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Isotope fractionation during microbial metal uptake measured by
MC-ICP-MS
L. E. Wasylenki,*a A. D. Anbar,ab L. J. Liermann,c R. Mathur,d G. W. Gordona
and S. L. Brantleyc
Received 11th April 2007, Accepted 28th June 2007
First published as an Advance Article on the web 16th July 2007
DOI: 10.1039/b705476a
High-precision isotopic analyses by MC-ICP-MS were used to investigate the mass-dependent
fractionation of Mo and Fe isotopes during bacterial metal assimilation in experiments with
Azotobacter vinelandii. A. vinelandii is a diazotroph with high demand for both Mo and Fe during
nitrogen fixation. Our results demonstrate that the growth medium became progressively enriched
in heavier isotopes of Mo during bacterial growth, indicating preferential assimilation of lighter
isotopes. In contrast, for Fe, the medium become isotopically lighter as Fe was removed from
solution. The experimental data can be interpreted in terms of Rayleigh fractionation, yielding
fractionation factors of 0.9997 and 1.0011 for Mo and Fe, respectively. Hence, we infer
D97/95Mocells-medium = �0.3% and D56/54Fecells-medium = 1.1%. Fractionation of Mo isotopes
could result from simple kinetic effects during assimilation, but may also be affected by
complexation with high-affinity metal binding ligands. Kinetic effects cannot easily account for
the sense of Fe isotope fractionation, and so equilibrium effects, possibly between different Fe
complexes, are implied. Adsorption of Mo and Fe onto cell surfaces may also play a role and
requires further examination. Isotope fractionation studies using MC-ICP-MS may provide new
constraints on the processes by which microbes extract metals from their surroundings, ultimately
yielding insights into the mechanisms of metal assimilation into the metallome.
Introduction
The development of multiple collector inductively coupled
plasma mass spectrometry (MC-ICP-MS) has opened many
new research avenues that require high-precision isotopic
analyses. Among these, there has been particularly explosive
growth in research into variations of transition metal isotope
compositions arising from mass-dependent fractionation.1–11
Biological isotope fractionation was among the initial areas of
inquiry, focusing on the fractionation of Fe isotopes during
dissimilatory reduction.3,12 From a metallomics standpoint,
however, it is more interesting to consider the isotope fractio-
nation that might occur during transition metal assimilation or
intracellular manipulation. If such effects occur, they could
provide novel insights into the mechanisms of metal assimila-
tion and the pathways of metal storage, distribution and
incorporation into enzymes inside cells. These effects could
also provide a novel means of looking for evidence of biolo-
gical use of metals in modern or ancient environments. In fact,
fractionation during uptake of both Fe5,13 and Mo14 by micro-
organisms has been identified. Applied to ancient sedimentary
rocks, identification of such phenomena might even yield
information on the evolutionary history of the metallome.15
To demonstrate the potential of MC-ICP-MS for such
research, here we report initial results from an experimental
investigation into Fe and Mo isotope fractionation during
assimilation by Azotobacter vinelandii. This bacterium con-
verts N2 to NH3 (‘‘nitrogen fixation’’) using the Fe- and Mo-
dependent nitrogenase enzyme complex. As a result, A. vine-
landii has a high metabolic demand for these particular
elements. These elements are also useful targets of investiga-
tion because of a relatively large and growing body of research
carried out by geochemists interested in their environmental
isotope chemistry.2,16–26 Some of this research provides a
context for understanding effects observed in our experiments.
In turn, the results of this experimental study may help to
better understand the isotope systematics of Mo and Fe in
some environmental settings.
Experimental methods
In order to measure stable isotopic fractionation of Mo and Fe
by A. vinelandii, bacteria were grown for several days in acid-
cleaned, sterilized, 500 mL, polycarbonate bottles each con-
taining B200 mL of medium. Two batches of culture medium
were prepared, both comprising 0.8 mM MgSO4, 0.7 mM
CaCl2, 0.6 mM KH2PO4, 0.7 mM K2HPO4, 50 mM citric acid,
50 mM MES buffer (C6H13NO4S �H2O), and B1 mMNa2MoO4 � 2H2O (B96 ng g�1 Mo). One batch was amended
a School of Earth and Space Exploration, Arizona State University,Tempe, AZ 85287, USA. E-mail: [email protected]
bDepartment of Chemistry and Biochemistry, Arizona StateUniversity, Tempe, AZ 85287, USA
cDepartment of Geological Sciences, Pennsylvania State University,University Park, PA 16802, USA
dDepartment of Geology, Juniata College, Huntingdon, PA 16652,USA
This journal is �c The Royal Society of Chemistry 2007 J. Anal. At. Spectrom., 2007, 22, 905–910 | 905
PAPER www.rsc.org/jaas | Journal of Analytical Atomic Spectrometry
with 14 mM ammonium acetate to produce nitrogen-rich
conditions (designated NR in Table 1). The pH of each batch
was adjusted to 6 by addition of NaOH before autoclaving.
Glucose and Fe-citrate were sterilized separately and added at
concentrations of 1% (w/v) and 21 mM (1.2 mg g�1 Fe),
respectively, to both media. These media differ slightly from
those used previously.14 Because Fe failed to stay in solution in
previous studies, here we replaced much of the phosphate with
MES buffer and included more citrate to discourage loss of Fe
by precipitation of (oxyhydr)oxides. No visible precipitates
formed in any of the cultures.
The nitrogen-rich and nitrogen-poor media were each di-
vided into six culture replicates. Each bottle was then inocu-
lated with A. vinelandii (strain OP) at late-log to stationary
growth phase. The starting cell density was B106 cells mL�1,
based on a plate count of a 10�6 dilution of the starting
inoculum. After inoculation, all bottles were placed on a
shaker table and incubated at 30 1C for 2–7 days.
At the end of the incubation period, small aliquots of each
culture were removed for cell density measurement. The
remaining cultures were centrifuged to pellet most cells, then
the supernatants were filtered through membranes with 0.2 mmpores and both solutions and centrifuge-pelletted cells were
recovered for analysis.
Analytical methods
Aliquots of recovered, filtered medium were acidified, and
concentrations of Fe and Mo were measured by ICP-MS.
Separate aliquots for Fe and Mo were digested with nitric acid
and hydrogen peroxide and run through ion exchange chem-
istry to purify the element of interest. To avoid isotope
fractionation artifacts during sample handling we used diges-
tion and purification procedures tested to ensure near-quanti-
tative (495%) yields.
Solution aliquots for isotope analysis were weighed and
digested in screw-top, Teflon vials (Savillex Corp.) with hot,
slightly pressurized HNO3 and H2O2 until they no longer
visibly reacted. Mo aliquots were dissolved in 6MHCl, loaded
on 2 mL of Biorad AG1-X8 resin that had been cleaned with
1 M HCl and conditioned with 6 M HCl, rinsed with 30 mL of
6 M HCl, then eluted with 40 mL of 1 M HCl. The eluents
were dried and then passed through a cation resin (Biorad
AG50W-X8) in 0.5 M HCl to remove Fe.
After digestion, the Fe samples were dissolved in 7 M HCl
for Fe purification. A small fraction of each sample was
reserved for yield determination, and the rest was loaded onto
0.5 mL of Biorad AGMP-1 M resin that had been cleaned in
0.5 M HCl and pre-conditioned with 7 M HCl. The sample
was rinsed with 5 mL of 7 MHCl and then eluted with 5 mL of
0.5 M HCl. The eluent was dried down and dissolved in 7 M
HCl for a second column pass with fresh resin. After the
second elution was dried down, a post-chemistry yield aliquot
was drawn from each sample, and the remainders were
dissolved in 0.32 M HNO3 for isotopic analysis. Yield samples
were diluted with 0.32 M HNO3 and analyzed by quadrupole
ICP-MS (X Series, Thermo Scientific).
Isotope compositions were measured by MC-ICP-MS (Nep-
tune, Thermo Scientific) using methods similar to those de-
scribed in Barling et al.2 and Anbar et al.,27 adapted to the
Neptune. Briefly, for Mo, samples and standards were mea-
sured in low-resolution mode at 200 mg L�1 Mo in 0.32 M
HNO3 and spiked with 100 mg L�1 Zr of known isotopic
composition for internal mass bias correction. We compen-
sated for drift in the relative fractionation behaviors of Mo
and Zr using sample–standard bracketing. Since no interna-
tional standard yet exists for Mo isotopes, the standard used
was a commercial ICP solution (‘‘RochMo,’’ Alfa Aesar
Specpure Lot# 802309E). Data quality control was ensured
by measuring several isotope pairs (92Mo/95Mo, 96Mo/95Mo,97Mo/95Mo, 98Mo/95Mo, 100Mo/95Mo) and verifying that all
ratios varied in accordance with mass-dependent fractiona-
tion. Specifically, data were accepted only when (1) d97/95Mo/2
and d98/95Mo/3 differed by o0.05% per amu and (2) the
maximum and minimum of d97/95Mo/2, d98/95Mo/3, and
Table 1 Experimental results
Sample[Fe] in medium, mgL�1 � 103 (%RSD)
Fraction ofFe remainingin medium
d56/54Fe medium(%) (2s), relativeto IRMM014
[Mo] in medium, mgL�1 � 106 (%RSD)
Fraction ofMo remainingin medium
d97/95Mo medium(%) (2s), relative toRochMo
N-rich medium 1.22 (2.1)a — 0.52 (—)bc 79.4 (1.8)a — 0.07 (0.07)b
N-poor medium 1.26 (0.7)a — 0.52 (—)bc 108.7 (4.0)a — 0.07 (0.07)b
NR_1 1.07 (1.8) 0.87 0.42 (0.01) 37.0 (5.4) 0.47NR_2 0.98 (2.4) 0.80 0.42 (0.09) 38.8 (3.2) 0.49NR_3 1.07 (5.4) 0.87 41.6 (2.3) 0.52 0.31 (0.04)NR_4 0.93 (0.8) 0.76 0.41 (0.04) 45.7 (1.2) 0.58 0.23 (—)c
NR_5 0.96 (2.1) 0.78 46.9 (3.3) 0.59 0.28 (—)c
NR_6 1.06 (2.3) 0.87 0.40 (0.01) 55.9 (1.4) 0.70 0.25 (0.07)NP_1 0.51 (2.3) 0.41 �0.51 (0.08) 103.9 (3.4) 0.96NP_2 0.67 (1.2) 0.53 0.09 (0.01) 102.5 (2.3) 0.94 0.14 (0.01)NP_3 0.88 (2.2) 0.70 0.08 (0.08) 96.7 (4.1) 0.89 0.18 (0.06)NP_4 0.77 (4.0) 0.61 �0.13 (0.06) 98.8 (4.9) 0.91 0.14 (0.04)NP_5 0.94 (1.7) 0.74 0.07 (0.03) 104.2 (3.3) 0.96 0.16 (0.04)NP_6 0.51 (2.5) 0.41 �0.47 (0.04) 102.9 (2.1) 0.95 0.19 (0.02)
a Concentrations measured in media prior to growth. b Initial isotopic compositions were measured on Fe citrate and Na2MoO4 reagents.c Where
no value for 2s is given only one good analysis was available.
906 | J. Anal. At. Spectrom., 2007, 22, 905–910 This journal is �c The Royal Society of Chemistry 2007
d100/95Mo/5 differed by r0.05% per amu, where
d9x=95Mosample ¼
9xMo�95Mo
� �sample
9xMo=95Mo
� �standard
� 1
0B@
1CA� 1000 ð1Þ
Data were also rejected if the mass bias correction on a
particular sample was too large, i.e., if the difference between
the uncorrected and mass-bias-corrected values exceeded
0.5%. Such large differences reflect the effects of sample matrix
on instrument mass bias and indicate inadequate sample
purification. In addition, in-house rock and gravimetric stan-
dards were run repeatedly in each session to verify agreement
with long-term averages. Analytical precision with this method
is generally �0.10% for d97/95Mo. Our quality control criteria
result from a few years’ experience and hundreds of analyses
of RochMo, SDO shale, and gravimetric standards; analyses
satisfying the criteria above reproduce long-term averages
accurately and precisely.
Iron isotope compositions were measured using procedures
similar to those reported by Arnold et al.,1 which take
advantage of the high resolution capabilities of the Neptune
to resolve interferences from 40Ar16O+, 40Ar14N+ and40Ar16OH+. Samples were diluted to 3 mg L�1 in 0.32 M
HNO3 and spiked with 3 mg L�1 Cu standard for internal
mass bias correction. Samples were bracketed and measured
relative to an in-house ICP Fe standard (Ultra Scientific, Lot#
F00196). Data were accepted (1) if the absolute standard
deviation on 56Fe/54Fe was o10�4 (to verify stability of
interference resolution), (2) if d56/54Fe/2 and d57/54Fe/3 differedby r0.05% per amu, where
d5x=54Fesample ¼
5xFe�54Fe
� �sample
5xFe=54Fe
� �standard
� 1
0B@
1CA� 1000 ð2Þ
and (3) the difference between the uncorrected and mass-bias-
corrected d56/54Fe values was r0.5%. We also analyzed
gravimetric standards and a rock standard to ensure that the
values attained in each analysis session were consistent with
long-term averages. In general our method yields internal
precision of 0.05% in d56/54Fe.
Results
Concentrations of Mo and Fe in the initial media and remain-
ing in each medium at the end of the experiments are given in
Table 1. Also listed are the fractions of initial Mo and Fe
remaining in the media, computed by dividing the concentra-
tion in each sample by that in the corresponding initial
medium. All Mo and Fe not in the filtered media is assumed
to be associated with the cell pellets. The Mo concentration in
the N-rich medium was 79 mg L�1 (�1.8% relative standard
deviation, %RSD), and the fraction remaining in solution
varied from 0.47 to 0.70. The N-poor medium had 109 mg L�1
Mo (�4.0% RSD), and the fraction in the inoculated repli-
cates ranged from 0.89 to 0.96. The Fe concentration in the N-
rich medium was 1.22 mg L�1 (�2.1% RSD), and the fraction
remaining in solution in the inoculated bottles ranged from
0.76 to 0.87. The N-poor medium had 1.26 mg L�1 Fe (�0.7%
RSD), and the fraction remaining in solution ranged from 0.41
to 0.74. Table 1 also reports isotopic results as d56/54Fe and
d97/95Mo. Where numbers are not reported, the ion exchange
column yields were unacceptable (o92%).
Mo isotopic compositions of the solutions are plotted in
Fig. 1 as a function of the fraction of Mo remaining in solution
for each of the replicates. Error bars indicate analytical error
at the 2s level for two or more measurements on separate days
of the same sample. Where not shown, error bars are smaller
than the symbol, although in a few cases we had enough
solution for only one good measurement. These samples lack a
value of 2s in Table 1. Values of d97/95Mo for the filtered
media from inoculated replicates range from 0.14% in the
cultures with the most Mo remaining in solution to 0.31% in
that with the least. Thus, the media became progressively
heavier, apparently because isotopically light Mo was removed
from solution.
Fe isotopic compositions are shown in Fig. 2. Although
samples were run against an ICP Fe standard, all values are
reported relative to IRMM-014, which was analyzed multiple
times in each session with an average value of d56/54Fe =
+0.27% relative to the ICP standard. Filtered media from the
inoculated replicates had values from 0.42% for the solution
with the highest concentration of Fe to �0.47% for that with
the lowest concentration of Fe remaining. Thus, the media
became depleted in heavy Fe isotopes as the amount of Fe
associated with the cells increased.
Discussion
Molybdenum
Our experiments indicate that lighter isotopes of Mo are
preferentially associated with the cells, while heavier isotopes
tend to remain in solution. It is likely that the Mo associated
with the cell mass in our experiments was assimilated into the
Fig. 1 Isotopic composition (d97/95Mo, in permil, relative to an in-
house standard, RochMo) of filtered media in A. vinelandii growth
experiments versus fraction of Mo remaining in media. Error bars are
analytical errors at the 2s level, except where only one good analysis
was available (see Table 1). Curve is best-fit Rayleigh curve to
experimental data (see eqn (4)), indicating a fractionation between
medium and cell pellets of D97/95Mocells-medium =�0.3% (Bequivalent
to a = 0.9997).
This journal is �c The Royal Society of Chemistry 2007 J. Anal. At. Spectrom., 2007, 22, 905–910 | 907
cells. Another possibility is adsorption onto cell membranes.
Adsorptive processes are unlikely to dominate, however,
because aqueous Mo occurs as the negatively charged molyb-
date ion, MoO42�, and cell walls are also negatively
charged.28,29 However, Fe (oxyhydr)oxides, which have posi-
tively charged surfaces, could possibly precipitate on or near
cell walls, followed by subsequent adsorption of Mo. Experi-
mental studies to date are inconclusive as to whether Fe
(oxyhydr)oxides precipitate on cell membranes in otherwise
undersaturated solutions (Wightman and Fein,30 and refer-
ences therein), so Fe (oxyhydr)oxide formation may be possi-
ble in the microenvironment surrounding cells. Also unknown
is whether Mo isotopes fractionate during adsorption to Fe
(oxyhydr)oxides, although preliminary experimental work by
Malinovsky et al.21 indicates little or no fractionation during
adsorption to ferrihydrite. Despite negatively charged sur-
faces, Mo is known to adsorb to Mn oxyhydroxides, with
lighter isotopes preferentially adsorbed,18,31 but the current
system contains no Mn.
In order to estimate the fractionation factor (a) between Mo
in the cell pellet and Mo remaining in the media for our
experiments, we modeled the data using the Rayleigh fractio-
nation equation
97=95Momedium
97=95Momedium0
¼ fða�1Þmedium ð3Þ
where fmedium is the fraction of Mo remaining in the medium,97/95Momedium is the ratio 97Mo/95Mo in the medium,97/95Momedium is 97Mo/95Mo in the starting medium (at
fmedium = 1), and
a ¼97=95Mocells
97=95Momedium
ð4Þ
This equation assumes irreversible loss of reaction product
from the reactant pool, as expected for metal assimilation. The
best-fit curve, shown in Fig. 1, shows a very good correspon-
dence with Rayleigh fractionation if acells-medium = 0.9997 (or
Dcells-medium = d97/95Mocells � d97/95Momedium B�0.3%, given
that d97/95Mocells � d97/95Momedium B1000 ln acells-medium).
This result is in good agreement with the isotope effects
observed in our previous pilot study on A. vinelandii as well
as by other workers studying Trichodesmium sp. IMS 101.14,32
This fractionation may result from changes in Mo coordi-
nation that occur during assimilation.14 A. vinelandii can
produce several siderophores (molecules secreted by micro-
organisms for the purpose of binding Fe), but previous work
suggests that some of these molecules can also act to aid in Mo
acquisition.33–36 Liermann et al.14 demonstrated that in Fe-
replete, Mo-deficient medium the presence of A. vinelandii
strain OP (able to produce siderophores) resulted in more Mo
release from a Mo-doped glass than presence of a mutant
strain (P100) unable to produce catecholate siderophores,
strongly suggesting that at least one catecholate molecule
was behaving as a ‘‘molybdophore’’.14,37 The fractionation
observed by Liermann et al.14 (d97/95Mocells � d97/95Momedium
B�0.4%) was hypothesized to involve coordination changes
as Mo initially chelated by a molybdophore, possibly amino-
chelin, was subsequently bound to the ModA transport pro-
tein for uptake.38 Catecholate ligands produced by A.
vinelandii such as aminochelin likely bind MoO22+ such that
Mo is in six-fold coordination.39 The equilibrium formation
constant for this complex is very large, of the order of 1035,35
indicating a strong bonding environment. Mo must subse-
quently be transferred to the ModA protein, which binds Mo
in four-fold coordination, as the first step in cellular uptake.
This change in immediate bonding environment has the
potential to drive a significant isotope effect such as we ob-
serve. The stronger bonding environment of the aminochelin–
Mo complex will tend to retain the heavier isotopes, while
lighter ones preferentially partition to ModA and are assimi-
lated into the cell.
In our current experiments, however, all media were Fe-
replete and Mo-replete, at least initially, so production of
siderophores should have been absent or limited. However, A.
vinelandii also produces dihydroxybenzoic acid, regardless of
Fe concentration.40,41 This molecule forms stable complexes
with Mo in six- or seven-fold coordination.42,43 Thus, even in
the absence of true siderophores, it is possible that partitioning
of Mo between a chelator such as dihydroxylbenzoic acid and
the ModA transport protein could cause the observed isotope
fractionation.
Another, simpler, explanation for our results may be a
kinetic isotope effect due to relatively faster rates of reaction
for light isotopes of Mo compared with heavy isotopes. Many
organisms have been shown previously to fractionate isotopes
of carbon and nitrogen kinetically during uptake, with lighter
isotopes preferentially being assimilated (reviewed by
Hoefs44). Further investigation is necessary to differentiate
between these possibilities and to constrain the mechanism
behind the fractionation we report.
Iron
In contrast to the sense of fractionation for Mo, we observed
the media becoming depleted in heavy isotopes of Fe as Fe was
Fig. 2 Isotopic composition (d56/54Fe, in permil, relative to IRMM-
014) of filtered media in A. vinelandii growth experiments versus
fraction of Fe remaining in media. Error bars are analytical errors
at the 2s level. The curve is the best-fit Rayleigh curve (see eqn (4)) to
experimental data, indicating a fractionation between medium and cell
pellets of D56/54Fecells-medium = 1.1% (Bequivalent to a = 1.0011).
908 | J. Anal. At. Spectrom., 2007, 22, 905–910 This journal is �c The Royal Society of Chemistry 2007
removed (Fig. 2). Thus, heavier isotopes of Fe are preferen-
tially associated with the cell pellets, as also observed or
inferred by Brantley et al.5,13 for other heterotrophic soil
micro-organisms. We modeled the Fe data collected here with
the Rayleigh equation, as explained for Mo, and the best-fit
curve is shown alongside the data in Fig. 2. The fractionation
factor corresponding to this curve is acells-medium = 1.0011;
(thus Dcells-medium = d56/54Fecells–d56/54Femedium E +1.1%).
We note that this is similar in sense and magnitude to the
isotope fractionation observed during microbially mediated,
dissimilatory reduction of Fe oxides3,45 and during oxida-
tion of ferrous Fe by anoxygenic photoautotrophs.46
However, such Fe metabolisms were not active in our experi-
ments. Because the sense of fractionation is opposite to
that observed for Mo, a simple kinetic fractionation during
biological uptake cannot explain the Fe isotope results.
Rather, it is possible that at least one step in the Fe uptake
process involves an equilibrium Fe isotope effect. Although
siderophore molecules are not necessary for Fe acquisition in
our experiments, if they are being produced, then some
Fe will certainly be strongly bound by those molecules.
Furthermore, A. vinelandii is known to produce at least
one constitutive chelator (always produced, regardless of
Fe concentration), dihydroxybenzoic acid, with a high bind-
ing affinity of 1025.40,41,47 Fe was introduced as Fe-citrate in
our media. Equilibrium fractionation between citrate-bound
Fe and Fe bound to stronger chelators may result in an
isotopically light pool of Fe-citrate and a heavier pool of Fe
bound to stronger chelators, because of the expectation that
isotopically heavy Fe partitions into the stronger binding
environment. Subsequent uptake of strongly-bound Fe che-
lates would then result in isotopically heavy Fe in the
cells, while lighter Fe remained in the medium, as observed
in the current study and by Brantley et al.5,13 This mechanism
requires that Fe-citrate and other Fe–ligand complexes
can equilibrate isotopically, a hypothesis that can be tested
experimentally.
Again, we must consider the possibility that at least some Fe
is adsorbing to cell walls rather than being assimilated.
Although most Fe will be chelated, a very small amount of
free Fe is present as cations in solution, and these could be
electrostatically attracted to the negatively charged outer
membranes of A. vinelandii. Fe might also adsorb to Fe
(oxyhydr)oxides. Indeed, Icopini et al.48 measured a fractiona-
tion D56/54Feadsorbed-dissolved = +0.8% between Fe2+ ad-
sorbed to goethite surfaces and Fe2+ remaining in solution.
In those experiments, Icopini et al. observed sorption of Fe2+
both to Shewanella cells and to goethite, but the extent of
sorption to cells was not large enough to produce observable
isotopic fractionation. As with any adsorbent, the extent of
sorption is a function of the aqueous concentration of adsor-
bate, the surface area of adsorbent, and the nature of interac-
tion between adsorbate and adsorbent. However,
fractionation during sorption to goethite was attributed to
effects specific to the goethite surface and may even have been
affected by diffusion of ferrous iron into the goethite under
anaerobic conditions. It is important to note that in our
experiments reported here, very little of the Fe would have
been Fe2+.
Concluding remarks
These experiments represent the first steps in a systematic
effort to study Mo and Fe isotope fractionation during
microbial metal assimilation of these bioessential metals.
Our findings demonstrate that current-generation MC-ICP-
MS instrumentation can detect variations in metal isotope
composition during microbial growth which occur when mi-
crobes remove metals from solution and that this fractionation
may be a consequence of assimilatory mechanisms. In the case
of Mo, these results extend a very small set of prior data on
nitrogen-fixing bacteria.14,32 In the case of Fe, the data are
novel; all prior studies of biologically associated Fe isotope
fractionation involved bacteria that actively oxidized or re-
duced Fe as part of their energetic metabolism.
At this stage we cannot yet unambiguously interpret the
fractionation mechanisms in these experiments. In particular,
careful work is needed to differentiate between effects arising
from metal uptake due to active microbial assimilation versus
passive adsorption of metals on cell walls, particularly if Fe
(oxyhydr)oxide coatings are present. However, it is clear that
high precision metal isotope fractionation measurements point
the way toward a new means of studying microbial metal
processing in the environment. Such research is also likely to
provide novel tools to study the mechanisms by which organ-
isms incorporate metals into their metallomes.
Acknowledgements
This work was supported by the National Science Foundation
Grant EAR-0519347 to ADA, LEW, and SLB. Isotopic
analyses were conducted in the W. M. Keck Foundation
Laboratory for Environmental Biogeochemistry at Arizona
State University.
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