Isotope fractionation during microbial metal uptake measured by MC-ICP-MS

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: laura.w@asu.edu

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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