culture £uid)31 ) or acetate (5 mM) was added as electron donor. An
unde¢ned, non-reduced bicarbonate-bu¡ered medium [22]
with a 100% CO2 gas phase and a ¢nal pH of 6.8 was used
for culturing acetogens; H2 (15 mmol (l culture £uid)31 )
was added as electron donor. All substrates were provided
as sterile anoxic stock solutions or as sterile gas. Tubes
were scored positive for acetate- or H2 -utilizing Fe(III)
reducers based on the formation of Fe(II) and the consumption of either acetate or H2 , respectively. Tubes were
scored positive for H2 -utilizing acetogens based on the
consumption of H2 and the formation of acetate. Unde¢ned, non-reduced bicarbonate-bu¡ered medium without
supplemental H2 was inoculated as a control to determine
the production of acetate from yeast extract. The average
2. Materials and methods
2.1. Field sites, sampling, and preparation of soil
microcosms
Soils were obtained from O’ahu and Kaua’i Hawaii;
these soils are developed from volcanic rocks. Field site
and soil characteristics are outlined in Table 1. The upper
A horizon (0^5 cm) was collected in sterile vessels. For
each soil microcosm, 20 g (fresh wt) soil was placed in a
sterile 150-ml infusion bottle (Merck ABS, Dietikon, Switzerland) inside a Mecaplex H2 -free chamber (100% N2 gas
phase). Soil was diluted with 40 ml anoxic sterile deionized
water to facilitate sampling; bottles were then closed with
rubber stoppers and screw-cap seals, £ushed with sterile
argon for 15 min, and put on an end-over-end shaker for
Table 1
Pedogeochemical characteristics of Hawaiian soilsa
Field site
Vegetation
Dry
pH
weight
(%)
Corg
(%)
Ntotal
(%)
Fed
(g kg31 )b
Feo
(g kg31 )c
Fetotal
(g kg31 )
Fe3 O4
(g kg31 )
((Fed 3Feo )+ Alo
Surface
(g kg31 )d area
Fe3 O4 )/Feo
(m2 g31 )
Kaua’i, Koke’e State
Park
O’ahu, near
Kaena State Park
O’ahu, Mokuleia
near Air¢eld
O’ahu, Waipahu
forest
65.9
4.9
12.3
0.9
102.2
70.7
128.3
9.2
0.6
4.25
28.0
grassland
78.6
5.9
4.1
0.3
49.0
6.5
115.9
49.6
14.2
2.24
42.2
grassland
96.2
7.4
3.6
0.2
6.6
1.7
33.5
12.3
10.1
0.85
10.7
sugar cane
plantation
98.2
7.7
1.4
0.3
131.4
117.6
153.3
42.0
0.5
1.18
32.7
a
Presented are the averages from duplicate soil samples.
Fed refers to the total amount of pedogenic iron oxides, iron in organic compounds, and exchangeable iron.
c
Feo refers to poorly crystallized iron oxides, hydroxides, and associated gels, iron in organic compounds, and exchangeable iron.
d
Alo refers to poorly crystallized aluminum oxides and aluminum-containing organic compounds.
b
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K. Ku«sel et al. / FEMS Microbiology Ecology 40 (2002) 73^81
75
Fig. 1. Consumption of supplemental H2 ^CO2 and formation of acetate, CO2 , and CH4 in anoxic microcosms of soils obtained from Koke’e forest (A)
or a grassland near Kaena State Park (B). Presented are the average values of duplicate microcosms. Symbols: (b) acetate, (E) CO2 , (O) CH4 , (R),
H2 .
H2 to acetate ratios in all positive dilution series approximated 5:1, a value indicative of H2 -dependent acetogenesis (the theoretical ratio is 4:1) [24].
2.3. Analytical techniques
Growth in media lacking Fe(III) was monitored as optical density at 660 nm with a Spectronic 501 spectrophotometer (Bausch and Lomb, Rochester, NY, USA). The
rate of Fe(III) reduction in microcosms was estimated by
determining the amount of Fe(II) formed [25]. To determine the formation of Fe(II), aliquots (0.2 ml) of the soil
suspension or of the medium were withdrawn using sterile
syringes connected to wide needles and transferred to 9.8
ml of 0.5 N HCl and incubated for 1 h at room temperature [26]. The soil suspension was shaken vigorously to
ensure sampling of a representative homogeneous soil suspension including soil particles. Fe(II) was measured by
the phenanthroline method [27]. Headspace gases (H2 ,
CO2 , and CH4 ) were measured with Hewlett Packard
Co. (Palo Alto, CA, USA) 5980 series II gas chromatographs [16]. Gas values included the total amounts in both
the liquid and gas phases and are reported in mM (i.e.
mmol (l soil suspension)31 ). Aliphatic acids and alcohols
were determined with Hewlett Packard 1090 series II high
performance liquid chromatographs [16]. The detection
limit for acetate approximated 100 WM. Nitrate and sulfate were analyzed by ion chromatography [16]. Soil pH
was measured with an Ingold (Steinbach, Germany) U457S7/110 combination pH electrode. The carbon and nitrogen content of oven-dried (60‡C), homogenized soil was
quantitated with an element analyzer (CHN-O-rapid,
Foss-Heraeus, Hanau, Germany). Pedogenic iron (Fed )
was extracted with dithionite^citrate^bicarbonate solution
[28]. Poorly crystallized iron oxides, hydroxides, and associated gels (Feo ) were extracted with acidic ammonium
oxalate solution [29]; aluminum (Alo ) was measured in
the same extract. Both methods assess organically bound
and exchangeable iron. Magnetite (Fe3 O4 ) was determined
by magnetic susceptibility (Magnetic Susceptibility Meter
MS2, Barrington, Oxford, UK). Total iron (Fetotal ) includes both soil- and rock-derived iron, and was determined by ion-catched plasma with atomic emission spectroscopy (XMP; GBC, Australia). Extracted Al and Fe
were measured by atomic absorption spectrometry (Unicam 939 spectrometer, ThermoNicolet GmbH, O¡enbach,
Germany). Surface area measurements [30] were carried
out by the BET method (Quantachrome Autosorb-1,
Odelzhausen, Germany). All soil chemical parameters
were analyzed in duplicate using sieved soil that was less
than 2 mm in diameter.
3. Results
3.1. Formation of acetate in Hawaiian soils
Hawaiian Koke’e forest soil spontaneously formed CO2
and acetate when incubated under anoxic conditions (data
not shown). Supplemental H2 was rapidly consumed by
anoxic Koke’e forest soil, and additional amounts of acetate were formed in response to H2 (Fig. 1A), indicating
that acetogenic bacteria were at least partially involved in
the consumption of H2 . Additional amounts of acetate
were also formed by Mokuleia grassland soil and by sugar
cane plantation soil supplemented with H2 (data not
shown). These results were similar to those obtained
with other forest and grassland soils [16,18]. In contrast,
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K. Ku«sel et al. / FEMS Microbiology Ecology 40 (2002) 73^81
Fig. 2. Consumption of endogenous nitrate and sulfate and the formation of Fe(II) and CH4 in anoxic microcosms of grassland soil obtained near
3
23
a
Kaena State Park. Presented are the mean values ( U S.D.) of triplicate microcosms. Symbols: (F) NO3
3 , (E) NO2 , (S) Fe(II), ( ) SO4 , (b) CH4 .
Hawaiian grassland soil obtained near Kaena State Park
formed negligible amounts of acetate (up to 300 WM) when
incubated under anoxic conditions (data not shown), and
the consumption of supplemental H2 by this soil was also
not coincident with either the formation or accumulation
of acetate (Fig. 1B). Only small amounts of acetate (up to
1.3 mM) were transiently detected during the period of H2
consumption, and CH4 was not detected (Fig. 1B).
Kaena State Park (Fig. 3) and yielded additional amounts
of CO2 compared to controls lacking acetate (data not
shown). Acetate-supplemented soil consumed nitrate
more rapidly than did unsupplemented soil. In acetatesupplemented soil microcosms, (i) Fe(II) was formed at a
3.2. Redox processes in Hawaiian grassland soil
Under anoxic conditions, grassland soil obtained near
Kaena State Park rapidly consumed endogenous nitrate,
and nitrite was a trace, transient product (Fig. 2). When
the concentration of nitrate decreased to levels below
0.4 mM, the formation of Fe(II) increased at a rate of
2 mmol Fe(II) (l soil suspension)31 day31 to a ¢nal concentration of approximately 50 mM Fe(II) after an incubation period of 25 days. Although the initial concentration of sulfate (0.4 mM) was stable for 15 days of
incubation, sulfate subsequently, and slowly, decreased
to negligible levels during the next 10 days of incubation
(Fig. 2). Formation of trace amounts of CH4 was detected
after 13 days of incubation.
3.3. E¡ect of supplemental acetate on redox processes in
Hawaiian grassland soil
Supplemental acetate was rapidly consumed under anoxic conditions by Hawaiian grassland soil obtained near
Fig. 3. E¡ect of supplemental acetate on the consumption of endogenous nitrate and the formation of Fe(II) in anoxic microcosms of grassland soil obtained near Kaena State Park. Presented are the mean values ( U S.D.) of triplicate microcosms. Symbols: (S) acetate, (F) NO3
3 ,
a
(E) NO3
3 in control lacking supplemental acetate, (b) Fe(II), ( ) Fe(II)
in controls lacking supplemental acetate.
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K. Ku«sel et al. / FEMS Microbiology Ecology 40 (2002) 73^81
Fig. 4. Formation of Fe(II) in anoxic microcosms of various Hawaiian
soils. Presented are the mean values ( U S.D.) of triplicate microcosms.
Symbols: (b) grassland soil obtained near Kaena State Park, (a) Mokuleia grassland soil, (F) sugar cane plantation soil (andisol), (E) Koke’e
forest soil (andisol).
rate of 4.3 mmol Fe(II) (l soil suspension) day , (ii)
sulfate was not consumed, (iii) methane was not detected,
and (iv) 53% of the reducing equivalents obtained from
the oxidation of acetate were theoretically recovered in
Fe(II). Under anoxic conditions, grassland soil obtained
near Kaena State Park also rapidly consumed supplemental formate (20 mM), and 44% of the reducing equivalents
obtained from the oxidation of formate were theoretically
recovered in Fe(II) (data not shown). Acetate appeared to
be a transient product during the period when formate
was consumed.
31
31
77
conditions, sugar cane plantation soil consumed 43% of
supplemental acetate (5 mM), but only 6% of the reducing
equivalents obtained from the oxidation of acetate were
theoretically recovered in Fe(II). In contrast to these soils,
Hawaiian Koke’e forest soil did not consume supplemental acetate within 12 days of incubation under anoxic conditions (data not shown). Formation of CH4 was not detected in Mokuleia grassland soil, sugar cane plantation
soil, and Koke’e forest soil supplemented with acetate.
However, the concentrations of nitrate and sulfate were
not determined. These results indicated that in these Hawaiian soils, (i) electron acceptors other than Fe(III) were
primarily responsible for the anaerobic consumption of
supplemented acetate, and (ii) the amount of unidenti¢ed
electron acceptors was limited relative to the amount of
acetate added. Although the abundance of Mn(IV) oxides
in Hawaiian soils was not measured, the reduction of
Mn(IV) might also be partially responsible for the oxidation of supplemental acetate.
3.5. H2 -consuming Fe(III) reducers and acetogens in
Hawaiian grassland soil
Under anoxic conditions, grassland soil obtained near
Kaena State Park rapidly consumed supplemental H2 and
formed more Fe(II) than did soil lacking H2 (Fig. 5). Sim-
3.4. Formation of Fe(II) by Hawaiian soils
Grassland soils displayed the highest Fe(II)-forming activity under anoxic conditions (Fig. 4). After a lag phase
of 6^10 days of incubation, the formation of Fe(II) was
relatively linear, and rates of formation approximated 0.4,
0.7, 1.4, and 7.2 Wmol Fe(II) (g dry weight soil)31 day31
for sugar cane plantation soil, Koke’e forest soil, Mokuleia grassland soil, and grassland obtained near Kaena
State Park, respectively.
Under anoxic conditions, Mokuleia grassland soil consumed 38% of supplemental acetate (5 mM) within 3 days
of incubation, and approximately 23% of the reducing
equivalents obtained from the oxidation of acetate were
theoretically recovered in Fe(II). The amount of Fe(II)
formed in acetate-supplemented microcosms and control
microcosms lacking acetate approximated 14 and 8 mmol
Fe(II) (l soil suspension)31 , respectively. After 3 days of
incubation, the concentration of acetate did not decrease
during the following 10 days of incubation. Under anoxic
Fig. 5. E¡ect of supplemental H2 on the formation of Fe(II) in anoxic
microcosms of grassland soil obtained near Kaena State Park. After 11
days of incubation, three of the six microcosms that were initially supplemented with H2 were resupplemented with H2 . Presented are the
mean values ( U S.D.) of triplicate microcosms. Symbols : (R) H2 , (F)
3
NO3
3 , (E) NO3 in controls lacking H2 , (8) Fe(II) in controls lacking
H2 , (b) Fe(II), (a) Fe(II) in H2 -resupplemented microcosms, (P) acetate in H2 -resupplemented microcosms (no acetate was detected in control microcosms lacking supplemental H2 ).
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K. Ku«sel et al. / FEMS Microbiology Ecology 40 (2002) 73^81
Table 2
MPN values of Fe(III) reducers and acetogens in Hawaiian grassland
soil (Kaena State Park)
Metabolic type
MPN (g dry weight soil)31
H2 -utilizing Fe(III) reducers
Acetate-utilizing Fe(III) reducers
H2 -utilizing acetogens
1.5U105 (3.2U104 ^7.1U105 )a
2.0U105 (4.2U104 ^9.3U105 )
5.0U105 (1.1U105 ^2.3U106 )
a
Values in parentheses represent the con¢dence limits of the MPN values.
ilar to results described above, acetate was detected as a
transient product during the period of H2 consumption.
The rapid consumption of resupplemented H2 was coincident with the continued formation of Fe(II) (Fig. 5). Acetate was formed as a stable end product in microcosms
resupplemented with H2 , and approximately 42% and 35%
of the reducing equivalents obtained from the oxidation of
H2 were theoretically recovered in Fe(II) and acetate, respectively. Trace amounts of CH4 (120 WM) were detected
at the end of incubation in microcosms resupplemented
with H2 (data not shown). The cultured numbers of H2 utilizing Fe(III) reducers, acetate-utilizing Fe(III) reducers,
and H2 -utilizing acetogens were similar (Table 2).
4. Discussion
When O2 is depleted in soils, terminal electron acceptors
other than O2 are utilized for the oxidation of organic
matter [2,3]. The change from aerobic to anaerobic metabolism occurs at O2 concentrations of less than 1% [31]. In
theory, the sequence in which electron acceptors are utilized is determined by the redox potentials of the half-cell
reactions [31]. Thus, aerobic respiration (E0 P = +0.81 V)
should be followed sequentially by the reduction of nitrate
to N2 by denitri¢ers (E0 P = +0.75 V), the reduction of an
Fe(III) oxide to Fe(II) by iron reducers (E0 P = 30.1 V; see
following clari¢cation), the reduction of sulfate to sul¢de
by sulfate reducers (E0 P = 30.22 V), the reduction of CO2
to methane by methanogens (E0 P = 30.25 V), and the reduction of CO2 to acetate by acetogens (E0 P = 30.29 V).
The Fe(III)/Fe(II) half-cell covers a broad range of redox
potentials (E0 P), from +0.1 V for ferrihydrite to 30.3 V for
goethite, hematite, and magnetite [32]. When Hawaiian
soils were incubated under anoxic conditions, the consumption of nitrate was followed by an increase of
Fe(II) and, eventually, the consumption of sulfate. However, soils are very heterogeneous, and the presence of
terminal electron acceptors can vary spatially and temporally [31]. The localization of easily degradable organic
matter, the spatial arrangements of microbial communities, the size of microbial populations, and pH can also
in£uence microbial activities. Thus, a process that is theoretically disadvantaged might be favored under certain in
situ conditions.
Fe(III), in its various chemical forms, is the most abun-
dant electron acceptor for dissimilatory metal-reducing
microorganisms [33,34]. A large number of phylogenetically diverse bacteria and archaea are capable of dissimilating Fe(III) [35]. However, most of the Fe(III) in soils
and sediment is in the form of insoluble oxides. Most iron
oxides are formed by the aerobic weathering of surface
magmatic rocks [36]. Sixteen di¡erent iron oxides have
been identi¢ed, and their availability for microbial reduction decreases with increasing crystallinity and decreasing
surface area [37,38]. In the absence of iron chelators or
electron shuttles (e.g. humic acids), it is anticipated that
microorganisms must come into contact with insoluble
iron oxides in order to utilize this form of iron [39].
Thus, many soils and sediments are readily depleted of
microbiologically reducible Fe(III), even though they are
rich in Fe(III)-containing minerals [38,40]. The soils examined in the present study are of volcanic origin and contained high amounts of Fetotal , dithionite-extractable pedogenic iron oxides (Fed ), and oxalate-extractable poorly
crystallized iron oxides (Feo ). Magnetite is poorly extracted by dithionite and oxalate, and is also typical for
soils of volcanic origin. The (Alo +0.5 Feo ) contents of soils
from Koke’e forest and the sugar cane plantation were
greater than 2%, indicating that these soils were andisols
[41]. Andisols contain high amounts of ‘amorphous’ minerals, including poorly crystallized allophane, imogolite,
and ferrihydrite [42]. Soils from the two grassland sites
were not andisols.
In general, poorly crystallized Fe(III) oxides are the
favored reducible forms of Fe(III) for microbial reduction
[37]. For the soils used in the present study, the ratios of
well-crystallized, dithionite-extractable Fe(III) oxides and
magnetite ((Fed 3Feo )+Fe3 O4 ) to poorly crystallized
Fe(III) oxides (Table 1) indicate that the Fe(III)-reducing
capacities of the two andisols should be greater than those
of the grassland soils. However, Fe(III)-reducing activities
were not positively correlated to increasing amounts of
poorly crystallized Fe(III) oxides. Lack of electron donors
was apparently not the cause of the low Fe(III)-reducing
capacities of the andisols (e.g. Koke’e forest soil contained
high amounts of soil organic carbon and produced high
amounts of acetate and CO2 under anoxic conditions). In
addition, supplemental acetate or H2 did not stimulate the
reduction of Fe(III) in Koke’e forest soil. In andisols,
amorphous oxidic Al- and Fe-hydroxy polymers interact
with water soluble organic compounds to form metal^humus complexes leading to insolubilization and stabilization against biodegradation [42,43]. Due to this inorganic
and/or inorganic^organic polymeric structure, cellular accessibility to Fe(III) might be physically obstructed. Inhibited microbial reducibility of Fe(III) oxides can also be
caused by adsorption and/or surface precipitation of biogenic Fe(II) during the microbial reduction of Fe(III) oxides [44]. If the Koke’e forest and the sugar cane plantation soil tended to accumulate surface-bound Fe(II) more
readily than the grassland soil obtained near Kaena State
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K. Ku«sel et al. / FEMS Microbiology Ecology 40 (2002) 73^81
Park, then this could be part of the explanation for why
Fe(III) oxides in andisols were not subject to extensive
reduction. Thus, the chemical extractability of iron is not
an absolute indicator of the availability of Fe(III) for microbial reduction. However, further experiments with pure
cultures of dissimilatory Fe(III)-reducing bacteria are necessary to verify or further evaluate the apparent limited
reducibility of the Fe(III) oxides in andisols.
When grassland soil that was obtained near Kaena State
Park was incubated under anoxic conditions, Fe(III) was
reduced at a rate of 7.2 Wmol (g dry weight soil)31 day31
as soon as nitrate was consumed. Higher rates of Fe(III)
reduction have only been observed in (i) a lactate-supplemented, activated sludge that contained high amounts of
Fe(III) oxides [45] and (ii) the upper zones of acidic mining-impacted sediments that contained high amounts of
schwertmannite (a poorly crystalline iron hydroxosulfate)
[46,47]. Approximately 270 Wmol Fe(III) (g dry weight
soil)31 (corresponding to 67 mM Fe(II) formed in soil
suspensions) appeared to be available for Fe(III)-reducing
bacteria in grassland soil that was obtained near Kaena
State Park, an amount that is one order of magnitude
higher than in most soils and sediments [32]. Although
the amount of citrate^dithionite-extractable Fe(III) (approximately 870 Wmol Fe(III) (g dry weight soil)31 ) is
more than su⁄cient to account for the observed amount
of Fe(II) production, magnetite might be also partly subjected to microbial reduction in Hawaiian grassland soil
that was obtained near Kaena State Park due to the
slightly acidic pH of the soil. Magnetite can be readily
reduced by Shewanella at pH values below 6 [48].
Short-chain organic acids, alcohols, or H2 , products indicative of fermentative activities [11], were not detected in
Hawaiian grassland soils when incubated under anoxic
conditions without supplemental substrates. In anoxic environments, the microbial degradation of organic matter
occurs via a complex network of primary and secondary
fermentors that link the turnover of acetate, one-carbon
compounds, and H2 to a terminal electron-accepting process [49]. Acetate can be detected in the porewater of
freshwater and marine sediments, habitats where either
methanogenesis or the reduction of sulfate is the normal
terminal electron-accepting process, respectively [50]. Acetate can be utilized by a variety of dissimilatory Fe(III)reducing bacteria (e.g. Geobacter metallireducens [51]).
Acetate was not detected in Hawaiian grassland soil obtained near Kaena State Park, suggesting that soil organic
matter was directly oxidized by Fe(III) reducers, or, more
likely, the activities of acetate-producing bacteria were
rate-limiting to acetate-oxidizing Fe(III) reducers. Thus,
acetate might constitute an important trophic link in
Fe(III)-rich Hawaiian soils, despite its negligible pool
size. Acetate is likewise not readily apparent in Fe(III)reducing freshwater sediments and rooted rice paddy soils,
but is nonetheless thought to be an important trophic link
in these habitats [23,52].
79
Small, transient amounts of acetate were detected during the consumption of supplemental H2 or formate by
grassland soil obtained near Kaena State Park, indicating
that acetogens were at least partially involved in the oxidation of supplemental H2 . Since supplemental acetate
was rapidly consumed by this soil, it is unclear to what
extent acetogenesis was involved in the consumption of
supplemental H2 and formate. The ability of soil acetogens
to both tolerate and consume low concentrations of O2
likely contributes to their stability in aerated soils
[53,54]. MPN estimates of H2 -utilizing acetogens and
H2 -utilizing Fe(III) reducers were similar for grassland
soil obtained near Kaena State Park. Since acetogens
have a high threshold value for H2 when they use CO2
as a terminal electron acceptor [55], acetogens might not
be as competitive as Fe(III) reducers for low in situ levels
of H2 . However, in spatially heterogenic habitats like soils,
acetogens could be more competitive for H2 by positioning
themselves near H2 -producing organisms that are not localized proximal to insoluble iron oxides. In contrast,
Fe(III) reducers must be attached to, or loosely associated
with Fe(III) oxides. Thus, due to the microscalic heterogeneity of soils, acetogens might compete successfully with
H2 -utilizing Fe(III) reducers. The subsequent oxidation of
acetate derived from acetogenesis and also from fermentation seemed to be tightly linked to the microbial reduction
of Fe(III) in Hawaiian soils rich in bioavailable Fe(III). A
shift to long-term anoxic conditions in such soils might
stimulate the activity of Fe(III)-reducing bacteria leading
to environmental implications, both in terms of soil carbon £ow and the mobility of phosphate, trace metals, and
other organic and inorganic compounds whose behavior is
in£uenced by sorption/desorption reactions with particulate Fe(III) oxides.
Acknowledgements
The authors express appreciation to Carola Matthies
and Steven L. Daniel for helpful discussions, to Bettina
Popp for technical assistance, and to Ewald Komor for
obtaining some of the soils. This study was supported by
the German Ministry for Education, Research, Science,
and Technology (BMBF).
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