ELSEVIER
FEMS Microbiology Ecology 16 (1995) 233-248
Limiting factors for microbial Fe( III) -reduction in a landfill
leachate polluted aquifer (Vejen, Denmark)
Hans-Jgrgen Albrechtsen *, Gorm Heron, Thomas H. Christensen
Institute of Environmental
Science and Engineering/
Groundwater Research Centre, Technical Unioersity of Denmark, Bldg. 115, DK-2800
Lyngby, Denmark
Received 8 July 1994; revised 7 November 1994; accepted 9 November 1994
Abstract
Aquifer sediment samples from two locations within the anaerobic leachate plume of a municipal landfill were compared
with respect to microbiology
(especially Fe(III)-reduction)
and geochemistry.
The samples close to the landfill were
characterized by low contents of Fe(III), whereas samples from the more distant cluster were rich in Fe(III)-oxides. The
active microbial population seemed to be less dense in samples more distant from the landfill (measured by ATP and
phospholipid fatty acids (PLFA)), but the microbial communities were very similar in the two sample clusters according to
the composition of PLFA. Very little, if any, Fe(III)-reduction was observed close to the landfill, but all the more distant
samples showed evident microbially mediated Fe(III)-reduction. After amendment with both acetate and Fe(III), all the
samples showed a potential for Fe(III)-reduction, and the in situ Fe(III)-reduction seemed to be limited by the lack of
Fe(III)-availability. It was suggested, that Fe(III)-reducing populations might be facultative, surviving by use of other
electron-acceptors than Fe(III), when Fe(II1) is not available for reduction.
Keywords:
Fe(III)-reduction; Leachate plume; Aquifer; Iron-speciation
1. Introduction
The leaching of organic matter and reduced inorganic components
from landfills may dramatically
change the redox conditions in aquifers. Typically, a
sequence of redox zones develops [l-3] as reviewed
by Christensen et al. [4]. Lately, studies have focused
on Fe(III)-reduction,
e.g. [5-71, since Fe(II1) oxides
and hydroxides are abundant in aerobic freshwater
aquifer sediments and theoretically make up a very
significant redox buffer [8,9]. In addition, field ob-
* Corresponding
author. Fax: ( + 45) 45932850.
0168-6496/95/$09.50
0 1995 Federation
SSDI 016%6496(94)00087-5
of European
Microbiological
servations have indicated that dissolved organic matter and organic chemicals
may degrade in the
Fe(W)-reducing
part of a landfill leachate plume [lo]
and sediments from this zone have been shown to
host microorganisms
degrading some organic chemicals [ll].
In the leachate plume downgradient of the Vejen
Landfill, a very large zone of Fe(III)-reduction
was
proposed [2], (Fig. 11, based on elevated concentrations of dissolved Fe(U) along with low contents of
dissolved sulfide, nitrate and oxygen in the groundwater. However, the amount of dissolved Fe(U) may
not be controlled only by the on-going Fe(III)-reduction, but also by migration, dilution, ion-exchange
and precipitation reactions.
Societies.
All rights reserved
234
H.-J. Albrechtsen
et al. / FEMS Microbiology
As a result of microbial and geochemical processes during 15 years of leachate entrance into the
aquifer, the sediment geochemistry changed substantially along a central flowpath through the proposed
redox zones in the Vejen Landfill leachate plume
[12]. The Fe(II1) oxide content as addressed by the
Oxidation Capacity method [9] was high in the more
distant part of the proposed Fe(III)-reducing
zone
(150-300 m downgradient) but low in the first 150
m of the plume, approaching
the detection limit
corresponding to an oxide content of 20 pg Fe/gdw.
This part of the aquifer is strongly reduced, indicated
by low redox potentials [2] and elevated contents of
dissolved methane and sulfide, precipitated pyrite
(FeS,) and acid volatile sulfur (mainly FeSl, which
were all shown to be below detection limits further
downgradient in the plume and in an adjacent unpolluted part of the aquifer [12]. In the reduced section
elevated contents of dissolved and ion-exchangeable
Fe(I1) were recorded. It is not known whether the
low amount of Fe(II1) oxides and the more stable
residual Fe(II1) fraction found in the reduced sediment [12] are available for microbial reduction or if
the observed dissolved Fe(I1) is actually due to slow
release from the sediment of Fe(H) produced during
an earlier stage in the plume development. Thus, it is
evident that studying the geochemistry provides insights into the reactive dissolved and solid pools, but
the factors controlling
the actual Fe(III)-reduction
rates and the extent of the Fe(III)-reducing
zone in
the plume should be addressed by other approaches.
Microbially mediated Fe(III)-reduction
has been
0
Distance from landfill [m]
200
100
Ecology 16 (1995) 233-238
observed in enriched sediment samples from several
locations in the proposed Fe(III)-reducing
zone in
the Vejen aquifer, and unamended
incubations
of
sediment
samples have also revealed microbial
Fe(III)-reduction
[7]. In this study, the actual
Fe(III)-reduction
rates in two parts of the proposed
Fe(III)-reducing
zone were addressed. The study included one (northern) sample cluster close to the
landfill (135 ml and one (southern) sample cluster
further downgradient
(255 m). It was investigated
whether microbial Fe(III)-reduction
could be detected, if lack of organic carbon, Fe(II1) or Fe(III)-reducing bacteria were limiting for the Fe(III)-reduction, and to which degree the Fe(III)-reduction
was
related to the geochemistry. Since substantial spatial
variability
with respect to both groundwater
and
sediment associated parameters has been observed in
the plume, several samples from the two clusters
were included. Each of 15 separate sediment samples
with adjacent groundwater samples were individually
characterized with respect to geochemical (e.g. Fe
speciationl and microbial parameters and incubated
to measure microbial mediated Fe(III)-reduction
and
factors potentially limiting Fe(III)-reduction.
2. Materials
and methods
2.1. Sampling
The samples were obtained from the proposed
Fe(III)-reducing
zone of the Vejen Landfill leachate
300
E38
7ii
5
-I 34
I
6-J
y 30
piezometer
Sediment
z
$ 26
COK3S
5
B 22
U<1.5mgC
I
Fig. 1. Map of the investigated
anaerobic
pollution plume (dissolved
I
F&I) concentration)
and the sample locations.
H.-J. Albrechtsen et al. / FEMS Microbiology Ecology 16 11995)233-248
plume in Denmark (Fig. 1). The shallow sandy
aquifer is unconfined, and consists mainly of glacial
meltwater sand with clay and silt inhomogeneities.
The sediments were deposited in the Weichselian
glacial period (10 000-70 000 years ago) in the late
Quaternary.
The samples were collected in two clusters, one
(northern cluster) 135 m downstream
the landfill,
and one (southern cluster) 255 m downstream (Fig.
1). Each cluster consisted of two or three sediment
cores within a horizontal distance of 1 m from a
l-inch drive point piezometer used for groundwater
sampling. The piezometer was screened from 5.00 to
5.10 meter below surface (mbs). The groundwater
samples were collected, and the chemistry in terms
of dissolved Fe and Mn, Cl-,
NH:, NO;, NO;,
SO:-, Sz-, CH,, O,, total alkalinity, dissolved organic matter (DOC) and pH was determined
as
previously described [2]. The sediment samples were
collected with a Waterloo piston sampler [13] and
handled as previously described [9]. The cores were
kept at 10°C for maximally 5 days, then cut into
three 23 cm long segments with centres of mass
around 5.1, 5.3 and 5.6 mbs. Subsamples for characterizing the sediment and for Fe(III)-reduction
assays
were collected inside an anaerobic glovebox (Coy
Laboratory Products Inc., Grass Lake, MI) using
aseptic techniques. Traces of 0, were removed from
the box by palladium catalysts in the presence of HZ,
and therefore the atmosphere in the box typically
contained 2% of H,. Hydrogen can be used as an
electron donor by many bacteria, also Fe(III)-reducers, but the present amount of H, was not enough
to explain the observed Fe(I1) development.
2.2. Sediment analysis
All wet extraction techniques involved mixing of
sediment with the extractant and centrifugation
in
order to remove particles greater than 0.25 pm. The
concentration
of Fe(I1) in the extracts was determined using ferrozine in the presence of an acetate
buffer at pH 5 [14], and the total amount of Fe in the
extract was determined by atomic absorption spectrophotometry (AAS). All presented values are means
of 5 replicate extractions.
The amount of Fe(I1) soluble in 1 M CaCl, at pH
7.0 (ion-exchangeable
Fe(I1)) was determined on wet
235
sediment samples by an anaerobic 24 h extraction at
20°C followed by Fe(I1) quantification [15].
Fe(B) and Fe(II1) soluble in 0.5 M HCl were
determined using a 24 h 0.5 M HCl extraction at
20°C [15]. Fe(II1) was calculated as total-Fe minus
Fe(I1) measured in the same extract. Note that 0.5 M
HCl does not dissolve a specific mineral fraction and
thus yields an operationally defined amount of iron.
Fe(I1) and Fe(II1) soluble in 5 M HCl were determined using a 21 days 5 M HCl extraction at 20°C
[15]. Fe(II1) was calculated as total-Fe minus Fe(I1)
measured in the same extract. After 21 days, Fe
concentration had stabilized indicating that no more
Fe would dissolve in 5 M HCl at this temperature.
Oxidation capacity (OXC) and Ti(III)-EDTA
extractable Fe and Mn (iron and manganese oxides and
hydroxides) in the sediment was determined by a
0.008 M Ti(III)-0.05 M EDTA extraction followed
by redox titration with dichromate and determination
of the extracted amount of Fe and Mn by AAS [9].
2.3. Microbial analysis
Subsamples of the sediment were preserved by
adding phosphate-buffered
(pH = 7) formaldehyde
(final concentration,
2%) for bacteria counting or
frozen at -80°C for ATP analysis.
The total amount of bacterial cells was counted by
the acridine orange direct count (AODC) as modified
by Albrechtsen and Winding [16]. Dilutions of the
samples were mixed thoroughly, filtered onto a black
0.2 pm Nucleopore filter and stained by acridine
orange (final concentration:
10 mg/l). Fading of the
fluorescence was reduced by washing the filter with
0.3 M DABCO (1,4-diazabicyclo[2,2,2]octane)
for
20 s. All liquids were filtered (0.2 pm) before use.
The bacteria were counted using an epifluorescence
microscope.
The ATP content in the sediment samples were
determined by the luciferin-luciferase
method after
extraction by the phosphorous acid-detergent extraction modified after Webster et al. [17,18]. The sediment samples (5 g wet weight) were shaken (300
rpm, reciprocal motion, 15 min) in triplicate with 2.5
ml of extractant: detergent (Lubrol PX (Sigma),
0.5%), H,PO, (2 M), urea (2 M), DMSO (28 mM),
adenosine (0.75 PM) and EDTA (ethylenediaminetetraacetic acid, tetrasodium salt, 4 H,O (BHD); 20
236
H.-J. Aibrechtsen ef al. / FEMS Microbiology Ecology 16 (1995) 233-248
mM). After centrifugation
(30000 g at 4°C for 20
min), 0.5 ml of the supernatant was neutralized by
4.5 ml of 0.1 M Tricine buffer and pH was adjusted
to 7.8 with 5 M ethanolamine.
In a Biocounter
M2010
(Lumac)
enzyme
reagent
(Boehringer
Mannheim
GmbH) was added to the neutralized
extract (100 ~1) and the recorded light output was
converted to ATP on the basis of an internal standard
and a standard curve prepared for each batch of
enzyme reagent with ATP purchased from Lumac
BV.
Samples for phospholipid fatty acids (PLFA) for
each core were prepared by pooling 10 g wet weight
(gww) from the three segments of each core. The
samples were frozen, lyophilized and analyzed for
total content of PLFA as an estimate of microbial
biomass, specific PLFA to describe the community
structure and nutritional status, and content of storage lipid (poly P-hydroxy butyric acid, PHB) by
Microbial Insights Inc., TN. The PLFA’s were extracted with a one-phase chloroform:methanol
extractant, and fractionated into neutral lipid, glycolipid and polar lipid fractions with silicic acid. After
derivatization,
the eubacterial PLFA were analysed
by capillary gas chromatography/mass
spectrometry
[19,20]. PHB were extracted and analyzed as described by [21].
from the cluster. For each cluster a set of four bottles
were set up as described above and 150 ~1 of
chloroform was added (to near saturation).
During the incubation period of 165 days, at the
actual groundwater temperature (lO”C), subsamples
of 1.5 ml were collected from the suspension by
syringe and needle for analysis of dissolved Fe(I1).
After filtration (0.45 pm Minisart SRP 15, Sartorius)
the concentration
of dissolved Fe(I1) was measured
by the ferrozine method [14]. Unfiltrated subsamples
of the suspension (fines) were at intervals extracted
by 0.5 M HCl for 1 h at 20°C to include Fe(I1)
associated with the suspended fine grained material
(e.g. ion-exchanged
or precipitated). The dissolved
Fe(I1) typically made up 9-64% of the iron recovered by these extractions in the samples from the
northern cluster and 2-20% in the samples from the
southern cluster.
At the end of the incubation, pH in all the bottles
were measured in an anaerobic glovebox. The neutral and the acetate enriched bottles were left
overnight to settle, before the water phase was removed, and the amount of ion-exchangeable
and 0.5
M HCl-extractable
Fe(II) was measured on the remaining sediment.
3. Results and discussion
2.4. Assays for microbial Fe(M)-reduction
Sediment (20 gww, approximately 16 g dry weight
(gdw)) was transferred to 58 ml autoclaved serum
bottles, sealed with 1 cm thick butyl rubber stoppers,
and 30 ml sterile-filtered
groundwater
(0.2 pm,
Minisart NML, Sartorius) was added from the respective cluster.
For each sediment core segment, four bottles were
set up: one as described above, one enriched with
sodium acetate (to a final concentration
of 250 mg
C/l), one enriched with amorphous Fe(II1) (to a
final concentration of 158 mg/l Fe(II1)) and finally
one enriched with both acetate and amorphous
Fe(II1). The synthetic amorphous Fe(III) oxide (ferrihydrite) was prepared as described by Lovley and
Phillips [22].
Control experiments were prepared for each cluster by subsampling from a pool of all the sediment
samples for each cluster and adding groundwater
3.1. Field observations:
chemistry
Groundwater
and sediment
The composition of the groundwater collected in
the centre of each cluster (Table 1) showed that the
northern cluster was clearly affected by the landfill
leachate, since chloride, ammonium and DOC were
elevated relative to background levels. The southern
cluster, which is located twice as far downgradient of
the landfill, was less affected. The chloride concentration of 76 mg/l was elevated relative to 15-20
mg/l in the unpolluted aquifer [23]. According to
the groundwater
criteria proposed for the Vejen
Landfill plume for assigning redox status [2], both
clusters were located within the Fe(III)-reducing
zone.
The iron and manganese chemistry of the sediment samples is given in Table 2. The content of
both ion-exchangeable
and solid Fe varies as much
H.-J. Albrechtsen
et al. /FEMS
Microbiology Ecology I6 (1995) 233-248
Table 1
Groundwater
composition
at the two sampling clusters in the
Fe(Ill)-reducing
zone of the Vejen Landfill leachate plume. All
values except pH are mg/l
PH
Cl0,
NO;
NO;
NH:
Mn
Fe(H)
SO:SC-II)
CH4
DOC
Northern cluster
135 m from landfill
Southern cluster
255 m from landfill
5.7
213
< 0.5
< 0.2
< 0.2
18
0.67
27
44
< 0.1
3.1
23
6.2
76
< 0.5
< 0.2
< 0.2
1.0
0.46
5
72
< 0.1
0.15
6.1
as 2 orders of magnitude within the southern cluster.
The large variations both over depth and horizontally
by far exceed the analytical uncertainties
(standard
deviations were typically lo-15% of the mean of 5
replicate extractions). Despite these variations, substantial differences in the geochemistry between the
two clusters were observed.
The Fe(II1) content was very low in the northern
cluster. This is in agreement with the findings of
Heron and Christensen [12] that Fe(II1) oxides and
Table 2
Sediment characterization
Core
Depth
mbs
with respect to iron and manganese.
Fe(H)
ion-exchangeable
Fe(H)
0.5 M HCl
Fe(H)
5 M HCl
hydroxides were nearly depleted in this aquifer section. The sediments contained significant amounts of
Fe(III), which mostly were measured in the poorly
defined residual fraction (extracted by 5 M HCl)
[12], which supposedly was unaffected by redox
reactions within the time scale of our experiments.
The southern cluster contained more Fe(III1 oxides
as seen by the higher oxidation capacity and Fe
extracted by Ti(III)-EDTA
(Table 2). It also contained more solid manganese, indicating less reducing conditions in this cluster, since Mn oxides may
not be completely
depleted (the used extractions
were not able to distinguish between reduced and
oxidized manganese).
The distribution of Fe(U) between the dissolved,
ion-exchangeable
and precipitated
phases differed
for the two clusters, with the northern cluster having
more dissolved and ion-exchangeable
Fe(I1). The S3
core from the southern cluster contained more ionexchangeable
Fe(I1) than the neighbouring
cores.
The Fe(I1) distribution among the phases is discussed
in the section on microbial Fe(III)-reduction.
3.2. Sediment microbiology
The total bacterial population enumerated as acridine orange direct counts ranged from 7.1 X lo6 to
55 X lo6 cells/gdw
(Table 3) and thus was within
Values are means of 5 replicates
Fe(II1)
0.5 M HCl
Fe Ti(III)-EDTA
t-e/gdw Northern cluster
Nl
5.1
6.7
5.3
37
5.6
69
N2
5.1
44
5.3
90
5.6
26
Southern cluster
Sl
5.1
5.4
5.3
0.3
5.6
0.0
s2
5.1
2.6
5.3
0.1
5.6
0.2
s3
5.1
23
5.3
26
5.6
9.0
231
Mn Ti(III)-EDTA
Total Fe
5 M HCl
a/g
pg/g dw w/gdw
dw
27
107
312
194
506
243
204
282
696
418
1345
606
53
2
0
3
0
16
63
58
115
107
223
110
0.2
0.4
1.1
1.1
2.4
1.2
870
840
1830
1290
2850
1610
552
464
374
498
405
449
541
661
477
1400
1242
848
904
722
933
1122
1270
833
1032
508
114
581
246
100
852
811
623
1955
782
255
879
490
285
1395
1480
1191
19.1
5.2
2.9
7.7
3.6
4.1
11.5
10.5
5.5
6650
4890
2430
2740
2070
1920
5090
6200
4230
Oxidation
Capacity (OXC)
35
16
7
17
10
6
22
27
21
H.-J. Albrechtsen
238
et al. / FEMS Microbiology
the range of values previously reported [24-27,161
from similar aquifers. The AODC showed no clear
variation with depth, between individual
cores or
between the two clusters (Table 3). ATP and PLFA
are considered to be accurate measures of viable
sedimentary biomass [26]. The ATP-content
ranged
from 28 to 1447 pg/gdw in accordance with other
observations
of 650 k 80 pg/gdw
and 18 f 1
pg/gdw [181, 70-500 pg/gdw [28] and 1390 & 420
pg/gdw [26] although different extraction methods
were used. The ATP and PLFA contents were higher
in the samples from the northern cluster. In all the
cores the ATP-content was the highest in the upper
sample, and in several cores the content decreased
with depth.
The PLFA-content
(Table 3) generally was low
compared to other aquifers. PLFA-contents of 350 i
80 pmol/gdw were recorded in an unpolluted aquifer
at Lula, Oklahoma, USA [26] and 50-300 pmol
PLFA/g
was found in a shallow uncontaminated
aquifer in Wisconsin,
USA [29]. In a shallow
wastewater
contaminated
aquifer 10-2000
pmol
PLFA/g was found [29]. Relatively high metabolic
activity in the Vejen aquifer was indicated by an
ATP/PLFA
ratio of 37-151 g/mol compared to 4
g/mol observed by Balkwill et al. [26] in a shallow
uncontaminated
aquifer.
Table 3
Microbial
characterization
Core
Northern cluster
Nl
N2
Southern cluster
Sl
S2
s3
Ecology 16 (1995) 233-248
The ATP and PLFA-content
indicated that the
population
of active bacteria was smaller in the
samples from the southern cluster than in the samples from the northern cluster. However, the Fe(III)reducing population might only make up a small part
of the living population, and this sub-population was
not enumerated.
The nutritional status of the microbial community
was evaluated by the PLFA-analysis:
The tram/&
ratio (so-called
stress biomarkers)
of these monoenoic acids usually increases during metabolic
stress, either due to starvation [30,31], overfeeding
[31] or presence of toxic pollutants like phenol in
high concentrations [32], but hardly no tram isomers
of 16:lw7 or 18:107 were observed (Fig. 2). The
absence of tram isomers of PLFA’s and of storage
lipid (poly /3-hydroxy butyric acid, PHB) [21,33]
suggests that the microbial community was not under
severe stress or nutritional
limitation,
and as expected from the relatively oligotrophic environment,
the populations seem to be growing but not in the
logarithmic growth phase.
Cyclopropyl fatty acids (cy#:O) as cy19:O were
found in all cores, whereas cy17:O only was found in
the cores from the northern cluster indicating slightly
more stressed conditions there maybe due to unbalanced growth as seen under overfed conditions [31]
of the sediment samples
X
ATP pg/gdw
PLFA a pmol/gdw
21+8
52 + 22
28 f 12
10 f 3
29+ 10
42 i_ 14
745 k 138
NM
206 f 13
1447 &-69
1258 f 4
978 + 120
6.2
36+
55 f
16 f
38 f
30*
30 f
21 f
7+3
7f2
122 + 16
91+ 34
97 * 30
90* 12
28+12
58 + 6
116 k 12
109 f 20
93 f 4
2.8
Depth mbs
AODC
5.1
5.3
5.6
5.1
5.3
5.6
5.1
5.3
5.6
5.1
5.3
5.6
5.1
5.3
5.6
12
14
5
14
17
8
7
NM: not measured.
a: measured for samples pooled for the whole core.
lo6 cells/gdw
33.0
2.0
0.7
H.-J. Albrechtsen et al. / FEMS Microbiology
3.3. Microbial
or environmental
stress [34]. The monoenoics
(#l w7c) are also known to change to cyclopropyl
fatty acids (cy#:O) in many bacteria when they
change from logarithmic to stationary growth conditions (i.e. when the community is aged) [35]. The
relatively
low
ratio
observed
(0.1-1.3
for
cy19:0/18:107c
and 0.1-0.4 for cy17:0/16:lw7c
(where cy17:O was present)) indicates stationary
growth conditions in the population.
20
40%
0
Normal Saturates
l&O
community
239
composition
Analysis of the PLFA composition indicated
sence of eukaryotes, since no polyenoics were
tected (Fig. 2) [36,37].
There was no consistent difference between
two clusters, although some variation among
samples was observed (especially core N2 and
differed from the others).
abdethe
the
Sl
N2
NI
0
Ecology 16 (1995) 233-248
20
40%
I
&i&&p
17:o
18:O
20:o
22:o
Terminally Branched Saturates
Mid-chain Branched Saturates
lOme18:O
1
Monoenoics
lfTlO5C
cy17:o
18:l
18:lw9c
18:107c
18:i o7t
cy19:o
Branched Monoenoics
i17:lw7c
1
I
T------
I
I
Polyenoics
T.
I--
1
Fig. 2. Community structure based on specific phospholipid fatty acid (PLFAJ analysis \m mot-% of total PLFA). The significant (> 2%, or
specific biomarkers) compounds are shown for the sediment samples combined for the samples from each core. Note that the total PLFA
content was very low in S3 (see Table 31, which might influence the detection of some of the specific PLFA only present at low mol-%. The
nomenclature used is as follows: the number of carbons; ‘:‘, the number of unsaturations, ‘w’, the distance of the first unsaturation from the
methyl end of the fatty acid; and ‘c’ or ‘t’ for the cis or tram geometric isomers of the unsaturation. The prefixes ‘i’ and ‘a’ denote iso- and
antiisobranching,
and ‘cy’ denote a cyclopropylmoiety.
240
H.-J. Albrechtsen
et al. / FEMS Microbiology
Except for normal saturates (38-76% of the total
PLFA content), which are indicative of almost anything organic, the monoenoics (19-44% of the total
PLFA content) were the most prominent
PLFA’s
detected in each sample (Fig. 2) and a principal
component analysis generated from the mol-%-values revealed that the monoenoics
were the most
descriptive of the data set. These PLFA’s are synthesized primarily by Gram-negative
microorganisms
via the biosynthetic pathway, called anaerobic desaturation (0, is not required but may be present)
[19,20]. In addition to the monoenoics,
terminally
branched saturates were detected which represented
ll-14%
of the total PLFA (except in S3, where only
very little PLFA was detected). These specific
PLFA’s are indicative of numerous Gram-positive
bacteria, but are also found in Gram-negative
sulfate
reducing organisms. Other indications of sulfate reducers were the dominance
by lOme16:O of the
mid-chain
branched saturates and the substantial
amounts of 16:107c. The lOme16:O is characteristic
of Desulfobacter
species [38], whereas i17: 07c,
characteristic
of Desulfouibrio
[39] was not observed. The 16: 1 w 7c is found in Thiobacillus species
[40] and in Desulfomonile
tiedjei [41] (but also in
type I methanotrophs [42] and in a number of Pseudomonas species [43]). Furthermore, the 16:lw7c is
the major PLFA in Geobacter metallireducens
[44]
and present in Shewenella, both capable of coupling
oxidation of organic compounds to Fe(III)-reduction
[45]. Thus, these data strongly indicate the presence
of sulfate- and Fe(III)-reducing
bacteria in the sediment.
3.4. Microbial
Fe(III)-reduction
Before turning to the observed Fe(III)-reduction,
some practical and analytical details deserve attention. Hypothetically,
the dilution of 20 gww sediment with 30 ml of water before the incubation
could alter the distribution
of Fe(I1) between the
dissolved and solid phases. However, this was not
the case, since in most incubations, the dissolved and
sediment associated Fe(I1) both increased during the
incubation
(data not shown). The distribution
of
Fe(I1) between the liquid phase (dissolved Fe(I1))
and solid phase (ion-exchangeable
or extractable by
0.5 M HCl) in the incubation flasks at the end of the
Ecology 16 (1995) 233-248
Fraction of Fe(ll) (%)
1001
90 -80 -70 -60 -50 -40 -30 -20ioO-E
.6 5.1 5.3
-N2-
-
1l dissolved
t
.6 5.1
.3‘5.6’!
il-
0 ion-exchangeable
q solid
t
0.5 M HCI-ext.
1
Fig. 3. The distribution of Fe(U) between the liquid phase (dissolved Fe(U)) and solid phase (ion-exchangeable
or extractable by
0.5 M HCl) in the incubation flasks containing 30 mL of water
and 20 gww of sediment. Values were obtained at the end of the
experiment.
neutral incubations is given in Fig. 3. In the northern
cluster, both dissolved and ion-exchangeable
Fe(I1)
occurred in significant amounts indicating that precipitation reactions were not dominant. In the southern cluster, more than 95% of the Fe(I1) in this
fraction was in the solid phase indicating that the
equilibrium was shifted more towards the solid phase.
This corresponds well with observations in related
studies on Fe(III)-reduction
in river sediments [5].
Measuring dissolved Fe(I1) is convenient since it
makes it possible to collect subsamples from the
bottles and to follow the development of Fe(I1) over
time. Now, since the Fe(I1) produced during microbial Fe(III)-reduction
will be distributed among the
different phases, the evolution of dissolved Fe(I1)
will indicate if Fe(III)-reduction
is occurring. However, the reacted Fe pools and thus the actual
Fe(III)-reduction
rates have to be addressed by including the ion-exchangeable
and precipitated Fe(I1).
Considering
the difficulty of collecting
sediment
subsamples
during the incubations,
the dissolved
Fe(I1) and the Fe(I1) associated with the fine-grained,
suspended material was used to assess the production
of Fe(I1) during the incubation period, and intact
sediment samples were only collected at the onset
and end of the experiments.
The groundwater-sediment
suspensions from the
southern cluster had the highest Fe(II1) oxide con-
241
H.-J. Albrechtsen et al. /FEMS Microbiology Ecology 16 (1995) 233-248
Northern cluster
rg Fe(ll)/gdw
?O ,
d
’
N2 5.1
+Fe(lll)
N2 5.3
Nt 5.3
-N2 5.6
Nl 5.6
a.
opmx.+=.-.y._
50
100
Days
150
200 0
100
Days
Southern cluster
I I
kg Fe(ll)/gdw
18 ,
It
18
Nl 5.1
Control
,
+ Acetate
0
I
I I
16
14
I2
IO
8
6
4
2
0
0
100
Davs
200 0
50
100
Davs
150
200
Fig. 4. Concentration of dissolved Fe(B) over time in suspensions of aquifer sediment (neutral: A, E). The different sediment samples were
collected from different cores and depths from a northern cluster 135 m and from a southern cluster 255 m downstream the landfill. The
development of dissolved Fe(B) after different amendments are also shown: B, F: addition of acetate; C, G: addition of amorphous Fe(II1);
D, H: addition of acetate and amorphous Fe(II1). The suspensions were incubated anaerobically at 10°C. The controls were inhibited by
chloroform.
242
H.-J. Albrechtsen
et al. /FEMS
Microbiology
wg Fe(ll)/gdw
8001
10
initial Fe(ll
Fig. 5. The amount of Fe(U) either dissolved or extractable by 0.5
M HCl in the incubation flasks at the onset of the incubations and
after 165 days of incubation. Solid FeUI) values are means of 5
replicate sediment extractions.
tent, relatively low dissolved Fe(U) and a pH of
6.7-7.0. During the incubation of these groundwater
sediment suspensions, the concentration of dissolved
Fe(U) increased without a lag period in all the 9
samples,
reaching
concentrations
(2-l 1 pg
Fe(II)/gdw)
consistently higher than in the chloroform-killed control (Fig. 4E), which strongly indicates that microbial Fe(III)-reduction
can occur under in situ conditions.
In some suspensions,
the
dissolved Fe(U) continued to increase during the
whole incubation, but in most of the suspensions a
plateau was reached after approximately
4 weeks.
The increase in dissolved Fe(N) was in accordance
with an increase in 0.5 M HCl-extractable
Fe(U)
from the sediment in incubations from the southern
cluster (Fig. 5) (average 81 pug/g dw, standard
deviation 40 kg/g dw). This increase was significant at a 5% significance
level when each Fe(U)
concentration
was considered to be the outcome of
independent normally distributed stochastic variables
with different mean values and identical variances.
Thus, the southern cluster showed an Fe(U) increase
during incubation in the order of 80 pg/g dw which
is typically lo-20 times the increase in dissolved
Fe(I1) alone. Variation within the samples from the
southern cluster was also observed:
the highest
reached concentrations
of dissolved Fe(U) (8-l 1
pg/gdw)
in the southern cluster were observed in
the samples from the S3 core. This core had relatively high contents of both Fe(II1) and ion-ex-
Ecology 16 (19951 233-248
changeable Fe(II), which might support that Fe(III)reduction is likely to happen in these samples.
Generally, the concentration
of dissolved Fe(U)
did not increase in the incubated samples from the
northern cluster, although the level of dissolved Fe(I1)
(21-36
pg Fe(II)/gdw)
was higher (Fig. 4A). In
support of this, no significant increase in the Fe(I1)
associated with the solids was observed (Fig. 5). The
average changes in this operationally defined Fe(U)
fraction was in fact negative (- 13 pg/g dw), but
statistically (on a 5% significance level) not different
from zero (standard deviation 55 pg/g
dw). In a
few samples, the concentration
of dissolved Fe(U)
increased at the same rate as in the southern cluster,
but these samples were characterized by low initial
Fe(I1) concentrations (21-25 pg/gdw)
and the highest content of 0.5 M HCl-extractable
Fe(II1) (Table
2). The concentration
of Fe(I1) in these samples
decreased at the start of the incubation, probably due
to oxidation of Fe(U) by oxygen intruding during
sampling and setting up the experiments. Since the
Fe(I1) resulting from microbial Fe(III)-reduction
in
these samples never exceeded the level in any of the
non-active
suspensions,
probably only the freshly
oxidized Fe(II1) was available for microbial Fe(III)reduction. Therefore, the incubations
representing
the northern cluster indicated that very limited, if
any, Fe(IIIZreduction
would occur in situ at this
location.
In conclusion, no on-going Fe(III)-reduction
was
seen in the northern cluster, despite the fact that the
groundwater samples contained significant concentrations of dissolved Fe(I1). The sediment geochemistry showed low Fe(II1) oxide contents and elevated
ion-exchangeable
and solid Fe(U). Thus, we speculate that the high dissolved Fe(I1) concentration (27
mg/l) actually was due to a substantial attenuation
of Fe(I1) produced during earlier stages in the plume,
and did not justify the proposed assignment to the
Fe(IIIl-reducing
zone.
Fe(III)-reduction
was shown in the southern cluster, which was richer in Fe(II1) oxides. Despite the
indications of lower bacteria content (ATP, PLFA),
lower DOC and supposedly a higher redox potential,
Fe(III)-reduction
lead to a substantial production of
Fe(U), of which the major part was associated with
the sediment. These sediment-groundwater
interactions were substantial and showed, that Fe(III)-reduc-
H.-J. Albrechtsen
et al. /FEMS
Microbiology
tion rates can only be addressed when ion-exchange
and precipitation reactions are accounted for.
Compared to previously reported Fe(III)-reduction
rates (1.4 pg/day/gdw)
for a two month incubation
of a crude oil polluted aquifer (Bemidji, MN, USA)
[46] the rates observed in our experiment (about 0.5
pg/day/gdw)
were lower if calculated as an average for the whole incubation period (165 days). But,
if the rates were calculated for the most active period
(2 months), the rates were of the same order of
magnitude as observed by Lovley et al. [46]. The
differences in Fe(III)-reduction
rates, and especially
the higher rates in the southern cluster may be due to
a higher Fe(II1) availability in these sediments, indicated by higher extractable Fe(III).
3.5. Limiting factors
Since distinct differences in Fe(III)-reduction
were
observed in the two clusters, although the microbial
communities could not be distinguished by the composition of PLFA’s (Fig. 2) we wanted to investigate some of the most obvious potentially limiting
factors for the Fe(III)-reduction:
the carbon source
(by adding acetate) and the electron-acceptor
(by
adding amorphous Fe(II1) oxides).
Hypothetically,
these manipulations
may change
the chemical environment
in the suspensions. Addition of acetate may change e.g. pH or chelate some
of the sediment bound Fe(I1) and thus increase the
concentration
of dissolved Fe(B). However, pH did
not change significantly
due to any of the additions
(data not shown) and no significant increase of Fe(I1)
was observed in the chloroform killed controls neither measured as dissolved Fe(I1) (Fig. 4A,E) nor as
0.5 M HCl-extractable Fe(B) of fines suspended after
shaking of the flasks.
By amending with both acetate and Fe(III), all the
investigated samples showed a potential for microbial Fe(III)-reduction,
since the concentration of dissolved Fe(B) (Fig. 4) and of Fe(B) extracted by 0.5
M HCl of water and fines increased. It is therefore
evident, that the in situ Fe(IlI)-reduction
was limited
either by the availability of the electron acceptor or
the carbon source, or both.
Comparing the two clusters, it is evident that the
potential for microbial Fe(III)-reduction
was largest
in the northern cluster. Dissolved Fe(I1) concentra-
Ecology 16 (1995) 233-248
243
tions, after amending with both acetate and Fe(III),
increased more in the northern (12-53
pg/gdw,
omitting one being inactive) than in the southern
cluster (4-17 pg/gdw)
during the incubation despite
the fact that hardly no Fe(III)-reduction
occurred in
the unamended sediments from the northern cluster.
The limiting factor could be available Fe(III), since
Fe(III)-reduction
also occurred when the sediment
was amended with Fe(II1) only (Fig. 4C). In the
southern cluster, amendment with Fe(II1) increased
the Fe(III)-reduction
relative to the neutral incubations (Fig. 4G) although substantial amounts of extractable (0.5 M HCl) Fe(II1) were present in these
sediments (Table 2). This indicates low availability
of even this Fe(III) fraction (supposedly poorly crystalline Fe(II1) [15]) for microbial reduction within the
given time-scale of 165 days. This low availability is
supposedly due to previous Fe(III)-reduction
in this
location removing the most readily reducible Fe(III),
since the Fe(II1) content is lower than in the surrounding aerobic aquifer [ 121.
It could be speculated that the carbon source in
terms of the diluted landfill leachate, besides being
lower in concentration,
was less available in the
more distant southern cluster [47]. Therefore, it was
expected that adding a carbon source such as acetate
would stimulate the Fe(III)-reduction.
As a consequence of acetate addition, the HCl-extractable Fe(I1)
(0.5 M) from fines and to some degree the dissolved
Fe(I1) (Fig. 4F), showed a distinct increase in the
Fe(III)-reduction
in the samples from the southern
cluster, when compared with the neutral incubations.
No effect was observed in the samples from the
northern cluster (Fig. 4B). Thus, lack of available
organic carbon seemed to be a limiting factor for
Fe(III)-reduction
in the southern cluster, where the
DOC concentration was low (Table 1).
The potential
for microbial
Fe(III)-reduction
seems to be related to the size of the metabolic
active microbial population
since the increase of
dissolved Fe(I1) during the incubation
in samples
amended with either acetate, Fe(II1) or both, correlated with the initial ATP-content when all the investigated samples were included (Fig. 6). This could
indicate that the Fe(III)-reducers
in our samples were
quite versatile, facultatively
Fe(III)-reducing,
and
able to shift to a more readily available terminal
electron-acceptor
as amorphous Fe(III), when added.
244
H.-J. Albrechtsen
et al. /FEMS
kg Fe(ll)/gdw
60 ,
Microbiology
1
-20
-30
0
200
400
600
600
1000
pg ATPlgdw
+ acetate
1200
1400
1600
Ecology 16 (1995) 233-248
ria e.g. Desulfouibrio desulfuricans [45] (although
no characteristic
biomarkers for this species was
found in our samples), and Thiobacillus at low pH
conditions
[51]. Only weak indications
of sulfate
reduction were observed at the northern cluster, but
since the PLFA profiles indicated the presence of
sulfate reducing bacteria in the samples and addition
of Fe(II1) have been reported to exclude sulfate
reduction e.g. [52,45], it is possible that the bacteria
performing the observed Fe(III)-reduction
may survive using sulfate as the electron-acceptor
when
Fe(II1) is not available for reduction. Presently, it is
not known if this Fe(III)-reduction
is energy-yielding
in these populations.
+ Fe(W)
Fig. 6. Relation between increases in the maximum reached
dissolved Fe(R) concentration
as an effect of different amendments during 165 days of incubation
and microbial biomass
measured as ATP-content.
For each experiment the difference
between the maximum increase of dissolved Fe(H) in the different
amendments and the maximum increase of dissolved Fe(R) in the
neutral incubation is shown.
This was further supported by the observation that
groundwater from the northern cluster became sulfate-reducing (i.e. sulfide was produced and precipitated) when incubated in absence of sediment and
thereby Fe(III) (data not shown).
Surprisingly, the Fe(lII)-reduction
in most of the
samples from the northern cluster amended with
acetate and Fe(II1) showed a lag phase of 50-60
days. Such lag phases were not observed when
amended with Fe(II1) only or in the samples from the
southern cluster, indicating a shift in the metabolism
in the microbial population in the northern cluster
during the incubation with both acetate and Fe(II1).
Fe(II1) may be reduced by organisms (e.g. BacilZus sp. and Clostridium sp.) [48,49] that are able to
use more complex carbon sources. Although Fe(III)reduction by these organisms is not stoichiometritally connected to carbon metabolism,
thermodynamic calculations
suggest that using Fe(II1) as a
minor electron sink can provide a slightly greater
energy yield than fermentation
alone [SO]. Fe(II1)
may also be reduced by some sulfate reducing bacte-
4. Conclusion
This combined geochemical and microbial characterization of 15 aquifer sediment samples from the
Vejen Landfill leachate plume showed that the in
situ microbially mediated Fe(III)-reduction
was limited by the availability or the amount of Fe(III) on
the aquifer sediment. Samples collected from the
northern part of the proposed Fe(III)-reducing
zone
of the plume were poor in Fe(II1) oxides and hydroxides (as evaluated by extraction by 0.5 M HCl or
Ti(III)-EDTA),
and only little, if any, Fe(III)-reduction was observed in these samples. A speciation of
the Fe in the sediment showed that the Fe(II1) present could be extracted only by 5 M HCl, and was
considered as residual and therefore relatively unavailable for reduction. Fe(II1) oxides and hydroxides were present in samples from the southern part
of the plume, and microbial mediated Fe(II1) reduction was observed in unamended incubations of these
samples. Despite the difference in geochemistry between the two clusters, no major differences could be
detected in the size (AODC) or composition (PLFA)
of the microbial populations.
The PLFA analysis
indicated presence of Fe(III)-reducers
at both locations and given an available Fe(II1) source, microbial
Fe(III)-reduction
was initiated in samples from the
northern cluster, confirming the presence of Fe(III)reducers. Acetate amendments indicated, given excess Fe(III), that the carbon source in terms of
dissolved organic matter in landfill leachate also may
be a limiting factor for the Fe(III)-reduction.
The
H.-J. Albrechtsen
et al./FEMS
Microbiology
association between ATP content in the samples and
the maximum increase in concentration of dissolved
Fe(R) after different amendments, indicated that the
capacity of Fe(III)-reduction
somehow was related to
the size of the active microbial population.
Finally it was stressed, that in a complex pollution
plume, elevated dissolved Fe(H) concentrations
cannot be taken as evidence for on-going Fe(III)-reduction, since the behaviour of Fe(R) in the plume may
be substantially
affected also by ion-exchange
and
precipitation. These results clearly show that a combination of geochemical characterization
and microbiological incubations is important in investigations
of Fe(II1) reduction in aquifers.
[7]
[8]
[9]
[lo]
[ll]
Acknowledgements
The technical assistance of Mette L. Andersen,
Karin Hansen, and Mona Refstrup is gratefully acknowledged. We thank Per Nielsen and Anja Foverskov for providing the sediment and groundwater
samples. We also thank Dr. D.C. White for valuable
comments. This study is part of a major research
program focusing on the effects of waste disposal on
groundwater. The program is funded by the Danish
Technical Research Council, the Technical University of Denmark and the Commission of the European Communities.
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