REVIEW

Opportunities in multidimensional trace metal imaging:taking copper-associated disease research to the next level

Stefan Vogt & Martina Ralle

Received: 9 July 2012 /Revised: 7 September 2012 /Accepted: 18 September 2012 /Published online: 19 October 2012# Springer-Verlag Berlin Heidelberg 2012

Abstract Copper plays an important role in numerous bio-logical processes across all living systems predominantlybecause of its versatile redox behavior. Cellular copperhomeostasis is tightly regulated and disturbances lead tosevere disorders such as Wilson disease and Menkes dis-ease. Age-related changes of copper metabolism have beenimplicated in other neurodegenerative disorders such asAlzheimer disease. The role of copper in these diseaseshas been a topic of mostly bioinorganic research efforts formore than a decade, metal–protein interactions have beencharacterized, and cellular copper pathways have been de-scribed. Despite these efforts, crucial aspects of how copperis associated with Alzheimer disease, for example, are stillonly poorly understood. To take metal-related disease re-search to the next level, emerging multidimensional imagingtechniques are now revealing the copper metallome as thebasis to better understand disease mechanisms. This reviewdescribes how recent advances in X-ray fluorescence mi-croscopy and fluorescent copper probes have started tocontribute to this field, specifically in Wilson disease andAlzheimer disease. It furthermore provides an overview ofcurrent developments and future applications in X-ray mi-croscopic methods.

Keywords Imaging . X-ray . Fluorescence . Copper .

Neurological disease

AbbreviationsAβ Amyloid βFTIRM Fourier transform infrared

microspectroscopyLA-ICPMS Laser ablation inductively

coupled mass spectrometryPIXE Proton-induced X-ray emissionSIMS Secondary ion mass spectrometryXANES X-ray absorption near-edge spectroscopyXFM X-ray fluorescence microscopyXRM X-ray microscopy

Introduction

Metals and their involvement in health and disease are of ever-growing importance in biomedical research. Lighter elementssuch as calcium participate in complex signaling pathwaysthat are still being unraveled, whereas transition metals areoften cofactors in proteins, where they undergo chemical statealterations (i.e., redox reactions) in the enzymatic reactionsthey facilitate. Although the reactivity of metals is of great usefor facilitating enzymatic reactions, it also requires strict con-trol to mitigate toxicity. Therefore, metal homeostasis is tight-ly managed by cells and any disturbance leads to severeconsequences. A comprehensive metallomic study would typ-ically entail choosing a pathway for a specific metal and theninvestigating metal concentration, distribution, and metal–protein binding as well as changes thereof in healthy individ-uals and in disease. Whereas measuring bulk metal concen-trations and exploring metal–protein interactions employestablished methods and instrumentation, reliable, user-friendly techniques to produce spatially resolved data onmetalcontent in biological specimens have largely emerged only

Published in the topical collection Metallomics with guest editors UweKarst and Michael Sperling.

S. VogtX-ray Science Division, Advanced Photon Source,Argonne National Laboratory,9700 S. Cass Avenue,Argonne, IL 60439, USA

M. Ralle (*)Department of Biochemistry and Molecular Biology,Oregon Health & Science University,3181 SW Sam Jackson Park Rd,Portland, OR 97239, USAe-mail: rallem@ohsu.edu

Anal Bioanal Chem (2013) 405:1809–1820DOI 10.1007/s00216-012-6437-1

during the past 10 years. Examples are synchrotron-based X-ray fluorescence microscopy (XFM), fluorescent metal sen-sors, secondary ion mass spectrometry (SIMS) and nano-SIMS, laser ablation inductively coupled mass spectrometry(LA-ICPMS), electron probe X-ray microanalysis (EPXMA),and proton-induced X-ray emission (PIXE) (for a recent re-view of these methods and their application to metallomicstudies in neurodegenerative diseases, see [1]).

This report will review recent progress in investigation ofthe metallome of mammalian copper with respect to unravel-ing the disease mechanisms of two human disorders: Wilsondisease (WD) and Alzheimer disease. We will specificallydiscuss contributions to this field made by imaging techni-ques, predominantly XFM studies and fluorescent coppersensors. We will furthermore describe new methods anddevelopments in X-ray fluorescence tomography, XFM imag-ing at the nanoscale, and provide a perspective on samplepreparation.

Imaging methods

X-ray-based microscopy

X-ray microscopy (XRM) and specifically synchrotron-basedXRM methods are increasingly used either alone or in com-bination with other correlative techniques to image cellular,organelle, or elemental structures in biology. The advantageof using X-rays in biological imaging is based on their pen-etration depth, which allows imaging of cells or tissue close toor in the native state rather than bulk or homogenized andfractionated samples. Advances in focusing optics haveresulted in microscopes with 20-nm resolution, and tomo-graphic reconstructions allow three-dimensional imaging.Some correlative approaches such as fluorescent microscopyallow targeting of regions of interest prior to measurement,whereas others, such as Fourier transform infrared microspec-troscopy (FTIRM), are used to complement XRM informa-tion with chemical properties.

Two main classes of XRM outline the two major appli-cations. Soft X-rays (2–5-nm wavelength) are used to mea-sure the intrinsic phase contrast of lighter elements, i.e.,detecting cellular and intracellular structures such as organ-elles. Detection is performed in transmission mode, with thedetector located in-line with the X-rays and the sample.Because of limited penetration of soft X-rays, samples haveto be about 10 μm. Hard X-rays in combination withZernike phase contrast techniques enable users to performX-ray tomography on thicker samples (more than 10 μm).The resulting hard X-ray phase contrast images are oftenused in combination with XFM, a technique that will yieldquantitative information with high spatial resolution on theelemental distribution (see later).

Chemical state mapping (X-ray absorption near-edgespectroscopy)

X-ray absorption near-edge spectroscopy (μXANES) is anattractive method to gain information about an element’sredox status when imaging biological material. To deter-mine oxidation states of metals, a stationary focused X-raybeam (usually 5μm × 5μm) is applied to a target cell ortissue sample and the absorption of X-ray photons is mon-itored while scanning the energy through the X-ray absorp-tion edge of the element under investigation. The position ofthe edge (its first inflection point) and the shape of the edge(specifically “pre-edge features”) contain information aboutthe oxidation state and often the coordination sphere. Dataanalysis is still almost exclusively performed in an empiricmanner by comparing experimental data with measuredmodel compounds. Several groups have successfully deter-mined the oxidation states of mostly iron and copper inbiological specimens [2–4] (for a recent review, see Ortegaet al. [5]). A nice example is a more recent publication byGlasauer et al. [2], who studied the oxidation state of iron incytoplasmic granules in Shewanella putrefaciens that areformed during anaerobic respiration and found iron to bein a mixed-valence state. Yang et al. [4] determined theoxidation state of copper in a mouse fibroblast cell line thathad been exposed to 200 μM CuCl2. Their results indicatedthat copper is predominantly present as Cu(I) in a linear totrigonal coordination arrangement with mostly sulfur.

Although the experimental setup and data analysis forμ-XANES is straightforward, literature examples are stillrare. Longer dwell times (the sample remains stationaryduring a 5–10-min scan) with highly brilliant, focused X-rays induce significant photoreduction and radiation dam-age, and XANES measurements therefore have to be evaluat-ed carefully. Lastly, even a small beam size of 5μm× 5μmwillcontain multiple proteins and the resulting XANES spectrumwill be a representation of the average oxidation state, which ismostly likely dominated by a metals bound to a high-abundance storage protein.

X-ray fluorescence microscopy

XFM is part of the XRM super group and is rapidly becomingthe method of choice for an ever-growing community ofbioanalytical researchers to probe trace elements in biologicalsystems. It is used for both, quantitative visualization andanalysis of the local chemical state of elements of interest(for in depth reviews of XFM, see [5–12]). Briefly, hard X-rays (10 keVor greater), with an incident energy chosen to beabove the relevant absorption edges of the elements of inter-est, are focused onto the specimen, inner (K- or L-shell)electrons are knocked out, and the subsequent relaxationprocess causes X-ray fluorescence characteristic for the

1810 S. Vogt, M. Ralle

given) for the transgenic mice compared with 12 μg/g in 18-month-old control mice. The opposite was found in thethalamus (20 μg/g in transgenic mice versus 60 μg/g incontrol mice). They did not explain how they identifiedthe hippocampus or the thalamus in their brain sections,and it seems likely that areas that are known to be higherin copper (such as the periventricular areas) were accidentlyincluded in some of the calculations. XFM experimentsfrom our laboratory that also used a transgenic mouse modeldid not result in elevated copper content of the hippocam-pus; however, we did see a moderate increase in copperconcentration from presymptomatic mice (5 μg/g) to symp-tomatic mice (7 μg/g) (unpublished results).

Conclusions

Multidimensional, high-resolution imaging methods are be-ginning to elucidate the metallome of copper and its changesduring diseases processes. Particularly, XFM methods arerapidly evolving in terms of resolution, three-dimensionalcapabilities, and user-accessibility, and publications fromthe past 6 years have established a central role for XFM inmetallomic research. The involvement of copper in complexdisorders such as Alzheimer disease can only be determinedby taking this research to the next level, i.e., from one to twoor three dimensions. In combination with correlative techni-ques such as copper-sensing fluorescent probes or lower-resolution techniques such as LA-ICPMS, XFM will likelybecome the method of choice in the future to study copperpathways in biological systems.

Acknowledgments The authors gratefully acknowledge the use ofthe facilities at the Advanced Photon Source. This work was supportedby the National Institutes of Health grant GM090016 to M.R. The useof the Advanced Photon Source was supported by the US Departmentof Energy, Office of Science contract DE-AC-02-6CH11357.

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Hippea jasoniae sp. nov. and Hippea alviniaesp. nov., thermoacidophilic members of the classDeltaproteobacteria isolated from deep-seahydrothermal vent deposits

Gilberto E. Flores,1 Ryan C. Hunter,2 Yitai Liu,1 Anchelique Mets,3

Stefan Schouten3 and Anna-Louise Reysenbach1

Correspondence

Anna-Louise Reysenbach

reysenbacha@pdx.edu

1Department of Biology, Portland State University, Portland, OR 97201, USA

2Howard Hughes Medical Institute and Division of Biological Sciences, California Institute ofTechnology, Pasadena, CA 91125, USA

3NIOZ Royal Netherlands Institute for Sea Research, Department of Marine OrganicBiogeochemistry, PO Box 59, 1790 B, Den Burg, The Netherlands

Thirteen novel, obligately anaerobic, thermoacidophilic bacteria were isolated from deep-sea

hydrothermal vent sites. Four of the strains, designated EP5-rT, KM1, Mar08-272rT and Mar08-

368r, were selected for metabolic and physiological characterization. With the exception of strain

EP5-rT, all strains were short rods that grew between 40 and 72 6C, with optimal growth at 60–

65 6C. Strain EP5-rT was more ovoid in shape and grew between 45 and 75 6C, with optimum

growth at 60 6C. The pH range for growth of all the isolates was between pH 3.5 and 5.5

(optimum pH 4.5 to 5.0). Strain Mar08-272rT could only grow up to pH 5.0. Elemental sulfur was

required for heterotrophic growth on acetate, succinate, Casamino acids and yeast extract.

Strains EP5-rT, Mar08-272rT and Mar08-368r could also use fumarate, while strains EP5-rT, KM1

and Mar08-272rT could also use propionate. All isolates were able to grow chemolithotrophically

on H2, CO2, sulfur and vitamins. Phylogenetic analysis of 16S rRNA gene sequences placed all

isolates within the family Desulfurellaceae of the class Deltaproteobacteria, with the closest

cultured relative being Hippea maritima MH2T (~95–98 % gene sequence similarity).

Phylogenetic analysis also identified several isolates with at least one intervening sequence within

the 16S rRNA gene. The genomic DNA G+C contents of strains EP5-rT, KM1, Mar08-272rT and

Mar08-368r were 37.1, 42.0, 35.6 and 37.9 mol%, respectively. The new isolates differed most

significantly from H. maritima MH2T in their phylogenetic placement and in that they were obligate

thermoacidophiles. Based on these phylogenetic and phenotypic properties, the following two

novel species are proposed: Hippea jasoniae sp. nov. (type strain Mar08-272rT5DSM

24585T5OCM 985T) and Hippea alviniae sp. nov. (type strain EP5-rT5DSM 24586T5OCM

986T).

Members of the class Deltaproteobacteria are physiologi-cally diverse with eight described orders and thermophilesisolated from all of them (Kuever et al., 2005). The orderDesulfurellales, however, is the only order whose membersare exclusively thermophiles. There is one family within

this order, the family Desulfurellaceae, represented by twogenera, Desulfurella and Hippea. Four species of the genusDesulfurella have been formally described from terrestrialthermal environments. All are neutrophiles that growoptimally between 52 uC and 60 uC, and conserve energyby sulfur or thiosulfate respiration or by pyruvatefermentation (Miroshnichenko et al., 1998). The genusHippea, on the other hand, is currently represented by onerecognized species, Hippea maritima MH2

T, which wasisolated from a shallow marine vent off the coast of PapuaNew Guinea (Miroshnichenko et al., 1999). H. maritimaMH2

T is neutrophilic to moderately acidophilic (pH 5.7–6.5) and requires salt, yeast extract and sulfur for growth.

Abbreviations: ELSC, Eastern Lau Spreading Center; EPR, East PacificRise; GB, Guaymas Basin; IVS, intervening sequence(s); MAR, Mid-Atlantic Ridge.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA genesequences reported in this paper are FR754496–FR754508.

Supplementary figures and tables are available with the online version ofthis paper.

International Journal of Systematic and Evolutionary Microbiology (2012), 62, 1252–1258 DOI 10.1099/ijs.0.033001-0

1252 033001 G 2012 IUMS Printed in Great Britain

without elemental sulfur, indicating that they are notfermentative. Yeast extract was not required for growth ifmedia was supplemented with vitamins. All isolates werealso capable of autotrophic growth with H2, CO2, sulfurand vitamins.

For morphological analysis by thin-section transmissionelectron microscopy, cells were prepared as previouslydescribed (Hunter & Beveridge, 2005). For negativelystained whole mounts, 20 ml drops of culture were allowedto adsorb onto the copper grids for 2 min and weresubsequently post-stained in 2 % uranyl acetate. IsolatesMar08-272rT (Fig. 2a), Mar08-368r (Fig. 2b) and KM1(Fig. 2c) were rod-shaped, while isolate EP5-rT was moreovoid in shape (Fig. 2d). The cell dimensions of eachisolate varied (Table 1). Strains were all Gram-negative and

the peptidoglycan sacculi were often difficult to visualize(white arrow, Fig. 2d). No intracellular membrane struc-tures were observed, however strains Mar08-272rT, Mar08-368r and KM1 contained electron-dense intracellulargranules adjacent to the cytoplasmic membrane (blackarrows, Fig. 2). These inclusions closely resembled theintracellular mixed-valence iron granules formed by She-wanella putrefaciens CN32 under anaerobic conditions(Glasauer et al., 2007). Consistent with this observation,when the new strains were centrifuged for DNA extrac-tion, small black precipitates were observed in cell pellets.Negative stains of each isolate revealed polar flagellaextending from the surfaces of most cells (Figs 2e, f).

The physiological and phylogenetic characteristics of thenewly described isolates were distinct from H. maritima

Fig. 2. Thin section transmission electronmicrographs of strains (a) Mar08-272rT,(b) Mar08-368r, (c) KM1 and (d) EP5rT.Negatively stained cells of strains Mar08-368r (e) and EP5rT (f) show flagella. Blackarrows in (a), (b) and (c) point to areas whereelectron-dense granules are evident. The whitearrow in (d) shows where the peptidoglycansacculus is visible. Bars: a, b, c, 200 nm; d,100 nm; e, f, 500 nm.

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1256 International Journal of Systematic and Evolutionary Microbiology 62

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Direct speciation analysis of inorganic elements in single cells using X-rayabsorption spectroscopy†

Richard Ortega*ab

Received 9th September 2010, Accepted 11th November 2010

DOI: 10.1039/c0ja00153h

Up to now, X-ray absorption spectroscopy (XAS), which includes XANES (X-ray absorption near

edge structure) and EXAFS (Extended X-ray absorption fine structure), was mainly used with

unfocused beams. The recent development of X-ray focusing optics, with spatial resolution down to the

micrometre level while maintaining high photon fluxes, has opened the field of XAS application to

single cell analysis. Micro-XAS enables the characterization of the local structure of the elements within

subcellular structures in terms of oxidation state, site ligation, and coordination. It has been mainly

applied to the determination of trace metal oxidation states using micro-XANES, and in some

favorable cases to identify the molecules binding to the metals in cells. Due to the minute quantity of

analytes in subcellular compartments, micro-EXAFS is still more challenging to perform than micro-

XANES as it requires higher signal to noise ratios. Micro-XAS has proven to be useful in various fields

of research such as metal-based neuro-degeneration, cellular pharmacology, trace element physiology

and metal toxicology.Micro-XAS presents unique capabilities over other speciation methods because it

can be performed in situ, directly in subcellular compartments, without cell fractionation which is prone

to modify the chemical element species. However, the stability of chemical element species all along the

analytical procedure, from sample preparation to storage and analysis should be tested systematically.

Introduction

Mechanisms of metal bioavailability, homeostasis, toxicity or

even pharmacology are governed by the chemical state of the

elements within cells. A challenging issue in speciation analysis is

the direct determination of chemical element species in single

cells. Such information is needed to understand the biochemistry

of trace metals in cells. The recent development of highly focused

hard X-ray synchrotron beams has opened the application of

X-ray absorption spectroscopy (XAS) to microscopic samples

such as single cells. Since the development of synchrotron

aUniversit�es de Bordeaux 1 & 2, Laboratoire de Chimie Nucl�eaireAnalytique et Bioenvironnementale, Groupe d’Imagerie ChimiqueCellulaire et Sp�eciation, Chemin du solarium, 33175 Gradignan, France.E-mail: ortega@cenbg.in2p3.fr; Fax: +33 557 120 900; Tel: +33 557 120907bCNRS, Laboratoire de Chimie Nucl�eaire Analytique etBioenvironnementale, Groupe d’Imagerie Chimique Cellulaire etSp�eciation, Chemin du solarium, 33175 Gradignan, France. E-mail:ortega@cenbg.in2p3.fr; Fax: +33 557 120 900; Tel: +33 557 120 907

† This article is part of a themed issue highlighting outstanding andemerging work in the area of speciation.

R: Ortega

Richard Ortega is research director at CNRS and Bordeaux University, France. His research focus is on

the development of analytical methods based on ion beams and synchrotron radiation for the imaging and

speciation of metals in cells and proteins. He obtained a Chemical engineer and Master Degree in

Chemistry from the University of Marseille (1991), and a Ph.D. in Physics at the University of

Bordeaux (1994). He was a Postdoctoral fellow with Pr. Bibudhendra Sarkar at the Research Institute

of the Hospital for Sick Children, Canada (1995). He was an invited scientist at the European

Synchrotron Radiation Facility (2001) and gained an award from the French Society of Chemistry in

Analytical Chemistry (2004).

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beamlines on most synchrotron radiation facilities. Micro-XAS

capabilities strongly depend on the characteristics of the X-ray

beamline where it is carried out. It can be estimated that in order

to perform chemical element speciation within single cells, the

beam size should be at least in the micrometre range size, or less,

while the intensity of the focused beam must be high enough to

perform X-ray absorption spectroscopy, with about 109 ph/s, or

more. For example, micro-XAS was performed on single cells at

ESRF on ID22 beamline with an experimental setup based on

Kirkpatrick-Baez X-ray focusing optics that maintains high flux

of incoming radiation (>1011 photons/s at 11.9 keV) at micro-

metric spatial resolution (1.5 � 4.0 mm2).5 With this experimental

setup, the limit of detection, as evaluated from the standard

deviation of eight blank measures, was 4.3 mg g�1. Similarly to the

concept of ‘‘limit of quantification’’ used for quantitative

analytical methods, a ‘‘limit of speciation’’ could be evaluated

and was about 13 mg g�1. These values are only indicative because

they strongly depend on the experimental setup (detection

system, distance from sample to detector,.) and also on the

element and the nature of the sample. However, the values are

helpful for a rough estimate of the feasibility of micro-XANES

experiments as similar detection levels are expected for elements

of atomic number between 20 and 40, because the cross section

for X-ray fluorescence and the detection efficiency do not vary

much for these elements. In this experiment, speciation analysis

was limited to the use of micro-XANES and not of micro-

EXAFS. The limit of speciation for micro-EXAFS has not been

measured exactly yet. The micro-XAS signal in single cells is low

compared to macroscopic analysis first because the elements of

interest, the trace metals, are usually diluted in the biological

samples, and second because of the minute quantity of matter in

a single cell. Therefore, XAS is often limited to the study of the

absorption edge region (XANES) in single cells giving informa-

tion about the oxidation state of the absorber element. The

EXAFS data treatment requires high signal to noise acquisitions

in order to fit the oscillations after the absorption edge which is

difficult to obtain in most cases on single cells. Only a few

examples of micro-EXAFS analysis in cells have been reported

so far (Table 1). Another important limitation of micro-XAS is

the influence of intense X-ray irradiation on element chemical

state stability during analysis. Photo-oxidation and photo-

reduction processes can occur during the irradiation. These

processes are variable depending on the element and its initial

oxidation state, and the analytical conditions: intensity of the

X-ray beam, temperature of the sample, type of environment i.e.,

presence of oxygen in air,. Low temperature analysis at liquid

nitrogen temperature is highly advocated but not necessarily

sufficient. A systematic analysis of proper reference material

must be performed, even when low temperature and neutral gas

environment is used. The reference material, usually chemical

compounds of known oxidation state, must be irradiated within

exactly the same analytical conditions as the samples to verify

that the element chemical state is not modified during analysis.5

The main application of micro-XAS in single cells is the study

of redox metals by micro-XANES. Oxidation state is an

important parameter that defines the bioavailability and intra-

cellular reactivity of biologically relevant metals. Micro-XANES

is probably the unique analytical method available to probe

in situ the oxidation state of trace elements within subcellular

compartments. One alternative method for oxidation state

determination in subcellular compartments is EELS (Electron

Energy Loss Spectroscopy) but very few examples of applica-

tions have been published,6 probably because sample prepara-

tion for EELS is very challenging. The size of the organelles that

can be probed using micro-XANES depends on the size of the

microbeam, typically with a 1 mm beam size it is possible to probe

the nucleus, cytoplasm, lysosomes, vesicles, and the mitochon-

drial network. This is a real advantage over other speciation

methods as it avoids cellular fractionation using differential

centrifugation that can lead to changes of chemical species

during sample preparation. The two first studies that demon-

strated the feasibility of micro-XANES on single cells were both

applied to the examination of Fe oxidation state.7,8 The redox

cycling of Fe, Fe(II)/Fe(III), is known to participate to the

oxidative stress through the Fenton reaction and could be

involved in a number of pathological processes such as neuro-

degenerative diseases9 or cancer.10 Since these early studies,

micro-XAS of single cells has been applied in studies of metal-

based neuro-degeneration, metal carcinogenesis, cellular phar-

macology of anticancer drugs, trace element physiology and

environmental metals speciation in prokaryotes. The next section

reviews the existing literature in order to illustrate with practical

cases what type of structural information can be obtained with

micro-XAS. The results are summarized in Table 1.

Speciation of redox metals in neuronal cells

Redox metals are suspected to be involved in the etiology of

number of neurodegenerative diseases such as Alzheimer’s

disease or Parkinson’s disease. A hyperoxidation process due to

Table 1 Applications of micro-XAS to element speciation in single cells

Element Chemical species identified Spectroscopy References

As As(III); As(V) m-XANES 5,19

Cr Cr(III); Cr(VI) m-XANES 16,17

Chromium phosphate m-XANES 27

Polynuclear Cr m-EXAFS 17

Cu Cu(I); Cu(II) m-XANES 15,23,25

Fe Fe(II); Fe(III) m-XANES 7,8,11,12,13,14,23,28,29

Fe in ferritin and magnetite m-XANES 14

Pt Pt(II); Pt(IV) m-XANES 21

V V(III); V(IV) m-XANES 24

Zn Zn(II) m-XANES 22,23

Zn binding to S m-XANES/EXAFS 26

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Faculteit Bio-ingenieurswetenschappen

Academiejaar 2010-2011

Unconventional electron donors andacceptors for microbial ecosystems

Frederiek - Maarten KerckhofPromotor: Prof. Dr. ir. Nico Boon en Prof. Dr. ir. Willy VerstraeteTutor: ir. Jan Arends

Masterproef voorgedragen tot het behalen van de graad vanMaster in de bio-ingenieurswetenschappen: optie Cel en Gen

biotechnologie

IV.7.3 EXPERIMENTAL TRACK 3: MICROBIAL FLEXIBILITY TOWARDS SOLID PHASE ELECTRONDONORS 129

protects metal surfaces from corrosion (Breur et al., 2002).Another hypothesis to explain the strange pattern of Fe+

2 accumulation removal after 90h might liewith cellular storage compounds. At first, although extensive washing was performed, some of thecells still contain storage (fermentable) organic compounds carried over from the seed culture thatserved as a source for initial nitrate reduction for the first 24 hours (as shown by nitrite accumu-lation, Figure 6.9). After depletion of the storage compounds the abiotically liberated dissolvediron was the next electron donor available and gets removed by the cells. The higher dissolvediron accumulation in biotic bottles than in abiotic controls after 24 hours could be explained byhydrogenotrophic denitrification in this scenario. One last possibility was the accumulation ofiron inside cells as ’iron lungs’ (Lovley, 2008a), storage precipitates for anaerobic respiration.The time necessary for ’iron lung’ accumulation in Shewanella species (72 hours, Glasauer et al.(2007)) seems in accordance with the dissappaerance of dissolved iron between 24 hours and 90hours. Analysis of an unfiltered sample with AAS might show if substantial amounts of iron accu-mulated inside the biomass.The fact that the electron balance could not be closed could reflect several removal effects of theformed Fe+

2 that are already described above. Apart from phosphate precipitation and the use aselectron donor for denitrification it is important to keep in mind that Fe(II) solubility is stronglyaffected by pH, and can readily precipitate in the nutrient medium (Shin & Cha, 2008). The mea-sured concentrations could hence not be accurate or account for all the Fe(II) liberated electrons,making it impossible to close the electron balance.

7.3.3 Further research

The use of energy-dispersive X-ray spectroscopy (EDX analysis) on the precipitate formed by thebiological samples could reveal the elemental composition and confirm if it consists of biologicalinduced iron phosphate precipitate (Breur et al., 2002). Microbial characterization of the biomassis necessary to determine community changes and diversity. The diversity within a microbial com-munity with ZVI as the only electron donor could be indicative for the microbial flexibility towardssolid phase respiration, even though the exact mechanisms (direct oxidation, abiotic hydrogen orFe(II) as intermediates) remain unclear in this approach. The follow-up of biomass concentra-tions and live/dead cell counts (by flowcytometry) in this test might also be interesting to assesssurvival of the community and formation of organic compounds by cell lysis that might serve aselectron donors for denitrification instead of the ZVI, the test should be conducted over a longertimeframe and pH should be assessed. As visual examination of the bottles did not show biofilmor aggregate formation, flowcytometry could also provide further insights in the suspended com-munity composition and be an ideal way to examine microbial flexibility in this setup because offast characterization and low sample volume requirements (Harnisch et al., 2011).

7.3.4 Perspectives

Using this experimental setup it is not possible to conclusively suggest direct solid phase respi-ration of ZVI. However if hydrogen can be considered as an electron shuttle indirect solid phase

REFERENCES 161

El-Naggar, M. Y., Wanger, G., Leung, K. M., Yuzvinsky, T. D., Southam, G., Yang, J., Lau, W. M.,Nealson, K. H. & Gorby, Y. A. (2010). Electrical transport along bacterial nanowires from She-wanella oneidensis MR-1. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCESOF THE UNITED STATES OF AMERICA, 107(42):18127–18131. ISSN 0027-8424.

Elias, D. A., Zane, G. M., Auer, M. A., Fields, M. W., Wall, J. D. & Gorby, Y. A. (2010). Candirect extracellular electron transfer occur in the absence of outer membrane cytochromes inDesulfovibrio vulgaris? GEOCHIMICA ET COSMOCHIMICA ACTA, 74(12, Suppl. 1):A263.ISSN 0016-7037. Conference on Goldschmidt 2010 - Earth, Energy, and the Environment,Knoxville, TN, JUN 13-18, 2010.

Ferrari, B. C. & Gillings, M. R. (2009). Cultivation of Fastidious Bacteria by Viability Staining andMicromanipulation in a Soil Substrate Membrane System. APPLIED AND ENVIRONMENTALMICROBIOLOGY, 75(10):3352–3354. ISSN 0099-2240.

Finkelstein, D. A., Tender, L. M. & Zeikus, J. G. (2006). Effect of electrode potential on electrode-reducing microbiota. ENVIRONMENTAL SCIENCE & TECHNOLOGY, 40(22):6990–6995.

Flemming, H.-C. & Wingender, J. (2010). The biofilm matrix. NATURE REVIEWS MICROBIOL-OGY, 8(9):623–633. ISSN 1740-1526.

Fornero, J. J., Rosenbaum, M. & Angenent, L. T. (2010). Electric Power Generation from Munic-ipal, Food, and Animal Wastewaters Using Microbial Fuel Cells. ELECTROANALYSIS, 22(7-8):832–843. ISSN 1040-0397.

Franks, A. E. & Nevin, K. P. (2010). Microbial Fuel Cells, A Current Review. ENERGIES,3(5):899–919. ISSN 1996-1073.

Franks, A. E., Nevin, K. P., Jia, H., Izallalen, M., Woodard, T. L. & Lovley, D. R. (2009). Novelstrategy for three-dimensional real-time imaging of microbial fuel cell communities: monitoringthe inhibitory effects of proton accumulation within the anode biofilm. ENERGY & ENVIRON-MENTAL SCIENCE, 2(1):113–119. ISSN 1754-5692.

Fredrickson, J. K., Romine, M. F., Beliaev, A. S., Auchtung, J. M., Driscoll, M. E., Gardner,T. S., Nealson, K. H., Osterman, A. L., Pinchuk, G., Reed, J. L., Rodionov, D. A., Rodrigues, J.L. M., Saffarini, D. A., Serres, M. H., Spormann, A. M., Zhulin, I. B. & Tiedje, J. M. (2008).Towards environmental systems biology of Shewanella. NATURE REVIEWS MICROBIOLOGY,6(8):592–603. ISSN 1740-1526.

Glasauer, S., Langley, S., Boyanov, A., Lai, B., Kemner, K. & Beveridge, T. J. (2007). Mixed-valence cytoplasmic iron granules are linked to anaerobic respiration. APPLIED AND ENVI-RONMENTAL MICROBIOLOGY, 73(3):993–996. ISSN 0099-2240.

Gonzalez Lopez, C. V., Ceron Garcia, M. d. C., Acien Fernandez, F. G., Bustos, C. S., Chisti,Y. & Fernandez Sevilla, J. M. (2010). Protein measurements of microalgal and cyanobacterialbiomass. BIORESOURCE TECHNOLOGY, 101(19):7587–7591. ISSN 0960-8524.

Gorby, Y. A. (2006). Bacterial nanowires: electrically conductive filaments and their implicationsfor energy transformation and distribution in natural and engineered systems. In Proc. Bio Microand Nanosystems Conf. BMN ’06.

Microbial Ecology of Active Marine Hydrothermal Vent Deposits: The Influence of

Geologic Setting on Microbial Communities

by

Gilberto Eugene Flores

A dissertation submitted in partial fulfillment of the

requirements for the degree of

Doctor of Philosophy in

Biology

Dissertation Committee: Anna-Louise Reysenbach, Chair

Michael Bartlett Radu Popa

Parmely H. Pritchard Mircea Podar

Dirk Iwata-Reuyl

Portland State University © 2011

104

negatively-stained whole mounts, 20 μL drops of culture were allowed to adsorb onto

the copper grids for 2 min and were subsequently post-stained in 2% uranyl acetate.

Mar08-272rT (Figure 4.3a), Mar08-368r (Figure 4.3b), and KM1 (Figure 4.3c) were rod

shaped, while EP5-rT was more ovoid in shape (Figure 4.3d). Cell dimensions of each

isolate varied (Table 4.3). Strains were all Gram-negative and the peptidoglycan sacculi

were often difficult to visualize (white arrow, Figure 4.3d). No intracellular membrane

structures were observed, however strains Mar08-272rT, Mar08-368r, and KM1

contained electron dense intracellular granules adjacent to the cytoplasmic membrane

(black arrows, Figure 4.3). These inclusions closely resemble intracellular mixed-

valence iron granules formed by Shewanella putrefaciens CN32 under anaerobic

conditions (Glasauer et al., 2007). Consistent with this observation, when these strains

were centrifuged for DNA extraction, small black precipitates were observed in cell

pellets. Negative stains of each isolate revealed polar flagella extending from the

surfaces of most cells (Figures 4.3e, f).

The physiological and phylogenetic characteristics of the newly described

isolates are distinct from H. maritima MH2T and each other. Unlike H. maritima which

was isolated from shallow (<40m) marine thermal environments, our strains were all

isolated from deep-sea vents (>1500m). All new strains have very different pH and

temperature ranges for growth from H. maritima MH2T which grows at pH 5.7 to 6.5

and 40 to 65°C (Miroshnichenko et al., 1999). In contrast, the newly described isolates

are obligately acidophilic growing at pH 3.5 to 5.5 (Mar08-272T only up to pH 5.0) and

temperatures between 40 and 72°C (EP5-rT grows between 45 and 75°C). The new

149

Glasauer, S., Langley, S., Boyanov, M., Lai, B., Kemner, K., and Beveridge, T.J. (2007)

Mixed-valence cytoplasmic iron granules are linked to anaerobic respiration.

Appl Environ Microbiol 73: 993-996.

Götz, D., Banta, A., Beveridge, T.J., Rushdi, A.I., Simoneit, B.R., and Reysenbach,

A.L. (2002) Persephonella marina gen. nov., sp. nov. and Persephonella

guaymasensis sp. nov., two novel, thermophilic, hydrogen-oxidizing

microaerophiles from deep-sea hydrothermal vents. Int J Syst Evol Microbiol

52: 1349-1359.

Hafenbradl, D., Keller, M., Dirmeier, R., Rachel, R., Roßnagel, P., Burggraf, S. et al.

(1996) Ferroglobus placidus gen. nov., sp. nov., a novel hyperthermophilic

archaeum that oxidizes Fe2+ at neutral pH under anoxic conditions. Archives of

microbiology 166: 308-314.

Hamady, M., Lozupone, C., and Knight, R. (2010) Fast UniFrac: facilitating high-

throughput phylogenetic analyses of microbial communities including analysis

of pyrosequencing and PhyloChip data. ISME J 4: 17-27.

Harmsen, H., Prieur, D., and Jeanthon, C. (1997) Distribution of microorganisms in

deep-sea hydrothermal vent chimneys investigated by whole-cell hybridization

and enrichment culture of thermophilic subpopulations. Appl Environ Microbiol

63: 2876-2883.

Hoek, J., Banta, A., Hubler, F., and Reysenbach, A. (2003) Microbial diversity of a

sulphide spire located in the Edmond deep-sea hydrothermal vent field on the

Central Indian Ridge. Geobiology 1: 119-127.

17 Accumulation of Heavy Metals by Micro-organisms 485

further to the net negative charge of the cell wall and enhance its metal-binding capacity (Beveridge 1989; Urrutia Mera et al. 1992).

The accumulation of metals in both Gram-positive and Gram-negative bacteria described above is a passive process and it is not controlled by the living cells. Moreover, it was demonstrated that spores (Beveridge 1989) and dead cells of some Gram-positive bacteria can be also implicated in metal binding and that in some cases this binding is even more effective than in the case of the active, metabolizing cells (Urrutia Mera et al. 1992). The reason for this is that the metabolizing cells continuously pump protons into the cell wall which compete with the metal ions for the negatively charged carboxylic and phosphate groups. In many Gram-negative bacteria it is suggested that the metal-saturated parts of their outer cell membranes can bleb off from the cells, forming vesicles which are responsible for the further precipitation of metals and development of minerals (Beveridge 1989).

The periplasm , as a space component in the cell wall mainly in Gram-negative bacteria, plays an important role in metal precipitation and biomineralization due to the enzymes harbored there, such as oxido-reductases and hydrogenases involved in bio-transformation, immobilization, and consequent biomineralization of a large variety of metals (De Luca et al. 200 I; Elias et al. 2004; Glasauer et al. 2007; Lloyd 2003). Because of the limited space, the biominerals formed in the periplasm are often nanoscale (Creamer et al. 2007). When metal reductases are situated on the outer membranes of the Grmn-negative bacteria, they are involved in extracellular metal accumulation and nucleation with consequent formation of larger biominerals (DiChristina et al. 2002; Marshall et al. 2006). This kind of biomineralization, which involves direct redox transformations of the accumulated metals, is referred as active bacteria-induced mineralization (Frankel and Bazylinski 2003).

The cell walls of Archaea are very different chemically from those of Bacteria, and they vary significantly between the phylogenetic groups of this prokaryotic kingdom, possessing various electrochemical charges and metal-binding properties. The interactions of these micro-organisms with metals are poorly studied (Kashefi et a1. 200 I, 2008).

Most archaea and a large number of bacteria possess proteinaceous layers external to their cell walls (Sleytr et al. 1996; see also Chap. 3 of this volume). These layers are in some cases glycosylated (Sleytr et al. 1996), possess a large number of carboxylated amino acids (Sleytr et al. 1996; Pollmann et al. 2005), and can be also phosphorylated (Merroun et al. 2005). As a result, they have an affinity to metal cations (Fahmy et al. 2006; Pollmann et al. 2006; Sleytr et al. 1996). S-layers of some photosynthetic bacteria can be implicated in processes of active biomineralization (Schulze-Lam et al. (992). The authors demonstrated that at moderate photosynthetic activity the S-layer of a Synechococcus sp. strain serves as a template for formation of gypsum (CaS04.2H20). In strong light, however, an alkalization of the surrounding cells takes place due to photosynthesis and as a result calcite (CaCO)) is formed.

Other surface layers such as capsules, slimes, and sheaths as outermost cell structures also play an important role in the biomineralization processes of many •

· ·

· ! , , · .

486 S. Selenska-Pobell and M. Merroun

prokaryotic organisms due to their richness in carboxylic and phosphate groups (Beveridge 1989; Bauerlein 2003; Hanert 2006).

A combination of passive metal sorption by the negatively charged cell wall extracellular polymers and active secretion of phosphate groups due to phosphatase activity was described as an interesting kind of biomineralization by Macaskie et al. (2000); it was confirmed for many bacterial isolates recovered from extreme (Martinez et al. 2007; Merroun et al. 2006; Nedelkova et al. 2007) and non-extreme habitats (Jroundi et al. 2007).

There are many interesting cases of microbe-controlled intracellular biominer­alization which are not discussed in this chapter but can be found in other reviews and publications (Bazylinski and Frankel 2003; Glasauer et al. 2002, 2007).

The next two sections of this chapter will focus,firstly, on bacterial and archaeal cell wall-dependent accumulation and biomineralization of metals (mainly iron and uranium) and, secondly, on the cell wall-supported fonnalion of metallic palladium bio-nanocatalysts by particular Gram-positive and Gram-negative bacteria.

17.3 Prokaryotic Cell Wall-Dependent Accumulation and Biomineralization of Iron and Uranium

The biologically induced mineralization processes by prokaryotes include passive (Beveridge 1989, 2005; Brown et al. 1998) or active (Fortin et al. 1996; Hanert 2006; Lloyd 2003; Lovley 2002; Macaskie et al. 1992, 2000) interactions with and accumulation of a large variety of metals on the cell wall. The metal accumulates can serve as nucleation sites for growth of cell minerals under suitable biogeochem­ical conditions (Bauerlein 2003; Beveridge 1989, 2005; Frankel and Bazylinski 2003; Macaskie et al. 2000; Martinez et al. 2007; Renninger et al. 2004).

The largest and best studied group of biominerals is based on iron. This is due to the wide distribution of this element, which is the fourth most abundant element in the earth's crust (after oxygen, silicon, and aluminum), and also because of its importance for life. It is supposed that some of the earl iest inhabitants of the anoxic prebiotic Earth were able to conserve energy via Fe(ITI) reduction with H2 as an electron donor and were implicated in this way in the earliest biological deposition of magnetite (Lovley 2002; Vargas et al. 1998).

Fe(IIl) reduction is an ubiquitous process in modern Earth habitats and is performed by a large variety of psychrophylic, mesophilic, thermophilic, and even hyperthermophilic bacteria and archaea (Kashefi et al. 2008; Lloyd 2003 ; Roh et al. 2006).

In a large variety of Gram-negative bacteria, the dissimilatory Fe(IIl) reduction is supported by peri plasmic, outer-membrane, or extracellularly located c-type of cytochromes which are responsible for direct transfer of electrons to Fe(III) oxides which are highly insoluble at circum-neutral pH (DiChristina et al. 2002; Elias et al. 2004; Lloyd 2003). Part of the resultant ferrous iron is usually up taken by the cells

u.· ."_~ __ ' ~... " " "" "",.. "or" " ,," """ ..... , '''' -" , . ' ,,.,. "',","'"""""",,,,,, . , ,. " . ~'_" '_'" _::: ~:: _, _: "'_'I11 ',_""."'_p"·.'.~'.·'."·'_"""._·"'.'"\ ""·" .. ' .. ""··.".,,_ •. ·..,·.""'.,.'."""""'=~m>mmm'~'_._._' •• ,," " ",,,. .~'''m' ""'''.".,_,,~,'',,,''',,'','' .. __ '' .. "' ... _"·'w __ " ,,_~.~~.~ ....... ,_ 1" ',',''''''' ' ."'" . " . =.~".'~,. .-, "~' ... _. _~ '" ''''H'''·''A' .~,. __ ''.~"'.'.

17 Accumulation of Heavy Metals by Micro-organisms 4g7

and used for the metabolic needs of the organisms. A significant amount of the solubilized metal is adsorbed by the negatively charged ligands (mainly carboxylic and phosphate groups) of the cell wall and usually stimulates an extracellular formation of iron minerals, Beveridge and colleagues were able to demonstrate that in one representative of these organisms, the gammaproteobacterial strain Shewanella putrefaciens CN32, not only does deposition of de novo formed magnetite precipitates on the cell surface occur, but also precipitation and even penetration and deposition of mineral nanocrystals from the environment into the periplasm and within the peptidoglycan layers (Glasauer et al. 2001), In the cell cytoplasm of this strain, intracellular magnetite-like granules were found which were more reduced than the extracellularly and cell-wall-deposited mineral parti­cles (Glasauer et al. 2002,2007), The authors relate the formation of these granules to an unique mechanism closely linked to the dissimilatory Fe(HI) reduction which is driven in the periplasm by enzymes located at the cytoplasm membrane. The latter also explains the release of mineral-containing blebs into the cytoplasm and not into the environment This process, however, differs significantly from the biologically controlled formation of magnetite in the magnetosomes of magneto­tactic bacteria (Glasauer et al. 2002; Bazylinski and Frankel 2003),

In natural environments the formation of mixed Fe(II)/Fe(IIl) minerals such as magnetite is not only a result of iron (hydr)oxides reduction but also 0 Fe(Il) oxidation under anaerobic conditions (Chaudhuri et al. 200 I) or in aerobic/micro­anaerobic environments with acidic pH, such as mine drainages, where Fe(H1) is highly soluble (Fortin et al. 1996). Moreover, in distinct micro-aerophilic sites of the latter environments, synergetic consortia consisting of iron-oxidizing and sul­fate/iron-reducing micro-organisms are established which provide iron in both oxidation forms and facilitate its mineralization. The initial precipitation of the iron minerals occurs onto the cell walls of these prokaryotic organisms (Fortin et al. 1996) and also in the acidic extracellular phosphate-containing lipopolysaccharide layers of the iron-oxidizing organisms, such as Acidithiobacillus ferrooxidans (Gehrke et al. 1998). A process of direct passive precipitation of regularly distributed iron arrays onto the highly ordered lattices of the S-layers enveloping some bacteria in natural biofilms was demonstrated by Brown et al. (1998).

[n addition to iron, prokaryotic cells are able to accumulate, in either active or passive ways, a large variety of other metals, such as Mn, V, Cr, Co, Pt, Pd, Au, and Ag, and even radionuclides such as Cm, Np, Pu, Tc, and U (Lloyd 2003). In natural conditions a co-precipitation of several metals and formation of mixed minerals can occur as well (Beveridge 1989; Lloyd 2003).

The bioaccumulation and biologically induced mineralization of uranium is a subject of particular interest due to the increased pollution of the environment with this rare element as a result of its mining and processing for nuclear fuel and military applications during recent decades. Bacteria, because of their fascinating resistance to heavy metals and radionuclides and also to their capability to bind metals from both concentrated and also highly diluted solutions, offer effective and cheap solutions for reclamation of polluted sites. The interaction mechanisms of prokaryotes with uranium, although dependent on the chemistry of this rare earth

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492 S. Selenska-Pobell and M. Merroun

U(VI) accumulation mechanisms in some Pseudomonas species of the Gamma subdivision of Proteobacteria which are widely distributed in heavily contaminated uranium wastes (Selenska-Pobell 2002; Geissler et at. 2009). It is noticeable that not all bacterial cells are equally loaded with uranium and that some of them were even free of uranium . This phenomenon might be connected with the different physio­logical state of the cells in the treated bacterial cultures which were not synchro­nized. Pseudomonas stutzeri DSMZ 5190, which possesses a thick layer of mucous extracellular polymers, accumulates U(Vl) initially onto its outer membrane, where at particular sites growth of bigger crystals is initiated which migrate from the cell surface into the surrounding mucous layer and continue to grow further, forming big needle-like crystals (Fig. l7.2a). According to Macaskie et at. (2000) similar extracellular lipopolysaccharide layers to those in P. stutzeri DSMZ 5190 play the role of a supporting matrix for further growth of uranyl phosphate crystals, due to the secretion of inorganic phosphate groups by the stressed bacteria.

Representatives of the species Pseudomonas rhodesie form fine uranium pre­cipitates on their cell wall (Fig. 17.2b), whereas in the case of Pseudomonas migulae a multilayer of fine uranium accumulates was formed which is associated with the outer membrane, with the thin peptidoglycan layer, and also with the cytoplasmic membrane (Fig. l7.2c). Occasionally, uranium associated with poly­phosphate bodies was also found in the cells of the latter strain. Renninger et al. (2004) have demonstrated that in Pseudomonas aeruginosa uranium is initially sorbed onto the surface in the form of uranyl hydroxides and then precipitated as uranyl phosphate due to the release of orthophosphate from the cells as a result of stress-activated degradation of the polyphosphate bodies. This mechanism seems also to be the case with the above-mentioned P. rhodesiae strain, because no intracellular accumulates of uranium have ever been found in its cells. The observed intracellular accumulation of uranium in the case of P. migulae is possibly due to the properties of its outer membrane which permits metal diffusion inside the cells where the metal is complexed into the polyphosphate bodies and detoxified. Similar observations were made with other Gram-negative bacteria such as Halo­monas sp. (Francis et at. 2004) and Acidithiobacillus ferrooxidans (Merroun et al. 2002). Another interesting case of uranium mineralization was found in the ura­nium mining waste isolate Stenotrophomonas maltophilia JG-2 (Fig. 17.2d). In most of the cells of this strain, uranium was associated with the cytoplasmic membrane and in some of the cells intracellular needle-like uranium crystals were formed. Such formations were reported earlier for a Pseudomonas sp. strain (Marques et at. 1991). The growth of such crystals may be associated with inter­actions with the polyphosphate bodies but they can be also attributed to the uranium precipitates formed onto the plasma membrane which were released into the cell cytoplasm and rearranged there. This case is similar to the case described above of formation of intracellular iron nanoparticles by S. putrefaciens CN32 (Glasauer et al. 2007).

The atomic structure of the uranyl precipitates formed by different bacterial strains recovered from both extreme and non-extreme habitats was characterized by using extended X-ray absorption fine structure (EXAFS) (Francis et at. 2004;

498 S. Selenska-Pobell and M. Merroun

Glasauer S, Landley S, Boyanov M, Lai B, Kemner K, Beveridge Tl (2007) Mixed valencc cytoplasmic iron granules arc linked to anaerobic respiration. Appl Environ Microbiol 73:993-996

Hanert HH (2006) The genus Siderocapsa (and other iron- and manganese-oxidizing Eubacteria). In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E (cds) The prokaryotes, vol 7. Springer, New York, pp 1005-1015

Istok lD, Senko 1M, Krumholz LR, Watson D, Bogle MA, Peacock A, Chang Y-l, White DC (2004) In situ bioreduction of technetium and uranium in a nitrate-contaminated aquifer. Environ Sci Technol 38:468-475

lroundi F, Merroun M, Arias 1M, Rossberg A, Selenska-Pobell S, Gonzalez-Munoz MT (2007) Spectroscopic and microscopic characterization of uranium biomineralization by Myxococcus xanthus. Geomicrobiol 1 24:441-449

Kasheli K, Tor JM, Nevin KP, Lovley D (2001) Reductive precipitation of gold by dissimilatory Fc(III)-reducing bacteria and archaea. Appl Environ Microbiol 67:3275-3279

Kasheli K, Shelobolina ES, Elliot WC, Lovley DR (2008) Growth of therlllophilie and hyperther­mophilie Fe(llI)-reducing mino-organisms on a ferruginous smectite as a sole electron acceptor. Appl Environ Microbiol 74:251-258

Kelly SD, Kemner KM, Fein JB, Fowle DA, Boyanov MI, Bunker BA, Yee N (2002) X-ray absorption line structure determination of pH-dependent U-bacterial cell wall interactions. Geochim Cosmochim Acta 65:3855-3871

Koban A, Geipel G, RoBberg A, Bernhard G (2004) Uranyl (VI) complexes with sugar phosphates in aqueous solution. Radiochim Acta 92:903-908

Lloyd JR (2003) Microbial reduction of metals and radionuclides. FEMS Microb Rev 27: 411-425

Lloyd lR, Yong P, Macaskie L (1998) Enzymatic recovery of elemental palladium by using sulphate-reducing bacteria. Appl Environ Microbiol 64:4607-4609

Lovley DR (1993) Dissimilatory Fe(lll) and Mn(IV) reduction. Microbiol Rev 55:259-287 Lovley DR (2002) Dissimilatory metal reduction: from early life to bioremediation. ASM News

68(5):231-237 Lovley DR, Phillips GY A, Landa ER (1991) Microbial reduction of uranium. Nature 350:413-416 Lovley DR, Widman PK, Woodward lC, Phillips E1P (1993) Reduction of uranium by cytochrome

C3 of Desulfovihrio vulgaris. Appl Environ Microbiol 59:3572-3576 Macaskie LE, Empson RM, Cheetham AK, Grey CP, Scarnulis Al (1992) Uranium bioaccumula­

tion by Citrohacter sp. as a result of enzymatically mediated growth of polycrystalline HU02P04 . Science 257:782-784

Macaskie LE, Bonthrone KM, Yong P, Goddart DT (2000) Enzymatically mediated bioprecipita­tion of uranium by a Citrohacter sp.: a concerted role for exocellular lipopolysaccharide and associated phosphatase in biomineral formation. Microbiology 146: 1855-1867

Macaskie LE, Baxter-Plant VS, Creamer Nl, Humphries AC, Mikheenko IP, Mikheenko PM, Penfold DW, Yong P (2005) Applications of bactcrial hydrogenases in waste decontamination, manufacture of novel bionanocatalysts and in sustainable energy. Biochem Soc Trans 33:76-79

Marques AM, Roca X, Simon-Pujol MD, Fuste MC, Congregado F (1991) Uranium accumulation by Pseudomonas sp. ESP-5028. Appl Microbiol Biotcchnol 35:406-410

Marshall Ml, Beliaev AS, Dohnalkova AC, Kennedy OW, Shi L, Wang Z, Boyanov MI, Lai B, Kemner K, McLean lS, Reed SB, Culley DE, Bailey VL, Simonson Cl, Saffarini DA, Romine MF, Zachara 1M, Fredrickson lK (2006) C-type cytochrome-dependent formation of U(VI) nanopar­tides by Shewanella onediensis. PLos 4(8): 1324-1333

Martinez R1M, Beazley 1, Teillefert M, Arakaki AK, Skolnick J, Sobecky PA (2007) Aerobic uranium (VI) bioprecipitation by metal-resistant bacteria isolated from radionuclide- and metal-contaminated subsurface soils. Environ Microbiol 9:3122-3133

Merroun M, Selenska-Pobell S (2001) Interactions of three eco-types of Acidithiohacillusferroox­idans strains with U(VI). BioMetals 14:171-179

FEATURE ARTICLE

Mineral-microbe interactions: a review

Hailiang DONG (✉)1,2,3

1 Key Laboratory of Biogeology and Environmental Geology of Ministry of Education, China University of Geosciences,Wuhan 430074, China

2 Geomicrobiology Laboratory, State Key Laboratory of Geological Processes and Mineral Resources, China University of Geosciences,Beijing 100083, China

3 Department of Geology, Miami University, Oxford, OH 45056, USA

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2010

Abstract The studies of mineral-microbe interactions lieat the heart of the emerging field of Geomicrobiology, asminerals and rocks are the most fundamental earthmaterials with which microbes interact at all scales.Microbes have been found in a number of the Earth’sextreme environments and beyond. In spite of the diversegeological environments in which microbes are found anddiverse approaches taken to study them, a common thread,mineral-microbe interactions, connects all these environ-ments and experimental approaches under the sameumbrella, i.e., Geomicrobiology. Minerals and rocksprovide microbes with nutrients and living habitats, andmicrobes impact rock and mineral weathering anddiagenesis rates through their effects on mineral solubilityand speciation. Given a rapid growth of research in thisarea in the last two decades, it is not possible to provide acomprehensive review on the topic. This review paperfocuses on three area, i.e., microbial dissolution ofminerals, microbial formation of minerals, and certaintechniques to study mineral-microbe interactions. Underthe first area, three subjects are reviewed; they includesiderophores as important agents in promoting mineraldissolution, microbial oxidation of reduced minerals (acidmine drainage and microbial leaching of ores), andmicrobial reduction of oxidized minerals. Under thesecond topic, both biologically controlled and inducedmineralizations are reviewed with a special focus onmicrobially induced mineralization (microbial surfacemediated mineral precipitation and microbial precipitationof carbonates). Under the topic of characterization, thefocus is on transmission electron microscopy (TEM) andelectron energy loss spectroscopy. It is the author's hopethat this review will promote more focused research on

mineral-microbe interactions and encourage more colla-boration between microbiologists and mineralogists.

Keywords interaction, microbe, mineral, oxidation,reduction, siderophores, transmission electron microscopy(TEM)

1 Introduction

The last decade has seen an extraordinary growth ofGeomicrobiology, the interdisciplinary field of Geologyand Microbiology. The studies of mineral-microbe inter-actions lie at the heart of Geomicrobiology, as minerals androcks are the most fundamental earth materials with whichmicrobes interact at all scales, from macroscopic tomicroscopic. Microbes have been found in a number ofthe Earth’s extreme environments (Dong and Yu, 2007),ranging from crystalline rocks of the deep subsurface(Boone et al., 1995; Stevens and McKinley, 1995;Fredrickson and Onstott, 1996; Fredrickson et al., 1997;Krumholz et al., 1997; Pedersen, 1997; Chapelle et al.,2002; Fredrickson and Balkwill, 2006), frozen ice fromthousands of meters under Antarctic ice sheets (Priscu andChristner, 2004), acid mine drainage (Edwards et al.,2000b; Baker and Banfield, 2003; Johnson and Hallberg,2009a), hot springs (Reysenbach et al., 2000a; Kashefi et al.,2002; Meyer-Dombard et al., 2005; Costa et al., 2009), anddeep ocean hydrothermal vent systems (Zierenberg et al.,2000; Reysenbach et al., 2000b; Reysenbach and Shock,2002). Even signatures of ancient life in Mars are beingrevealed ( Thomas-Keprta et al., 2000; Friedmann et al.,2001; Thomas-Keprta et al., 2002), although the subject hasbeen hotly debated (Buseck et al., 2001). In spite of thediverse geological environments in which microbes arefound and diverse approaches taken to study them, a

Received December 7, 2009; accepted February 1, 2010

E-mail: dongh@muohio.edu

Front. Earth Sci. China 2010, 4(2): 127–147DOI 10.1007/s11707-010-0022-8

of magnetosomes causes the cell to passively align alongthe geomagnetic field lines while it swims (magnetotaxis).Magnetotaxis is advantageous to motile microorganisms invertical concentration gradients because it increases theefficiency of finding and maintaining an optimal positionin vertical concentration gradients (such as oxygengradient) (Bazylinski and Frankel, 2003). The formationof these magnetosomes involves iron uptake into the celland some intracellular reduction/oxidation (Bazylinski andFrankel, 2004).There are other intracellular precipitates, i.e., Fe-, As-,

Mn-, Au-, Se-, and Cd-precipitates, but their biological andenvironmental functions remain unclear (Edwards andBazylinski, 2008). Of particular note is the intracellularformation of iron and manganese granules by a dissim-ilatory metal-reducing bacteria Shewanella putrefaciensstrain CN32 when this bacterium reduces Fe(III) inferrihydrite and Mn(IV) in birnessite or pyrolusite(Glasauer et al., 2002; Glasauer et al., 2004; Glasauer et al.,2007). The iron granules consist of ferric hydroxidepartially composed of small amounts of magnetite and/ormaghemite, and the identity of the manganese granules isnot determined. These intracellular granules have beenproposed to be "reservoir of oxidant," a source of energyduring the time periods of depletion of electron acceptorsfrom natural environments (Glasauer et al., 2007). It isemphasized that it is not currently known if these granulesare genetically controlled, because they have qualities ofminerals produced by both MCM and BIM.

4.2 Biologically induced mineralization (BIM)

Biologically induced mineralization, BIM, is a muchlooser term than BCM and refers to mineral precipitationinduced by microbial activity via alteration of local pH,removal of certain inhibitors, and mineral saturation state.Because of these diverse pathways and minerals formed,the presence of these biominerals cannot be used asbiosignatures. Below, we review a few examples of BIM.

4.2.1 Microbial surfaces as nucleation sites for mineralprecipitation

Living cells themselves can provide nucleation sites formineral precipitation on their surfaces, and they induce theformation of biominerals (Dove et al., 2003). The cellsurface has certain functional groups, mainly carboxyl,hydroxyl, amine, and phosphate groups. Most of thesefunctional groups are negatively charged and thus attractpositively charged metal cations from the natural environ-ments (Beveridge and Murray, 1976; Beveridge andMurray, 1980), the first step for authigenic mineralformation (Beveridge et al., 1983; Ferris et al., 1986;Ferris et al., 1987). Cell surfaces often contain manyappendages, including lipopolysaccharide (LPS) and

extracellular polymeric substances (EPS). These appen-dages further help bind metal ions and serve as nucleationsites. Because metal binding to cell surfaces not onlytriggers mineral precipitation but also affects metalmobility in natural environments, a great deal of researchhas been devoted to quantitatively assess and predict thekinetics and capacity of metal binding to cell surfaces (Feinet al., 1997; Daughney et al., 2001; Borrok et al., 2005).Mineral precipitation around cell surface eventuallyencrusts the cell and results in preservation of bacterialcells in sedimentary records (Beveridge et al., 1983).The newly formed precipitates are usually amorphous,

but with time, they undergo transformation to morecrystalline minerals. Many types of minerals have beenobserved on cell surfaces, including (hydr)oxides, silicates,amorphous silica, carbonates, phosphates, sulfates, andsulfides ( Rickard and Morse, 2005; Pósfai and Dunin-Borkowski, 2006; Konhauser, 2007). These minerals areusually fine-grained and lack particular morphology.Because of large surface areas, these precipitates areimportant sorbents for a number of metals, organiccompounds, and other agents in natural environments.

4.2.2 Microbial formation of carbonates

The study on microbial formation of carbonates has had along history, beginning in the late nineteenth century(Ehrlich and Newman, 2009) when extensive evidence waspresented for biogenic precipitation of CaCO3. The earlystudies demonstrated that CaCO3 precipitation was notspecific to any group of bacteria and depended onenvironmental conditions. Subsequent work focused onthe origin of massive carbonates in the Bahamas, and todate, it is clear that carbonates of the Bahamas are of bothbiogenic and chemical origins. Biogenic CaCO3 is usuallyin the form of aragonite. It is also clear that phototrophicbacteria, such as cyanobacteria, play an important role incarbonate precipitation from diverse environments (Ehr-lich and Newman, 2009). The precipitated carbonates areextracellular via diverse mechanisms, such as aerobic oranaerobic oxidation of carbon and nitrogen compounds,reduction of sulfate to sulfide by sulfate-reducing bacteria,hydrolysis, and removal of CO2 from bicarbonate-contain-ing solution (Ehrlich and Newman, 2009). More recentstudies have expanded depositional environment ofcarbonates from marine environments (the Bahamas andthe Gulf of Mexico) to caves (Barton and Northup, 2007)and saline lakes/lagoons (Wright, 1997; Wright andWacey, 2005). In cave environments, the role of micro-organisms has been demonstrated in the formation ofspeleothem and moonmilk (Barton and Northup, 2007).Despite the oligotrophic nature of caves, heterotrophicbacteria can be active in mediating calcium carbonateprecipitation by increasing pH and providing nucleationsites (Wang et al., 2010a). In addition to biogenic

134 Front. Earth Sci. China 2010, 4(2): 127–147

increases the concentration of CO2 through sulfatereduction, favoring dolomite formation, while methano-genesis increases the pCO2 of the pore waters, inhibitingdolomite formation (Moore et al., 2004). However, a directlink between a microbial function and dolomite formationpathways has not been established for important carbonreservoirs, such as gas hydrate and methane seep deposits.Moreover, dolomite formation under the in-situ conditionsof gas hydrate deposits (i.e., in-situ pressure, temperature,and water chemistry) has not been attempted.

5 Techniques for studying mineral-microbeinteractions

5.1 Transmission electron microscopy (TEM) andenvironmental cell TEM

TEM has been a primary tool to study mineral-microbeinteractions at high spatial resolutions ( Fredrickson et al.,1998; Dong et al., 2000; McLean et al., 2000; Dohnalkovaet al., 2001; McLean and Beveridge, 2001; Glasauer et al.,2001; Liu et al., 2002; Glasauer et al., 2002; Glasauer et al.,2007; Geesey et al., 2008), but the spatial relationship ofmineral-microbe interface for such studies may not bepreserved intact, partly due to sample preparationnecessary for conventional TEM observations. In conven-tional TEM, high vacuum is required not only to reducescattering of the electron beam by gas molecules but also tominimize corrosion of the electron source. In such a highvacuum, biological materials and hydrous minerals (suchas clays) dehydrate, resulting in bacterial cell rupture andmineral structure collapse. Thus, these materials have to beprocessed via fixation, dehydration, and embedding andsectioning prior to TEM observations (Kim et al., 1995;Fredrickson et al., 1998; Dong et al., 2000; McLean et al.,2000; Glasauer et al., 2001; Dohnalkova et al., 2001;McLean and Beveridge, 2001; Glasauer et al., 2002; Liuet al., 2002; Dong et al., 2003; Kim et al., 2004), assumingthat the structure and the composition of these materials arenot altered during sample preparation. However, theseassumptions are never verified. Even with these assump-tions, the standard procedures may not be adequate forobservations of hydrous and expandable clay mineralstructure, such as those of nontronite and smectite, becausewater-bearing nontronite and smectite tend to dehydrateunder the TEM vacuum. For example, despite the effort tokeep layer spacing of nontronite noncollapsed by impreg-nation with the Nanoplast resin, Kim et al. (2003) stillobserved collapse of (001) layers of nontronite in theconventional TEM electron column, making d(001) ofmicrobially reduced nontronite indistinguishable from thatof unreduced material. Stucki and Tessier (Stucki andTessier, 1991) also observed homogenous layer spacing forboth reduced and nonreduced smectite, illustrating thelimitations of conventional TEM.

Alteration of oxidation states of metals introducedduring the dehydration and embedding procedure forTEM observations is another issue, yet the preservation ofoxidation states is essential to quantitatively evaluate thecapabilities of bacteria to respire metals in mineral struc-tures. Bulk chemical extractions (Stookey, 1970; Stucki,1981; Stucki and Anderson, 1981; Amonette andTempleton, 1998) and Mössbauer spectroscopy (Donget al., 2000; Kukkadapu et al., 2001; Kukkadapu et al.,2004) have been used to measure the extent of microbialreduction of iron, where oxidation states are generallypreserved, but these techniques give no information on thespatial distribution of oxidized and reduced metals,relative to cell surface. Dohnalkova et al. (2001) attemptedto preserve anaerobic states of various minerals bydehydrating, embedding, and sectioning samples in ananaerobic glove box and by excluding most fixatives andall heavy metal stains. The authors successfully observedcrystalline biogenic minerals outside the outer membraneand amorphous or nanocrystalline material in cellperiplasm. This is consistent with the current under-standing of electron transfer mechanisms in dissimilatorymetal reducing bacteria, where the mineral reductaseactivity is associated with the cell membrane, periplasm,and outer membrane.Conventional TEM has not been used to probe time-

course change of oxidation state of metals during microbialreduction, but this information is critical to revealmechanisms of biooxidation and bioreduction of redox-sensitive metals. Lower et al. (2001) was probably the firstto quantify the attachment of Shewanella oneidensis togoethite and diaspore surfaces by modified atomic forcemicroscopy and demonstrated that the affinity of S.oneidensis for goethite was strongest under anaerobicconditions. The authors presented compelling evidencethat a 150-kD protein in the outer membrane of the cellspecifically interacted with the goethite surface to facilitateelectron transfer. To truly understand the two-wayinteractions between microbes and minerals, it is equallyimportant to simultaneously detect structural and composi-tional change of mineral surfaces as bacteria approachthem. In other words, it is critical to observe how microbeand mineral respond when these two entities approach andinteract with each other at the nanometer scale.To overcome the sample dehydration problem asso-

ciated with high vacuum required in the TEM electroncolumn, material scientists have modified conventionalTEM to study gas-solid interactions (Sharma and Weiss,1998; Sharma, 2001). Several modifications, either to thespecimen holder or to the microscope vacuum system,have been developed in order to restrict the gas path lengthto the sample area only. In the first design, the sample issandwiched between two thin windows. This type of cell isideally suited to the study of wet samples (biologicalmaterials) and is referred to as environmental cell (EC). Inthe second design, small differentially pumped apertures

136 Front. Earth Sci. China 2010, 4(2): 127–147

not only over the cell membrane but also across it, from theouter membrane, periplasm, into cytoplasm (Fig. 5).

It has been well documented that minerals heteroge-neously attach to cell surface (Grantham et al., 1997;Glasauer et al., 2001), and this may be due toheterogeneous distribution of negative functional groupson cell surface (Sonnenfeld et al., 1985; Sokolov et al.,2001). Heterogeneous mineral attachment may result in theextent of reduction and distribution of resulting biogenicsolids being heterogeneous. In conventional TEM obser-vations, artifacts associated with sample preparation (suchas by centrifugation) cannot be completely eliminated. It iscommon to observe features of mineral detachment fromcell surface, apparently associated with the centrifugationforce. A recent sample preparation technique developed inour laboratory, embedding cell suspensions in agar prior tothe dehydration step, appears to alleviate this problem.However, a better method is to observe cell-mineralassociation in-situ without any sample treatment. Forexample, heterogeneity in the extent of reduction over cellsurface has never been studied before (Fig. 5), but thesedata can provide important information revealing mechan-isms of microbial reduction of metals. With the advent ofEELS coupled with EC-TEM, it is now possible to determinethis heterogeneity at the nanometer scale. For example, thehigher ratio of Fe2+/Fe3+ should reflect the higher extent ofbioreduction at a particular location on cell surface, and thisvariability can be mapped at a nanometer scale.Minerals have also been found inside cells. In the

magnetotactic bacteria, the chains of magnetosomes havebeen found aligned parallel to the cell axis, and they arebelieved to play a role in cell navigation (Bazylinski andFrankel, 2000; Bazylinski and Frankel, 2003). Glasaueret al. (Glasauer et al., 2002; Glasauer et al., 2004; Glasaueret al., 2007) reported intracellular iron and manganeseminerals in Shewanella putrefaciens CN32, and Doh-nalkova et al. (personal communication) even found

nanocrystalline uraninite formation inside CN-32 cells.Cerussite (PbCO3) was even found inside yeast andbacterial cells (Seabaugh et al., 2003). The distributionof these intracellular minerals is heterogeneous. Except forthe well-documented magnetosomes, the formationmechanisms of other intracellular minerals have not beenunderstood. Real-time high-resolution TEM observationsof the dynamics of both extra- and intracellular mineralformation process, when coupled with EELS, couldprovide critical information on the mineral-microbeinteraction mechanisms and metal biogeochemistry.The value of the EELS technique is illustrated in the

following example. Figure 6 shows an Fe-M2,3 electronloss near edge structure (ELNES). The one on the left isFe-M2,3 ELNES of Fe standards (pyroxene solid solution)from van Aken et al. (1999), and the one on the upper rightis Fe-M2,3 ELNES of our standards hematite (100% Fe3+)and ilmennite (100% Fe2+). The one on the lower right isFe-M2,3 ELNES of unreduced and microbially reducednontronite. Van Aken (van Aken et al., 1999) demonstratedthat differences in the Fe-M2,3 ELNES are a result ofchanges in Fe oxidation state and that differences in thecrystal structure of Fe standards are of secondaryimportance. The authors further concluded that quantita-tive determination of Fe oxidation state in an unknownmineral can be accomplished by the energy differencebetween main maximum and prepeak (also called pre-edge). Using this criterion, the Fe3+/Fe ratio wasdetermined to be 90% and 30% for unreduced and biore-duced nontronite, respectively. These data are in goodagreement with bulk chemical results by HCl extraction,illustrating the reliability of the method. Using the sameprocedure, the change of Fe3+/Fe with the distance fromthe cell-magnetite interface from an earlier study (Donget al., 2000) was determined. The hypothesis was thatmagnetite was actively reduced at the cell-magnetiteinterface (Fig. 7(a)), eventually forming siderite in thecore encrusted by magnetite particles (Fig. 7(b)) by the endof reduction experiment (14 days) (Dong et al., 2000).Indeed, EELS detected the change of the Fe(III)/Fe ratiofrom siderite to magnetite (Fig. 8).

5.3 Other techniques

There are a suite of other techniques that are available toexamine mineral-microbe interactions, such as synchro-tron-based X-ray spectroscopy and X-ray microscopy(Templeton and Knowles, 2009) and nanosecondarymass spectroscopy (Herrmann et al., 2007), among others.These techniques greatly enhance our capability toinvestigate microbial growth, mineral dissolution, redoxtransformation, and biomineralization processes owning totheir high chemical sensitivity and spatial resolution.Interested readers are referred to these review articles forinformation.

Fig. 5 A schematic diagram showing heterogeneous distribu-tions of microbial functional groups on cell surface. Thisheterogeneity may result in heterogeneous mineral attachmentand metal reduction over and across the cell surface

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Intracellular Precipitation of Pb byShewanella putrefaciens CN32during the Reductive Dissolution ofPb-JarositeC H R I S T I N A M . S M E A T O N , *B R I A N J . F R Y E R , A N DC H R I S T O P H E R G . W E I S E N E R

Great Lakes Institute for Environmental Research, Universityof Windsor, Windsor, Ontario, Canada, N9B 3P4

Received June 3, 2009. Revised manuscript received August24, 2009. Accepted September 9, 2009.

Jarosites (MFe3(SO4)2(OH)6) are precipitated in the Zn industryto remove impurities during the extraction process andcontain metals such as Pb and Ag. Jarosite wastes are oftenconfined to capped tailings ponds, thereby creating potentialfor anaerobic reductive dissolution by microbial populations. Thisstudy demonstrates the reductive dissolution of synthetic Pb-jarosite (PbFe6(SO4)4(OH)12) by a subsurface dissimilatory Fereducing bacterium (Shewanella putrefaciens CN32) usingbatch experiments under anaerobic circumneutral conditions.Solution chemistry, pH, Eh, and cell viability were monitoredover time and illustrated the reduction of released structuralFe(III) from the Pb-jarosite to Fe(II). Inoculated samples containingPb-jarosite also demonstrated decreased cellular viabilitycoinciding with increased Pb concentrations. SEM imagesshowed progressive nucleation of electron dense nanoparticleson the surface of bacteria, identified by TEM/EDS asintracellular crystalline precipitates enriched in Pb and P. Theintracellular precipitation of Pb by S. putrefaciens CN32observed in this study provides potential new insight into thebiogeochemical cycling of Pb in reducing environments.

IntroductionPlumbojarosite (PbFe6(SO4)4(OH)12) was first described in1902 at Cook’s Peak, New Mexico and often forms during theoxidation of sulfide deposits (1, 2). In these environments,Pb mobility is limited by the low solubility of anglesite (PbSO4,log Ksp ) -7.7) and cerrusite (PbCO3, log Ksp ) -12.8),consequently plumbojarosite (log Ksp ) -16.8) is often thelast mineral to form (1, 3). Plumbojarosite is thermodynami-cally stable up to pH 5 under high Fe and sulfate concentra-tions typical of acid mine drainage (AMD) environments andis suggested to control aqueous Pb mobility at these sites(2-4). At pH values above 5.0, plumbojarosite is subject todissolution/reprecipitation reactions and may form Fe oxidesor hydroxides (5). Under these conditions, Fe hydroxidessuch as ferrihydrite become important scavengers of Pb.

Structurally, plumbojarosite or Pb-jarosite (i.e., syntheticanalog) is a member of the alunite-jarosite mineral supergroup with the general formula AB3(TO4)2(OH)6. The A sitesare occupied by monovalent (e.g., K and Na) and divalentcations (e.g., Ca(II), Pb(II), and Ag(II)). The B position

corresponds to cation sites with octahedral (O) coordination,typically Al(III) or Fe(III), while the T position correspondsto tetrahedral (T) coordination (e.g., S(VI), As(V)) (6). Overthe past 20 years, the Zn industry has capitalized on thestability of jarosite minerals and during the metal extractionprocess, synthetic jarosites are purposely precipitated toremove unwanted impurities such as Fe, sulfates, alkalis,and other heavy metals (e.g., Pb, Ag). Jarosite precipitationis advantageous because the precipitates are easily filteredand give low losses of Zn metal, therefore, this processaccounts for 80% of all Zn extracted worldwide (8 Mt/yr) (1).The majority of the precipitates contain Na and K jarosite,however, Pb-jarosite will form during the acid pressureleaching of Pb-containing Zn concentrates (1). While thejarosite process is a highly efficient and economical methodto optimize Zn recoveries, it also produces large volumes ofwastes. For example, a plant annually producing 150 000tonnes of metallic Zn will generate approximately 125 000tonnes of jarosite (1). Jarositic wastes are not easily recyclableand may contain elevated concentrations of toxic heavymetals such as Pb, therefore the safe disposal of these wastesis of paramount concern.

One of the most widely used storage method is disposalin lined ponds under circumneutral conditions (1). However,despite the environmental relevance of jarosite minerals, fewstudies have evaluated the abiotic or biotic dissolution ofjarosites under these storage conditions (5, 7-10). Therefore,the primary objective of this study was to assess the effectsof a model metal reducing bacterium, Shewanella putrefa-ciens CN32, on the dissolution of synthetic Pb-jarosite undercircumneutral anaerobic conditions. S. putrefaciens CN 32was chosen because it is a well characterized subsurfacechemoautotrophic anaerobe capable of using Fe(III) duringdissimilatory iron reduction (11-13). A secondary objectiveof this study was to evaluate the fate and toxicological effectsof the Pb released during the dissolution experiment on S.putrefaciens CN32. To the best of our knowledge, this is thefirst study to assess the fate of Pb during Pb-jarosite/microbeinteractions.

Materials and MethodsPreparation of the Pb-Jarosite/Cell Suspensions and Sam-pling Procedures. Pb-jarosite was synthesized as per themethod used by Dutrizac et al. (14, 15). (see SupportingInformation (SI) Section 1 for more details). Pure cultures ofS. putrefaciens CN32 were grown, harvested, and inoculatedinto a modified M1 minimal media (SI Section S2). Theinoculated minimal media was transferred to an anaerobicchamber (96% N2/4% H2) and 16 mL aliquots were added to20 mL polypropylene test tubes containing 0.0500 ( 0.0013g of Pb-jarosite (33 samples). Experimental controls contained16 mL of sterile minimal media in 20 mL polypropylene tubescontaining 0.0500 ( 0.0013 g of Pb-jarosite (33 samples). Tomonitor background elemental concentrations over time,an additional set of test tubes were prepared containing onlythe inoculated minimal media (10 samples) and sterileminimal media (10 samples). All samples were capped, sealed,and rotated end-over-end in the anaerobic chamber. Samplescontaining Pb-jarosite were removed in triplicate from therotator at 0, 6, 16, 24, 38, 48, 72, 120, 192, 408, 768 h, whereasminimal media samples (i.e., in the absence of Pb-jarosite)were removed from the rotator in duplicate at 0, 48, 192, 408,768 h. All samples were monitored for Eh and pH usingsemimicro electrodes, and aqueous concentrations of Pb,Fe, and S were determined using inductively coupled plasmaoptical emission spectroscopy (ICP-OES) (SI Section S3).

* Corresponding author phone: (519) 253-3000ext. 4246; fax: (519)971-3616; e-mail: smeato3@uwindsor.ca.

Environ. Sci. Technol. 2009, 43, 8086–8091

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changes in Fe(II) and Fe(III) concentrations over time. Basedon reaction path modeling, React (GWB 7.01) calculatedsaturation indices (log Q/K) over time using the minimalmedia composition and experimental data collected at 0, 48,and 768 h (SI Table S3) and predicted the initial precipitationof hematite > goethite > magnetite> carbonate green rust (SITable S4). However, as the reaction progressed and Fe(III)decreased and Fe(II) increased, React predicted the pre-cipitation of carbonate green rust > sulfate green rust>chloride green rust > hematite > magnetite (SI Table S4).Green rusts are mixed Fe(II)/Fe(III) hydroxides that typicallyform under weakly acidic and alkaline suboxic environmentsand were formed in previous studies by S. putrefaciens CN32during the dissimilatory Fe reduction of ferrihydrite andlepidocrocite (13, 19). However, the secondary precipitatesformed in this study do not exhibit the characteristichexagonal platelet morphology typical of green rusts. More-over, Fredrickson et al. (13) demonstrated magnetite forma-tion over the formation of green rusts in the absence of P byS. putrefaciens CN32 during dissimilatory Fe reduction.Therefore, it is unlikely that carbonate green rust formedduring these experiments and it is more likely that magnetiteor an Fe hydroxide such as goethite was formed.

The absence of P in the minimal media and the presenceof aqueous Pb may have played an important role in the lowconcentrations of aqueous Fe(II) observed throughout theexperiment as both have previously demonstrated inhibitoryeffects on Fe reduction in Fe(hydr)oxides by S. putrefaciens(20, 21). While dissimilatory iron reduction of varioussynthetic Fe (hydr)oxides has been studied extensively, fewstudies have examined the dissimilatory Fe reduction ofjarosite minerals. Recently, Weisener et al. (10) demonstratedFe reduction during silver jarosite, AgFe3(SO4)2(OH)6 dis-solution in the presence of S. putrefaciens CN32 undercomparable conditions and showed a maximum aqueousFe(II) concentration of 167.0 µM at 168 h. Similarly, Jones etal. (22) also demonstrated Fe reduction from jarosite,KFe3(SO4)2(OH)6, using Geobacter metallireducens GS-15 andGeobacter sp. ENN 1.

To evaluate the potential effects of the Pb released duringPb-jarosite dissolution on S. putrefaciens, cell viability wasmonitored in inoculated samples in both the presence andabsence of Pb-jarosite and reported as luminescence (RLU).Initially, luminosity was higher in the inoculated Pb-jarositesamples (1.49 × 106 ( 7.81 × 104 RLU) compared to theinoculated minimal media samples (8.81 × 105 ( 1.62 × 104

RLU) after 48 h thus suggesting greater cell viability at thebeginning of the experiment in samples containing Pb-jarosite (SI Figure S5). The greater cell viability initiallyobserved in the inoculated samples containing Pb-jarositecorresponded with increased Fe(III) release and subsequentFe(II) production and was likely due to increased metabolicactivity due to dissimilatory Fe reduction (Figure 1A).However at 72 h, cell viability in the inoculated samplescontaining Pb-jarosite (7.91 × 106(2.23 × 105 RLU) decreasedwhile cell viability in the inoculated samples without Pb-jarosite (6.92 × 106 ( 2.63 × 104 RLU) demonstrated largerluminescence (i.e., cell viability) and continued to increaseuntil 198 h (SI Figure S5). The decrease in cell viabilityobserved earlier in the inoculated samples containing Pb-jarosite was likely due to the presence of aqueous Pb releasedduring Pb-jarosite dissolution (Figure 1) (20, 21).

Fate of Pb. Back scattered electron (BSE)-SEM imagestaken within 2 h of sampling demonstrated progressivenanoparticle formation over time indicated by high contrastspots associated with the cell surface (Figure 2B, D, F) notobserved in the control experiments (Figure 2A, C, E). Thediameter of the electron dense particles associated with thecell surface were measured using the Scandium SEM imageplatform, and the mean diameter of the each particle was

113.20 nm (n ) 13, SD ) 29.59 nm).While BSE-SEM imagessuggest extracellular precipitation, TEM images collected ona cross section of an individual cell at 406 h (Figure 3A)demonstrated intracellular accumulation of electron densegranules within the cytoplasm and were observed in the manyof the cells imaged (SI Figure S6A). The appearance ofextracellular precipitation of the electron dense nanoparticlesduring BSE-SEM not seen in the TEM images is likely theresult of cellular dehydration under low vacuum (80 Pa). Theprogressive increase in electron dense nanoparticles overtime associated with the cells (Figure 2A, C, E) coincidedwith decreased aqueous Pb concentrations thus suggestingthe precipitation of an enriched Pb phase (Figure 1B). Inaddition to typical cellular components such as C and S,TEM-EDS analyses of the electron dense granules (i.e., spots1-3) showed enrichment of Pb (90.38-92.74 wt %) and P(1.76-2.26 wt %) (SI Table S5). EDS analyses also showedresidual Ca (0.46-0.56 wt %) not seen in any other cellularcomponents. The cytoplasm (i.e., spot 4) in the absence ofelectron dense particles was primarily composed of C (95.58wt %) and showed residual Pb (0.98 wt %) thus demonstratingtransport of Pb into the cytoplasm likely followed byprecipitation of the electron dense Pb rich granules. EDSanalysis of the cell wall (i.e., spot 5) showed Pb enrichment(5.71 wt %) and is likely due to the formation of adsorptioncomplexes with carboxyl and phosphoryl functional groupscommonly reported at neutral pH values (23, 24). In additionto Pb, the cell wall (i.e., spot 5) was also enriched in C (89.38wt %) and Fe (11.67 wt %) with lower concentrations of O(0.95 wt %) and P (2.85 wt %). The enrichment of Fe in thecell wall is likely the result of dissimilatory Fe reductionoccurring at the outer membrane by metal-reducing proteinscalled cytochromes, while the P signal is contributed bytypical cellular constituents such as DNA, RNA, and phos-pholipids (25). Therefore, the immediate precipitation of Pbenriched nanoparticles associated with S. putrefaciens CN32at the start of the experiment (Figure 2B) coupled with thepresence of Pb in the cell wall and cytoplasm suggests asimultaneous mechanism involving rapid Pb sorption ontothe cell wall and intracellular uptake and precipitation(24, 26, 27).

While Shewanella putrefaciens CN32 has previouslydemonstrated intracellular precipitation of mixed-valenceiron and manganese oxides, this is the first report ofintracellular Pb precipitation (11, 28, 29). Several bacterialspecies such as Citrobacter sp. and Staphylococcus aureushave demonstrated intracellular Pb precipitation (27, 30, 31),however, due to the analytical limitations imposed by thesmall size of the Pb inclusions (<100 nm), detailed analysesof intracellular Pb precipitates are rare. Aside from onereported case of PbS intracellular precipitation by Klebsiellasp (32), the majority of studies show enrichment of Pb andP within the intracellular granules (26, 27, 30) and areidentified as either amorphous Pb-polyphosphate (27) orcrystalline Pb phosphate (30, 33).

Polyphosphates (poly P) are linear orthophosphate poly-mers that serve as energy and phosphorus reservoirs in manymicroorganisms and are implicated to play a key role in thedetoxification of metals such as Cd, Cu, Pb, and U (34-36).While potassium polyphosphate formation was recentlysuggested in Shewanella putrefaciens CN32, to the best ofour knowledge it has not been confirmed microscopically(11, 37). In contrast to other studies demonstrating Pb-polyphosphate formation, HR-TEM images of the precipitatesin our study reveal a crystalline structure thereby suggestinginorganic lead phosphate formation rather than amorphouspolyphosphate formation (Figure 3B and SI Figure S6).Moreover, the relative concentration of P is lower than theamount of Pb thus conflicting with similar analyses ofbacterial polyphosphate inclusions where P is high (SI Table

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ES901629C

VOL. 43, NO. 21, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 8091

mboyanov

Oval

mboyanov

Oval

J. R. Soc. Interface (2009) 6, S649–S658

doi:10.1098/rsif.2009.0166.focus

Published online 15 July 2009

REVIEW

*Author for c

One contributlarge facilities

Received 30 AAccepted 19 J

Bio-metals imaging and speciation incells using proton and synchrotronradiation X-ray microspectroscopyRichard Ortega*, Guillaume Deves and Asuncion Carmona

Cellular Chemical Imaging and Speciation Group, CNAB, CNRS UMR 5084,University of Bordeaux, 33175 Gradignan, France

The direct detection of biologically relevant metals in single cells and of their speciation is achallenging task that requires sophisticated analytical developments. The aim of this article isto present the recent achievements in the field of cellular chemical element imaging, anddirect speciation analysis, using proton and synchrotron radiation X-ray micro- and nano-analysis. The recent improvements in focusing optics for MeV-accelerated particles andkeV X-rays allow application to chemical element analysis in subcellular compartments.The imaging and quantification of trace elements in single cells can be obtained usingparticle-induced X-ray emission (PIXE). The combination of PIXE with backscattering spec-trometry and scanning transmission ion microscopy provides a high accuracy in elementalquantification of cellular organelles. On the other hand, synchrotron radiation X-rayfluorescence provides chemical element imaging with less than 100 nm spatial resolution.Moreover, synchrotron radiation offers the unique capability of spatially resolved chemicalspeciation using micro-X-ray absorption spectroscopy. The potential of these methods inbiomedical investigations will be illustrated with examples of application in the fields ofcellular toxicology, and pharmacology, bio-metals and metal-based nano-particles.

Keywords: synchrotron; X-ray; cell; metal; imaging; speciation

1. INTRODUCTION

A eukaryotic cell is packed with internal compartments,the organelles, where specific chemical reactions takeplace. There is a growing need for in situ chemicalelement analysis in order to understand the biochemicalprocess involved directly in cell organelles (Navratilet al. 2006). Cell organelle sizes range from the micro-world to the nanoworld; for example, mitochondriaare one of the larger organelles in the eukaryotic cell,typically 0.3–1.0 mm, whereas neural vesicles can beonly a few tens of nanometres large. Among the ana-lytes to be determined are the metal ions involved inbiological processes referred to here as bio-metals. Thedetermination of intracellular distribution of bio-metalsis challenging because it requires analytical methodswith high sensitivity and high spatial resolution(Lobinski et al. 2006).

The chemical speciation analysis is also important tounderstand the reactivity of bio-metals. Chemicalelements can be present in cell organelles in variousforms, such as purely inorganic, or transformed into

orrespondence (ortega@cenbg.in2p3.fr).

ion of 13 to a Theme Supplement ‘Biological physics at’.

pril 2009une 2009 S649

coordination compounds after cellular metabolization.For redox metals, the determination of the oxidationstate will be of particular importance because theycan catalyse oxidative reactions. Ideally, microanalyt-ical methods should therefore combine high spatialresolution (in the nanometre to the micrometre range)with high detection sensitivity (ng g21 to mg g21) andchemical speciation capabilities such as the determi-nation of the oxidation state. The aim of this article isto present some recent achievements in the field ofcellular chemical element imaging, and direct speciationanalysis, using proton and synchrotron radiation X-raymicro- and nanoanalysis.

Chemical imaging can be defined as the spatialidentification and characterization of the chemicalcomposition of a given sample. In recent decades,numerous advances have been made in the understand-ing of fundamental biological processes at the cellularlevel. However, the ability to obtain subcellular datarelating to metal content is still challenging. X-raychemical imaging holds the potential for fundamentalbreakthroughs in the understanding of biologicalsystems because chemical interactions can be evidencedat the subcellular level. By examining the energies atwhich X-rays are emitted or absorbed, it is possible to

This journal is # 2009 The Royal Society

S656 Review. Bio-metals imaging and speciation R. Ortega et al.

up to now, there are still very few examples of X-rayabsorption spectrometry applied to cellular compart-ments, probably because this technique still requireslong acquisition times and beam damage is of particularconcern. In 2001, we demonstrated the feasibility ofm-XANES analysis on single cells by examining the ironoxidation state in cancer cells exposed to doxorubicin,an anti-cancer anthracycline known to complex iron(Ortega et al. 2001). We found that iron was present asFe(III) in the nucleus of cultured cancer cells with spec-tral features similar to that of Fe–doxorubicin complexes,but different from the main iron-storage protein, ferritin.m-XANES was also applied to the chemical speciation ofplatinum anti-cancer drugs (Hall et al. 2003), showingthat Pt(IV) complexes are readily reduced to Pt(II) incells, except some specific compounds such as iproplatinthat can stand in its oxidized form inside cells. The tech-nique was also used for the single cell determination ofmetal redox states in prokayotes (Kemner et al. 2004).m-XANES also revealed the existence of intracellulargranules containing ferric and ferrous iron formed inShewanella putrefaciens (Glasauer et al. 2007). In traceelement physiology of eukaryotic cells, m-XANES per-formed on a copper-loaded NIH 3T3 cell revealed at alltested subcellular locations a near-edge feature that ischaracteristic for monovalent copper (Yang et al. 2005).

Intracellular redox states are important parametersin understanding metal carcinogenesis and toxicology.The imaging of chromium oxidation states was com-pared in cells exposed to soluble and low solubilitychromate compounds, showing the intracellularreduction of Cr(VI) to Cr(III) in all cell compartmentsexcept in perinuclear structures from low solubilitycompounds (Ortega et al. 2005). The m-XANES tech-nique was also used to investigate the oxidation stateof iron (Bacquart et al. 2007; Chwiej et al. 2007) andcopper (Chwiej et al. 2008) in neurons. The comparisonof the absorption spectra near the iron K-edgemeasured in melanized neurons from the substantianigra of patients with Parkinson’s disease and ofcontrol samples did not show significant differences iniron or copper oxidation states. In order to accuratelyidentify intracellular compartments, we have recentlycoupled the tracking of cellular organelles in a singlecell by confocal and epifluorescence microscopy withlocal analysis of chemical species by m-XANES. Thisoriginal methodology enabled the direct speciationanalysis of arsenic in subcellular organelles with a10215 g detection limit (Bacquart et al. 2007).m-XANES shows that As(III) is the main form ofarsenic in the cytosol, nucleus and mitochondrialnetwork of HepG2 cells exposed to As(OH)3. In somecases, oxidation to a pentavalent inorganic form isobserved in nuclear structures of HepG2 cells (figure 3).

5. CONCLUSIONS AND PERSPECTIVES

Chemical imaging with X-ray microprobes offers ameans of investigating the distribution and speciationof bio-metals in single cells with a detection sensitivityand a spatial resolution well suited to explore subcellu-lar interactions. Several chemical nanoimaging

J. R. Soc. Interface (2009)

instruments are under development worldwide, princi-pally on third-generation synchrotron facilities in theUSA, Japan and also in Europe (France, Germany,Spain, UK, etc.). The same is true for the protonnanoprobe community. It is likely that fundamentalbreakthroughs in our understanding of basic chemicalprocesses in biology will be achieved in the next fewyears owing to these new instruments. Real break-throughs have already been obtained in the understand-ing of the physiological functions of trace elements suchas for the role of Cu in angiogenesis or selenium in sper-matogenesis. Important achievements have also beenreached in the fields of redox metal toxicology, neuro-chemistry, carcinogenesis and cellular pharmacology.The application to nanotechnology research is reallypromising as the X-ray microanalytical techniques arequite unique tools for validating intracellular targetsand for quantification of metal-based nanoparticles.One exciting research area where the X-ray nanoprobeswill continue to open new frontiers is the study of intra-cellular preferential localization of trace elements. Fromthe examples of subcellular chemical imaging listed inthis article, it can be concluded that in many cases cellu-lar organelles are characterized by a specific chemicalelement composition, which is related to the specificfunctions of the organelles (i.e. energy production in themitochondria requires Fe and Cu involved in metallo-enzymes). The same is true for the subcellular distributionof toxic metals that are stored within intracellular com-partments such as the Golgi apparatus or the lysosomesfor detoxification purposes. The relationship betweenthe specific cellular functions of organelles and theirchemical element composition can be considered aspart of the chemotype that characterizes each livingsystem, as suggested by Williams (2007).

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Available online at www.sciencedirect.com

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Geochimica et Cosmochimica Acta 73 (2009) 696–711

Iron biomineralization by anaerobic neutrophiliciron-oxidizing bacteria

Jennyfer Miota,*, Karim Benzeraraa, Guillaume Morin a, Andreas Kappler b, Sylvain Bernard c,Martin Obstd, Celine Ferard a, Feriel Skouri-Panet a, Jean-Michel Guigner a,

Nicole Posth b, Matthieu Galvez a, Gordon E. Brown Jr. e,f, Franc�ois Guyot a

a Institut de Mineralogie et de Physique des Milieux Condenses, UMR 7590, CNRS, Universites Paris 6 et Paris 7, et IPGP,

140, rue de Lourmel, 75 015 Paris, Franceb Geomicrobiology Center for Applied Geoscience (ZAG), University of Tuebingen, Sigwartstrasse 10, 72076 Tuebingen, Germany

c Laboratoire de Geologie, Ecole normale superieure, CNRS, 24 rue Lhomond, 75005 Paris, Franced BIMR McMaster University Hamilton & Canadian Light Source, 101 Perimeter Road, Saskatoon, Sask., Canada S7N OX4e Surface & Aqueous Geochemistry Group, Department of Geological & Environmental Sciences, Stanford University, Stanford,

CA 94305-2115, USAf Stanford Synchrotron Radiation Laboratory, SLAC, Menlo Park, CA 94025, USA

Received 2 June 2008; accepted in revised form 21 October 2008; available online 11 November 2008

Abstract

Minerals formed by bio-oxidation of ferrous iron (Fe(II)) at neutral pH, their association with bacterial ultrastructures aswell as their impact on the metabolism of iron-oxidizing bacteria remain poorly understood. Here, we investigated iron bio-mineralization by the anaerobic nitrate-dependent iron-oxidizing bacterium Acidovorax sp. strain BoFeN1 in the presence ofdissolved Fe(II) using electron microscopy and Scanning Transmission X-ray Microscopy (STXM). All detected mineralsconsisted mainly of amorphous iron phosphates, but based on their morphology and localization, three types of precipitatescould be discriminated: (1) mineralized filaments at distance from the cells, (2) globules of 100 ± 25 nm in diameter, at thecell surface and (3) a 40-nm thick mineralized layer within the periplasm. All of those phases were shown to be intimatelyassociated with organic molecules. Periplasmic encrustation was accompanied by an accumulation of protein moieties. Inthe same way, exopolysaccharides were associated with the extracellular mineralized filaments. The evolution of cell encrus-tation was followed by TEM over the time course of a culture: cell encrustation proceeded progressively, with rapid precip-itation in the periplasm (in a few tens of minutes), followed by the formation of surface-bound globules. Moreover, wefrequently observed an asymmetric mineral thickening at the cell poles. In parallel, the evolution of iron oxidation wasquantified by STXM: iron both contained in the bacteria and in the extracellular precipitates reached complete oxidationwithin 6 days. While a progressive oxidation of Fe in the bacteria and in the medium could be observed, spatial redox (oxi-do-reduction state) heterogeneities were detected at the cell poles and in the extracellular precipitates after 1 day. All thesefindings provide new information to further the understanding of molecular processes involved in iron biomineralization byanaerobic iron-oxidizing bacteria and offer potential signatures of those metabolisms that can be looked for in the geologicalrecord.� 2008 Elsevier Ltd. All rights reserved.

0016-7037/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.gca.2008.10.033

* Corresponding author. Fax: +33 144273785.E-mail address: miot@impmc.jussieu.fr (J. Miot).

1. INTRODUCTION

Iron-oxidizing bacteria and archeae may have a signifi-cant impact on the Earth’s surface geochemistry, by takingpart in the redox cycling of iron under both oxic and anoxic

708 J. Miot et al. / Geochimica et Cosmochimica Acta 73 (2009) 696–711

accumulation of proteins in iron-encrusted cells and anasymmetric pattern of cell mineralization at the poles.These results are both suggestive of protein aggregationand the possible relation with a preferential cell encrusta-tion would be interesting to study further. Alternatively,the preferential location of iron precipitates at the cell polesmight be reminiscent of iron oxide granules previously re-ported to accumulate at the poles of the iron-reducing bac-terium Schewanella putrefaciens (Glasauer et al., 2007).These authors proposed that this polar location could resultfrom a facilitated iron uptake at the poles.

4.2. Evolution of iron redox state in the periplasm

Proteins involved in the transfer of electrons during ironoxidation by anaerobic nitrate-dependent iron-oxidizingbacteria are not known nor localized yet. However, recentstudies of the anaerobic iron-oxidizing phototrophs Rhodo-

pseudomonas palustris strain TIE-1 and R. ferroxidans strainSW2 suggested that a soluble decahaeme c-type cyto-chrome, potentially playing a crucial role in iron oxidation,could reside in the periplasm (Croal et al., 2007; Jiao andNewman, 2007). Similarly, in the acidophilic iron-oxidizingbacterium Acidithiobacillus ferroxidans, the electron trans-fer system coupled to iron oxidation is expected to functionin the periplasmic space (Bruscella et al., 2005) and in par-ticular, the cytochrome c oxidase has been described as amembrane-bound protein (Yamanaka and Fukomori,1995). In the present study, significant amounts of Fe(III)within BoFeN1 periplasm were observed from the begin-ning of the culture, increasing over time. These resultsstrongly suggest that at least one of the steps of iron oxida-tion takes place in the periplasm of this strain as well.

Given the high permeability of the outer membrane ofGram-negative bacteria, it is likely that the higher theFe(II) concentration in the culture medium, the higher theFe(II) concentration in the periplasm. Hypothesizing thatFe(II) oxidation occurs within the periplasm of BoFeN1,the redox state of periplasmic iron phosphates is directlyinfluenced by the balance between soluble Fe(II) concentra-tions in the culture medium and Fe(III) formed by meta-bolic oxidation. The following scenario can thus beproposed: at time zero, there is a high concentration ofFe(II) in solution, and part of this iron is taken up and oxi-dized by the bacteria, leading to the precipitation of amixed-valence iron phase in the periplasm. Over the courseof culture growth, the concentration of dissolved Fe(II) insolution decreases and thus the average iron redox stateof the periplasmic precipitate increases. This scenario couldpartly explain how the redox state of the periplasmic pre-cipitate roughly follows the iron redox state of the bulksolution. Moreover, in a scenario where Fe oxidation stopsas soon as precipitation occurs within the periplasm, onecould expect to find a mixture of cells with periplasmic pre-cipitates exhibiting various iron redox states ranging from amixed-valence compound towards a pure Fe(III) com-pound in the latest stages of growth. In contrast, althoughthe number of analyses was inherently limited by themicroscopy approach, the iron redox state of the periplas-mic precipitates was constant from one cell to another at

a given growth stage. Two hypotheses could account forthese results: (1) a constant re-equilibration of the redoxstate of the precipitates after their formation, or (2) anincreasing fraction of biomineralizing cells at a given stagecompared to former stages (potentially caused by a combi-nation of growth and precipitation kinetics or by cell lysis).These hypotheses will have to be assessed in future studies.Overall, the homogeneity of the iron redox state and thecolocalization of Fe and P evidenced by EDXS (Fig. 6)both suggest the existence of a single Fe-phosphate phaseat a given stage of the culture. Some authors have proposedthe existence of an autocatalytic process consisting in theoxidation of Fe(II) in solution by Fe(III)-containing solids.Fakih et al. (2008) have observed a decreased autocatalyticeffect of the oxidation of Fe(II) associated with bacteria incultures of Bacillus subtilis. While this process may occur inBoFeN1 cultures, it should be noted that it would slowdown the rate of Fe(II) oxidation within the periplasm ofBoFeN1 cells. However, the main trend we observe is thereverse, i.e. faster oxidation of Fe(II) on the cells, whichlikely results at first order from the metabolic oxidationof Fe(II) by the bacteria.

4.3. Extracellular precipitation of iron phosphates

4.3.1. Role of organics in extracellular precipitation of iron

phosphates

Extracellular mineralized filaments consisting of ironphosphates were shown to be associated with exopolysac-charides (Fig. 7) in the BoFeN1 cultures. These precipitatesdisplayed a morphology (Fig. 4) and an organic contentsimilar to the polysaccharide-associated iron oxyhydroxidefilaments described by Chan et al. (2004) in a neutral-pHenvironment. These authors suggested that extrusion ofpolysaccharides localized iron precipitation in proximityto the cell membranes, thus harnessing the proton gradientfor energy generation. In addition, this study showed thatpolysaccharide strands triggered the nucleation of high as-pect ratio pseudo-single crystals of akaganeite. In contrast,iron phosphates formed by BoFeN1 are amorphous. How-ever, our results suggest that EPS may facilitate iron phos-phate nucleation or aggregation of iron phosphate colloidsoutside the cells, concomitantly with periplasmicencrustation.

4.3.2. Origin of extracellular Fe(III)

STXM analyses of the extracellular precipitates formedin BoFeN1 cultures highlighted a local heterogeneity ofiron redox state, with highly oxidized spots disseminatedwithin a mixed-valence iron phosphate matrix (Fig. 10).Furthermore, iron was almost completely oxidized in theextracellular precipitates after three days of culture(Fig. 11 and Table 2). These extracellular iron biomineralsare reminiscent of those described in neutrophilic iron-oxi-dizing phototroph cultures of R. palustris strain SW2 for in-stance (Kappler and Newman, 2004). Those authors havequestioned the mechanisms involved in the transfer ofFe(III) from the cells to the extracellular medium. Indeed,the presence of Fe(III) away from the bacteria is somewhatsurprising given the low solubility of Fe(III) at neutral pH

710 J. Miot et al. / Geochimica et Cosmochimica Acta 73 (2009) 696–711

chrotron radiation facilities and we thank Stephanie Belin, Emili-ano Fonda and Valerie Briois for their assistance in using theSAMBA beamline. We acknowledge Florian Hegler for his helpcultivating the nitrate-depending Fe(II)-oxidizing strain BoFeN1.The contribution from AK and NP was funded by an Emmy-Noe-ther fellowship and additional funding from the German ResearchFoundation (DFG) to AK. This study was supported by a grantfrom Agence Nationale de la Recherche (K.B. and J.M.). This isIPGP contribution No. 2441. This work was supported by ACI/FNS grant 3033.

APPENDIX A. SUPPLEMENTARY DATA

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.gca.2008.10.033.

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Available online at www.sciencedirect.comJournal of

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Journal of Structural Biology 161 (2008) 401–410

StructuralBiology

Combined structural and chemical analysis of the anammoxosome:A membrane-bounded intracytoplasmic compartment

in anammox bacteria

Laura van Niftrik a, Willie J.C. Geerts b, Elly G. van Donselaar b, Bruno M. Humbel b,Alevtyna Yakushevska b, Arie J. Verkleij b, Mike S.M. Jetten a, Marc Strous a,*

a Department of Microbiology, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlandsb Department of Molecular Cell Biology, Electron Microscopy group, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

Received 2 March 2007; received in revised form 18 May 2007; accepted 20 May 2007Available online 2 June 2007

Abstract

Anammox bacteria have unique intracellular membranes that divide their cytoplasm into three separate compartments. The largestand innermost cytoplasmic compartment, the anammoxosome, is hypothesized to be the locus of all catabolic reactions in the anammoxmetabolism. Electron tomography showed that the anammoxosome and its membrane were highly folded. This finding was confirmed bya transmission electron microscopy study using different sample preparation methods. Further, in this study electron-dense particles wereobserved and electron tomography showed that they were confined to the anammoxosome compartment. Energy dispersive X-ray anal-ysis revealed that these particles contained iron. The functional significance of a highly folded anammoxosome membrane and intracel-lular iron storage particles are discussed in relation to their possible function in energy generation.� 2007 Elsevier Inc. All rights reserved.

Keywords: Anammox bacteria; Compartmentalization; Anammoxosome; Electron tomography; Energy dispersive X-ray analysis (TEM-EDX); Intrac-ellular iron particles

1. Introduction

The anaerobic ammonium oxidizing (anammox) bacte-ria belong to a phylum with many unusual properties: thePlanctomycetes. Unlike most other bacteria, species withinthis phylum lack peptidoglycan and have a proteinaceouscell wall (Konig et al., 1984; Liesack et al., 1986; Strouset al., 2006). Further, all known Planctomycetes have acompartmentalized cytoplasm (Fuerst, 1995). The organi-zation of their cell envelope is very different from otherbacteria (Lindsay et al., 2001; Strous et al., 2006); thereare two membranes on the inner side of the cell wall andno outer membrane (Fig. 1). Of these two Planctomycete

membranes, the outermost is closely apposed to the inside

1047-8477/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.jsb.2007.05.005

* Corresponding author. Fax: +31 24 3652830.E-mail address: M.Strous@science.ru.nl (M. Strous).

of the cell wall. This membrane has been defined as thecytoplasmic membrane based on the finding of RNA onits inner side by RNase-gold labeling (Lindsay et al.,1997, 2001). The second membrane is therefore defined asan intracytoplasmic membrane. In the anammox case, athird and final membrane surrounds an anammoxosome;a bacterial ‘‘organelle’’ or vacuole without ribosomes.The anammox cytoplasm is thus divided into three separatecompartments bounded by individual bilayer membranes.This gives rise to the following cell plan (Fig. 1, and see alsoFig. 4A): proteinaceous and peptidoglycan-less cell wall,cytoplasmic membrane, paryphoplasm compartment,intracytoplasmic membrane, ribosome-, and nucleoid-con-taining riboplasm compartment, anammoxosome mem-brane and anammoxosome compartment.

In addition to the cell plan, the anammox membranelipids are also atypical. Anammox bacteria contain acombination of ether-linked (typical of the Archaea) and

L. van Niftrik et al. / Journal of Structural Biology 161 (2008) 401–410 409

levels of phosphorus compared to the other cell compart-ments, with an iron-to-phosphorus ratio of 0.83. So it ispossible that phosphate is a significant component of theiron complex here as well. Though the ratios of the otherelements do not seem to be in favor of another form ofiron, other complexes cannot be ruled out. The anam-moxosome particles, 16–25 nm in diameter, are somewhatlarger than the typical bacterioferritins which are 12 nmin diameter. Their appearance in electron microscopy isalso different from other known iron storage material, forexample, the bacterioferritins in E. coli (Yariv et al.,1981), the iron particles in Mycoplasma capricolum (Baum-inger et al., 1980), or the iron particles in Shewanella putre-

faciens (Glasauer et al., 2007). The anammox particlesdiffer in size (slightly bigger than the first two and smallerthan the last), their shape is more rounded and distinct,and the particles are not segregated at or associated witha membrane like in M. capricolum and S. putrefaciens. Alsounlike S. putrefaciens, where the iron particles are formedduring anaerobic dissimilatory iron reduction, or M. capri-

colum, where the iron particles are formed upon growth iniron-enriched medium, the anammox particles were alwayspresent; both in the two different anammox species and atdifferent cell cycle stages. Never in 3D was a cell observedwithout the iron particles. The heterogeneity in electron-density and varying iron levels of the particles couldpossibly reflect a large protein with an iron core. The‘‘Candidatus K. stuttgartiensis’’ genome was also examinedfor either of the two electron carriers that are hypothesizedto act as Bfr-specific reductases in the mobilization of ironfrom Bfr; Bfd (Bfr-associated ferredoxin) (Andrews et al.,2003) and Rd-2 (rubredoxin-type 2) (da Costa et al.,2001). But the ‘‘Candidatus K. stuttgartiensis’’ genome con-tained only type 1 rubredoxins (kustc0323, kustc1169, andkustd2038) and no Bfd. The presence of bacterioferritingenes in the ‘‘Candidatus K. stuttgartiensis’’ genome couldindicate that the iron particles in the anammoxosomecompartment, observed with electron tomography andidentified with TEM-EDX, are indeed bacterioferritins.

Only speculations can be made about the functional sig-nificance of these anammoxosome iron particles. Oneexplanation could be that the anammox iron storage islinked to iron respiration (Strous et al., 2006). Thoughanammox bacteria are capable of iron respiration, theenergy is mainly generated through anaerobic ammoniumoxidation with nitrite. Another possibility is that iron stor-age in anammox bacteria is a preconditioned response toiron scarcity in the environment. Iron storage is ofteninduced when its availability is scarce (Andrews, 1998).The anammox medium contains 6.25 mg/L iron sulfate,of which the anammox bacteria use only half (Strous,unpublished results), so it is unlikely that the anammoxbacteria are grown under iron-limiting conditions. How-ever, anammox bacteria do produce a large amount ofhaem-containing enzymes (>20% [w/w] of total cell pro-tein) so it could very well be that this organism scavengesand stores iron to have an excess supply of iron for future

haem synthesis. The location of the iron storage is consis-tent with the postulated location of the anammox haemproteins (also inside the anammoxosome), if iron wouldbe inserted into the protohaem inside the anammoxosome.In this way the iron particles would serve as an iron storagefacility for the haem-containing enzymes that are involvedin the electron transport chain or they might representaggregated iron-rich proteins themselves.

The anammoxosome membrane curvature fits thehypothesis that the membrane-bounded anammoxosomecompartment is used as an energy generator. The otheraspects of the anammoxosome described in this study,the iron particles and tubule-like structures, remain a puz-zle to be solved. All in all, this bacterial ‘‘organelle’’ is trulyunique in its kind and is very fascinating from an evolu-tionary perspective.

Acknowledgments

We thank Katinka van de Pas-Schoonen and BoranKartal for the anammox enrichment cultures, ChrisSchneijdenberg and Hans Meeldijk for technical instruc-tions, Prof. John Geus for discussions on the TEM-EDXanalysis and Prof. Gijs Kuenen for general discussionsand feedback.

Appendix A. Supplementary data

Supplementary data associated with this article canbe found, in the online version, at doi:10.1016/j.jsb.2007.05.005.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2008, p. 410–415 Vol. 74, No. 20099-2240/08/$08.00�0 doi:10.1128/AEM.01812-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Novel Combination of Atomic Force Microscopy and EpifluorescenceMicroscopy for Visualization of Leaching Bacteria on Pyrite�

Stefanie Mangold,1,2 Kerstin Harneit,1 Thore Rohwerder,1 Gunter Claus,2 and Wolfgang Sand1*University of Duisburg-Essen, Biofilm Centre, Aquatic Biotechnology, Geibelstr. 41, 47057 Duisburg, Germany,1 and

Mannheim University of Applied Sciences, Institute of Technical Microbiology,Windeckstrasse 110, 68163 Mannheim, Germany2

Received 3 August 2007/Accepted 24 October 2007

Bioleaching of metal sulfides is an interfacial process comprising the interactions of attached bacterial cellsand bacterial extracellular polymeric substances with the surface of a mineral sulfide. Such processes and theassociated biofilms can be investigated at high spatial resolution using atomic force microscopy (AFM).Therefore, we visualized biofilms of the meso-acidophilic leaching bacterium Acidithiobacillus ferrooxidansstrain A2 on the metal sulfide pyrite with a newly developed combination of AFM with epifluorescencemicroscopy (EFM). This novel system allowed the imaging of the same sample location with both instruments.The pyrite sample, as fixed on a shuttle stage, was transferred between AFM and EFM devices. By staining thebacterial DNA with a specific fluorescence dye, bacterial cells were labeled and could easily be distinguishedfrom other topographic features occurring in the AFM image. AFM scanning in liquid caused deformation anddetachment of cells, but scanning in air had no effect on cell integrity. In summary, we successfully demonstratethat the new microscopic system was applicable for visualizing bioleaching samples. Moreover, the combina-tion of AFM and EFM in general seems to be a powerful tool for investigations of biofilms on opaque materialsand will help to advance our knowledge of biological interfacial processes. In principle, the shuttle stage canbe transferred to additional instruments, and combinations of AFM and EFM with other surface-analyzingdevices can be proposed.

By combining atomic force microscopy (AFM) and fluores-cence microscopy, two techniques with complementarystrengths and weaknesses are joined to yield a powerful toolfor the investigation of biological samples. The atomic forcemicroscope (1, 2, 5) is a mechanical imaging device that re-quires minimal sample preparation and creates three-dimen-sional images with high spatial resolution. Since its introduc-tion in 1986 (2), AFM has widely been used in biology owing toits ability to visualize samples under physiological conditions.Consequently, this gives a great advantage over electron mi-croscopy where extensive sample preparation and measure-ment in high vacuum disallow investigations of native samples.Subnanometer resolution with AFM, for example, has beenachieved in the visualization of bacterial cell surface structuresconsisting of two-dimensional arrays of protein or glycoproteinsubunits under physiological conditions (25, 26, 35). The topo-graphic information for AFM images is acquired by scanning asurface with a sharp probe. Since the underlying tip-sampleinteraction forces are nonspecific, AFM is applicable to awide range of samples. However, at the same time this limitsthe capacity of AFM to discern different structures of a sampleunless they have a particular shape and size, which have to beknown a priori. In contrast, optical imaging techniques such asfluorescence microscopy are routinely used for the identifica-tion of various species comprising a sample. By fluorescencelabeling of DNA, RNA, proteins, polysaccharides, or lipids,

these biomolecules can be tracked in complex samples such asbiofilms, single cells, or membranes. However, the resolvingpower of optical imaging is fundamentally limited by diffrac-tion to about 250 nm or more precisely to half the wavelengthsof the light used (17). Consequently, both techniques comple-ment each other in that AFM has excellent spatial resolutionbut lacks identification capabilities, whereas fluorescence mi-croscopy offers poor resolution but excellent identificationproperties. Hence, the combination of both microscopy tech-niques provides AFM with the desirable identification ability.Therefore, the combined microscopy technique has been es-tablished for translucent samples within the last decade (9,15–17, 19, 21, 23, 28, 39). Indeed, the integration of both AFMand transmission fluorescence microscopy in one device isstraightforward since an inverted optical microscope can beused for imaging a sample from the back side and AFMmeasurements can be performed upon the sample. Hence,combinations of AFM with confocal microscopy (9, 16, 17,19) or with total internal reflection fluorescence microscopy(23, 28) have been reported. However, for the investigationof natural biofilms or other biotic, as well as abiotic, struc-tures on opaque substrata, this combination of both micro-scopes in one device is not applicable. Nevertheless, consid-ering the outstanding role of opaque materials in manyfields, e.g., in biocorrosion and biodeterioration, there is anessential demand for joint imaging with AFM and epifluo-rescence microscopy (EFM).

In particular, investigations of biofilms on opaque substratahave a high relevance for bioleaching research since specificmicrobe-mineral surface interactions are involved in the mi-crobially mediated release of heavy metal ions from insolublecompounds. In case of metal sulfides, the metal recovery is

* Corresponding author. Mailing address: University of Duisburg-Essen, Biofilm Centre, Aquatic Biotechnology, Geibelstr. 41, 47057Duisburg, Germany. Phone: 49 203 379 4475. Fax: 49 203 379 4495.E-mail: wolfgang.sand@uni-due.de.

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sample location by both techniques is thus possible. Imagingwith AFM of cells of A. ferrooxidans A2 on pyrite was possibleby scanning in contact mode in air or by intermittent contactmode in fluid after the sample was air dried. In previous stud-ies, it has been noted that planktonic bacteria that are notsufficiently immobilized to the surface might be moved by theAFM probe during imaging in fluid (5, 40). In the case ofplanktonic bacteria, simple physical adsorption of whole bac-terial cells to a substratum is often not sufficiently strong as thecontact area between cell and surface is small. To circumventthis problem, air drying, chemical fixation, or mechanical im-mobilization using membrane filters were suggested (5). Incontrast, the problem of a relocation of sessile bacterial cells,meaning not attached planktonic cells but the ones grown in abiofilm environment, by the interaction with the AFM probehas not yet been addressed (1). However, the important role ofbacterial EPS for attachment and the natural immobilizationof bacteria within biofilms are pointed out. In conclusion, theinvestigations of native biofilms without the need of additionalfixation are preferable.

Thus far, biofilms of A. ferrooxidans on metal sulfides havenot been visualized under physiological conditions, since AFMinvestigations have been carried out in air only (14, 24, 30).However, fully hydrated biofilms of other organisms have beenvisualized by using AFM (4, 18). In the study of Bremer et al.(4), 7-day-old freshwater biofilms on copper surfaces wereexamined by scanning in culture medium. These AFM imagesrevealed a heterogeneous multilayered biofilm and bacterialcells covered with EPS. Detachment or dislocation of bacterialcells within these biofilms is not reported. In contrast, the workof Kolari et al. (18) demonstrated that cells of Deinococcusgeothermalis in 1-day-old, hydrated biofilms are dislocated butnot detached by the AFM probe. These authors attribute thisbehavior to flexible, adhesive polymers that provide bacterialcells with a strong but flexible gluing to the surface. As shownin our study the attachment of A. ferrooxidans after 4 days ofbiofilm formation is not sufficiently strong to withstand thelateral forces caused by the AFM probe under some experi-mental conditions. As bacterial attachment grows strongerwith time, imaging of unfixed natural biofilms of A. ferrooxi-dans might be possible with older samples.

After bacteria were dislocated by AFM scanning, microbialfootprints were visible at the former attachment location onthe pyrite. In line with this observation are previous reportsindicating that bacteria are able to label surfaces with varioussubstances, mainly EPS (27). Most probably, the microbialfootprints of A. ferrooxidans A2 visualized here are such EPS,since these play a pivotal role in attachment and the so-calledcontact mechanisms in bioleaching (10, 12, 14, 32, 34). Usingthe new system, it becomes now possible to validate the role ofEPS and their composition by staining them with fluorescentlylabeled lectins (20, 38), followed by a combined visualizationwith AFM and EFM.

In the present study, we show that the new system for com-bined imaging with AFM and EFM on opaque samples isfeasible for the application to A. ferrooxidans on pyrite. Mostmaterials, whether they are manufactured or natural, are nottranslucent. For this great variety of materials, the novel sys-tem described here offers new opportunities for combined in-vestigation by AFM and EFM. As demonstrated here there

will be a great potential for combined AFM and EFM in theinvestigation of biofilms on opaque substrata. Biofilms are themost important mode of growth for prokaryotes, and they haveimplications in industry, infectious disease, and the naturalenvironment (1, 13, 37). Thus, it is important to learn moreabout this stage of prokaryotic life. In this connection, thenovel system can help to elucidate many aspects of biofilmsformed on their natural or synthetic opaque substrata. AFMimages of hydrated biofilms can provide information abouttheir spatial arrangement and thus give indications about masstransfer from bulk solution to single cells. In combination withEFM, various constituents of the biofilm and its matrix can beidentified. Possible applications are microbially influenced cor-rosion, biofilms formed on medical implants, or biofouling inindustrial plants. In addition, combined AFM and EFM cannow be applied to the investigation of opaque plant material.Moreover, not only are biological applications possible but alsoinvestigations in surface chemistry involving fluorescent poly-mers or coatings can profit from the combination of AFM andoptical microscopy. Thus, the combination of AFM and EFMis a powerful tool for the research on surface related phenom-ena and offers new opportunities for the investigation of allkinds of opaque samples. In principle, the employed shuttlestage can also be transferred to additional devices. Thus, acombination with other instruments, e.g., for measuring chargedifferences on the surface of materials by a Kelvin probe (10)or the quantitative mapping of elemental distributions by syn-chrotron X-ray fluorescence microscopy (6, 8, 11), may beachieved in the future.

ACKNOWLEDGMENTS

This study was carried out in the frame of COST D33 and BioMinE(European project contract NMP1-CT-500329-1). We acknowledgethe financial support given to this project by the European Commis-sion under the Sixth Framework Programme for Research and Devel-opment.

We also thank JPK Instruments for their cooperation.

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ORIGINAL ARTICLES

Extracellular electron transfer

Extracellular electron transfer: wires, capacitors, iron lungs, and more

DEREK R. LOVLEY

Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA

Received 7 October 2007; accepted 25 February 2008

Corresponding author: Derek R. Lovley. Tel: 413-545-9651; fax: 413-545-1598; e-mail: dlovley@microbio.umass.edu.

‘and by the help of microscopes, there is nothing so small, as toescape our inquiry; hence there is a new visible world discoveredto the understanding.’

Robert Hooke

Terry Beveridge’s broad and insightful studies of microbe–metal interactions, powered in large part by amazing microscopy,have served as an inspiration and provided guidance for manyof us working in this field. This perspective deals with just oneof the aspects of microbe–metal interactions to which Terrysignificantly contributed, extracellular electron transfer.

For purposes of this perspective extracellular electron transferis defined as the process in which electrons derived from theoxidation of electron donors are transferred to the outer surfaceof the cell to reduce an extracellular terminal electron acceptor.Extracellular electron acceptors may be soluble, yet reducedoutside the cell because they are too large to enter the cell, asis probably the case for humic substances (Lovley

et

al

., 1996,1998). Some soluble metal species, such as U(VI) complexedwith carbonate, or Fe(III) chelated with citrate, are often reducedextracellularly (Lovley

et

al

., 1991; Marshall

et

al

., 2006;Coppi

et

al

., 2007; Gralnick & Newman, 2007; Shelobolina

et

al

., 2007) as is dimethyl sulfoxide (Gralnick

et

al

., 2006).It has also been proposed that organisms involved in thesyntrophic degradation of organic compounds may make theappropriate connections to directly exchange electrons (Stams

et

al

., 2006). However, extracellular electron transfer tominerals, such as Fe(III) and Mn(IV) oxides (Lovley, 1991;Lovley

et

al

., 2004; Gralnick & Newman, 2007; Shi

et al.

,2007) and to electrodes (Lovley, 2006a,b), is probably of thegreatest interest at present because of their environmentalsignificance and practical applications. Oxidation of organicmatter coupled to the reduction of Fe(III) and Mn(IV) oxidesplays an important role in the carbon, iron, and manganese

cycles in sedimentary environments, and also influences thefate of a diversity of trace metals and phosphate (Lovley, 1993;Thamdrup, 2000). Anaerobic oxidation of organic contaminantswith the reduction of Fe(III) is important in groundwaterbioremediation (Lovley

et

al

., 1989; Lovley, 1997), and stim-ulating dissimilatory metal reduction has shown promise as amethod for immobilizing toxic metals in the subsurface(Anderson

et

al

., 2003; Lloyd

et

al

., 2003). Oxidation oforganic matter coupled to electron transfer to electrodes is apotential strategy for harvesting electricity from the environ-ment and waste organic matter (Lovley, 2006a).

The primary goal for many investigators of extracellularelectron transfer is to determine how electrons are transferredto the electron acceptors and the factors controlling the rateand extent of this process. With this information in hand, itmay be possible to optimize practical applications and bettermodel natural processes. Some of the potentially importantquestions still remaining in extracellular electron transfer arediscussed below in relation to key publications of Terry’s thathave led to the development of these questions and/or haveprovided the techniques that may lead to their answers.

WHAT CONSTITUTES A ‘MICROBIAL NANOWIRE’ AND ARE THEY THE FINAL CONDUIT FOR ELECTRON TRANSFER TO EXTRACELLULAR ELECTRON ACCEPTORS SUCH AS FE(III) OXIDES AND ELECTRODES?

Terry was involved in some of the earliest work on the conceptthat some structures emanating from microorganisms may beconductive and might serve as a conduit for extracellular electrontransfer (Gorby

et

al

., 2006). ‘Microbial nanowires’ (Reguera

et

al

., 2005, 2006; Gorby

et

al

., 2006) are certainly an attractive,easily visualized, explanation for extracellular electron transfer.

Extracellular electron transfer

227

© 2008 The AuthorJournal compilation © 2008 Blackwell Publishing Ltd

DO SOME DISSIMILATORY METAL-REDUCING MICROORGANISMS HAVE ‘IRON LUNGS’?

Another fascinating observation made by Terrry and colleaguesis that

Shewanella putrefaciens

CN32 has the novel capacityto store iron and manganese minerals intracellularly duringdissimilatory Fe(III) or Mn(IV) reduction (Glasauer

et

al

.,2002, 2004, 2007). Interestingly, the other

Shewanella

speciesthat have been evaluated do not appear to share this ability(Glasauer

et

al

., 2007). Terry and colleagues have suggestedthat the intracellular iron oxide deposits might function ‘as areservoir of oxidant’ because they are depleted under sustainedanoxic conditions (Glasauer

et

al

., 2007). This is an intriguingconcept.

We have yet to identify similar Fe(III) or Mn(IV) oxidedeposits in

Geobacter

species, but have discovered what webelieve is an alternative ‘iron lung’ for

Geobacter

species(Esteve-Núñez

et

al

., 2008). This is the abundance of iron inthe periplasm and outer membrane of

Geobacter

species, whichis found in the form of

c

-type cytochromes. The hypothesis isthat these extracytoplasmic cytochromes can act as capacitorsto store electrons in the periplasm and outer membrane whenexternal electron acceptors are not immediately available. Thereason that this is significant is that energy conservation fromorganic matter oxidation in

Geobacter

species is expected toresult solely from the transport of electrons across the innermembrane with associated proton-pumping, which generatesa proton-motive force that drives ATP formation, flagellarotation, etc. (Mahadevan

et

al

., 2006). Once the electronsare transferred to the periplasm, subsequent electron transfersteps to extracellular electron acceptors do not further contributeto the cellular energy yield. This is evident from the findingthat

Geobacter

cell yields are comparable with soluble andinsoluble forms of Fe(III), even though there are substantialdifferences in the redox potentials of these electron acceptors.The external electron acceptors merely function to oxidize theelectron acceptors earlier in the electron transfer chain topermit continued electron transfer across the inner membrane.

The electron-accepting capacity of the

c-type cytochromesin G. sulfurreducens, determined with two independent methods,was estimated to be c. 1.6 × 10−17 mol electrons per cell(Esteve-Núñez et al., 2008). This capacitance can, in theabsence of an extracellular electron acceptor, potentiallysupport enough inner membrane electron transfer/protonpumping for G. sulfurreducens to satisfy its maintenance energyrequirements for 8 min or to swim several hundred cell lengthsbefore all the electron-accepting capacity of the hemes isexhausted (Esteve-Núñez et al., 2008).

The ability to temporarily maintain active electron transferacross the inner membrane in the absence of an extracellularelectron acceptor may be key to the survival of Geobacter spe-cies in the subsurface and the often-noted ability of Geobacterspecies to outcompete a diversity of other dissimilatory Fe(III)reducers in such environments. This is because, in contrast to

Shewanella and Geothrix species that produce electron shuttles(Newman & Kolter, 2000; Nevin & Lovley, 2002a,b; Lieset al., 2005; Lanthier et al., 2008), Geobacter species mustdirectly contact Fe(III) oxides in order to reduce them (Nevin& Lovley, 2000). This presents a challenge to Geobacter speciesin subsurface environments in which Fe(III) sources, such asFe(III) oxides and structural Fe(III) in clays (Shelobolina et al.,2003, 2006), are heterogeneously dispersed. Once a cell depletesthe Fe(III) oxide in one locale it must find an alternativesource of the electron acceptor. The current model is thatGeobacter species are motile and hunt for Fe(III) oxides(Childers et al., 2002). This is consistent with an analysis ofthe microbial community during in situ uranium bioremedia-tion, which indicated that the metabolically active Geobacterspecies are highly planktonic in the subsurface during activeFe(III) oxide reduction (Holmes et al., 2007). However, itpresents the quandary of how cells can generate ATP requiredfor motility as well as cell maintenance when they are in aplanktonic state searching for a new source of electron acceptor.The electron-accepting capacity of the extracytoplasmic cyto-chromes provides a potential short-term solution. Respirationcan continue and then once a new source of Fe(III) is locatedthe electrons stored in the cytochromes can be dischargedfrom these mini-capacitors.

In addition to the direct measurements of the electronstorage capacity of the c-type cytochromes in G. sulfurreducensthere is substantial circumstantial evidence that is consistentwith this concept. For example, although genetic studies haveindicated that some c-types are essential for growth of G. sul-furreducens on Fe(III), there is remarkably little conservationof c-type cytochromes genes across the six Geobacter specieswhose genomes have been sequenced. This suggests that therehas not been evolutionary pressure to maintain specific struc-tures that might promote interaction of the cytochromes withelectron acceptors. However, there has apparently been evo-lutionary pressure for the Geobacter species to maintain anabundance of hemes. Not only are there an inordinatelyhigh number of c-type cytochrome genes in Geobacter species(Methé et al., 2003) (www.jgi.doe.gov), most of these cyto-chromes have multiple hemes (27 is the maximum yet pre-dicted) giving them relatively high electron-accepting capacity.The vast majority of the cytochrome genes are expressed(Ding et al., 2006) and the cells are intensely red due to theabundance of cytochromes. Further support for the capacitorconcept is the finding that the expression of many extracyto-plasmic c-type cytochrome genes is increased during growthon either synthetic Fe(III) oxides, sediment Fe(III) oxides orelectrodes versus growth on soluble electron acceptors (Hol-mes et al., 2006) (D.E. Holmes and K.P. Nevin, unpublisheddata). Such a high abundance of cytochromes is not requiredfor Fe(III) reduction, as other Fe(III) reducers, such asPelobacter species (Lovley et al., 1995; Haveman et al., 2006)and the Fe(III)-reducing hyperthermophiles (Vargas et al., 1998;Kashefi & Lovley, 2003; Lovley et al., 2004), reduce Fe(III)

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Glasauer S, Weidler PG, Langley S, Beveridge TJ (2003) Controls on Fe reduction and mineral formation by a subsurface bacterium. Geochimica et Cosmochimica Acta 67, 1277–1288.

Glasauer S, Langley S, Beveridge TJ (2004) Intracellular manganese granules formed by a subsurface bacterium. Environmental Microbiology 6, 1042–1048.

Glasauer S, Langley S, Boyanov M, Lai B, Kemner K, Beveridge TJ (2007) Mixed-valence cytoplasmic iron granules are linked to anaerobic respiration. Applied Environmental Microbiology 73, 993–996.

Gorby YA, Yanina S, McLean JS, Rosso KM, Moyles D, Dohnalkova A, Beveridge TJ, Chang IS, Kim BH, Kim SK, Culley DE, Reed SB, Romine MF, Saffarini DA, Hill EA, Shi L, Elias DA, Kennedy DW, Pinchuk G, Watanabe K, Ishii S, Logan B, Nealson KH, Fredrickson JK (2006) Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proceedings of the National Academy of Sciences of the USA 103, 11358–11363.

Gralnick JA, Newman DK (2007) Extracellular respiration. Molecular Microbiology 65, 1–11.

Gralnick JA, Vali H, Lies DP, Newman DK (2006) Extracellular respiration of dimethyl sulfoxide by Shewanella oneidensis strain MR-1. Proceedings of the National Academy of Sciences of the USA 103, 4669–4674.

Haveman SA, Holmes DE, Ding YR, Ward JE, DiDonato RJ, Lovley DR (2006) c-type cytochromes in Pelobacter carbinolicus. Applied Environmental Microbiology 72, 6980–6985.

Holmes DE, Chaudhuri SK, Nevin KP, Mehta T, Methe BA, Ward JE, Woodward TL, Webster J, Lovley DR (2006) Microarray and genetic analysis of electron transfer to electrodes in Geobacter sulfurreducens. Environmental Microbiology 8, 1805–1815.

Holmes DE, Mester T, O’Neil RA, Adams LA, Larrahando MJ, Glaven R, Sharma M, Ward JE, Nevin KP, Lovley DR (2008) Genes for two multicopper proteins required for Fe(III) oxide reduction in Geobacter sulfurreducens have different expression patterns both in the subsurface and on energy-harvesting electrodes. Microbiology (in press).

Holmes DE, Nevin KP, Lovley DR (2004) In situ expression of nifD in Geobacteraceae in subsurface sediments. Applied Environmental Microbiology 70, 7251–7259.

Holmes DE, Nevin KP, O’Neil RA, Ward JE, Adams LA, Woodward TL, Lovley DR (2005) Potential for quantifying expression of Geobacteraceae citrate synthase gene to assess the activity of Geobacteraceae in the subsurface and on current harvesting-electrodes. Applied Environmental Microbiology 71, 6870–6877.

Holmes DE, O’Neil RA, Vrionis HA, N’Guessan LA, Ortiz-Bernad I, Larrahando MJ, Adams LA, Ward JA, Nicoll JS, Nevin KP, Chavan MA, Johnson JP, Long PE, Lovley DR (2007) Subsurface clade of Geobacteraceae that predominates in a diversity of Fe(III)-reducing subsurface environments. ISME Journal 1, 663–677.

Hunter RC, Beveridge TJ (2005a) Application of a pH-sensitive fluoroprobe (CSNARF-4) for pH microenvironment analysis in Pseudomonas aeruginosa biofilms. Applied Environmental Microbiology 71, 2501–2510.

Hunter RC, Beveridge TJ (2005b) High-resolution visualization of Pseudomonas aeruginosa PAO1 biofilms by freeze-substitution transmission electron microscopy. Journal of Bacteriology 187, 7619–7630.

Kashefi K, Lovley DR (2003) Extending the upper temperature limit for life. Science 301, 934.

Lanthier M, Gregory KB, Lovley DR (2008) Growth with high planktonic biomass in Shewanella oneidensis fuel cells. FEMS Microbiology Letters 278, 29–35.

Lies DP, Hernandez ME, Kappler A, Mielke RE, Gralnick JA, Newman DK (2005) Shewanella oneidensis MR-1 uses overlapping pathways for iron reduction at a distance and by direct contact under conditions relevant for biofilms. Applied Environmental Microbiology 71, 4414–4426.

Lloyd JR, Lovley DR, Macaskie LE (2003) Biotechnological application of metal-reducing microorganisms. Advances in Applied Microbiology 53, 85–128.

Lovley DR (1991) Dissimilatory Fe(III) and Mn(IV) reduction. Microbiology Review 55, 259–287.

Lovley DR (1993) Dissimilatory metal reduction. Annals of Review of Microbiology 47, 263–290.

Lovley DR (1997) Potential for anaerobic bioremediation of BTEX in petroleum-contaminated aquifers. Journal of Industrial Microbiology 18, 75–81.

Lovley DR (2003) Cleaning up with genomics: applying molecular biology to bioremediation. Nature Review of Microbiology 1, 35–44.

Lovley DR (2006a) Bug juice: harvesting electricity with microorganisms. Nature Review of Microbiology 4, 497–508.

Lovley DR (2006b) Microbial fuel cells: novel microbial physiologies and engineering approaches. Current Opinions in Biotechnology 17, 327–332.

Lovley DR, Baedecker MJ, Lonergan DJ, Cozzarelli IM, Phillips EJP, Siegel DI (1989) Oxidation of aromatic contaminants coupled to microbial iron reduction. Nature 339, 297–299.

Lovley DR, Phillips EJP, Gorby YA, Landa ER (1991) Microbial reduction of uranium. Nature 350, 413–416.

Lovley DR, Phillips EJP, Lonergan DJ, Widman PK (1995) Fe(III) and S° reduction by Pelobacter carbinolicus. Applied Environmental Microbiology 61, 2132–2138.

Lovley DR, Coates JD, Blunt-Harris EL, Phillips EJP, Woodward JC (1996) Humic substances as electron acceptors for microbial respiration. Nature 382, 445–448.

Lovley DR, Fraga JL, Blunt-Harris EL, Hayes LA, Phillips EJP, Coates JD (1998) Humic substances as a mediator for microbially catalyzed metal reduction. Acta Hydrochimica et Hydrobiologica 26, 152–157.

Lovley DR, Holmes DE, Nevin KP (2004) Dissimilatory Fe(III) and Mn(IV) reduction. Advances in Microbial Physiology 49, 219–286.

Lovley DR, Mahadevan R, Nevin KP (2008) Systems biology approach to bioremediation with extracellular electron transfer. In Microbial Degradation: Genomics and Molecular Biology (Diaz, E., ed.), pp. 71–96. Norfolk, UK: Caister Academic Press.

Mahadevan R, Bond DR, Butler JE, Esteve-Nunez A, Coppi MV, Palsson BO, Schilling. CH, Lovley DR (2006) Characterization of metabolism in the Fe(III)-reducing organism Geobacter sulfurreducens by constraint-based modeling. Applied Environmental Microbiology 72, 1558–1568.

Marshall MJ, Beliaev AS, Dohnalkova AC, Kennedy DW, Shi L, Wang Z, Boyanov MI, Lai B, Kemner KM, McLean JS, Reed SB, Culley DE, Bailey VL, Simonson CJ, Saffarini DA, Romine MF, Zachara JM, Fredrickson JK (2006) c-Type cytochrome-dependent formation of U (IV) nanoparticles by Shewanella oneidensis. PLoS Biology 4, e26B. DOI. 10.1371/journal.pbio.0040268.

Mehta T, Coppi MV, Childers SE, Lovley DR (2005) Outer membrane c-type cytochromes required for Fe(III) and Mn(IV) oxide reduction in Geobacter sulfurreducens. Applied Environmental Microbiology 71, 8634–8641.

Mehta T, Childers SE, Glaven R, Lovley DR, Mester T (2006) A putative multicopper protein secreted by an atypical type II secretion system involved in the reduction of insoluble electron acceptors in Geobacter sulfurreducens. Microbiology 152, 2257–2264.

Geobiology (2008),

6

, 263–269 DOI: 10.1111/j.1472-4669.2008.00160.x

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263

Blackwell Publishing Ltd

ORIGINAL ARTICLES

Biogeochemical studies through electron microscopy

Resolving biogeochemical phenomena at high spatial resolution through electron microscopy

G. G. GEESEY,

1

T. BORCH

2

AND C. L. REARDON

1

*

1

Department of Microbiology and Center for Biofilm Engineering, Montana State University, Bozeman, MT 59717-3520, USA

2

Departments of Chemistry and Soil and Crop Sciences, Colorado State University, Fort Collins, CO 80523-1170, USA

Received 06 September 2007; accepted 28 March 2008

Corresponding author: G. G. Geesey. Tel.: 406-994-3820; fax: 406-994-4926; e-mail: gill_g@erc.montana.edu.

Our understanding of microbe-metal interactions has advanceddramatically since the mid-1970s when little was known aboutthe reactivity of bacterial cell wall components toward metalions in the extracellular milieu. Although certain metals suchas and Pb

+

were known to react with components ofbacterial cell walls and used to visualize their structure byelectron microscopy (Garland

et

al

., 1975), little physicochemicaldata were available on the specificity and sites of interactions(Humphrey & Vincent, 1966; Heptinstall

et

al

., 1970; Irvin

et

al

., 1975; Lambert

et

al

., 1975; Raymond & MacLeod,1975). Furthermore, there were no model systems to explorethe mechanisms of these interactions. This began to changewhen Beveridge and Murray used isolated cell walls of

Bacillussubtilis

to quantify metal ion binding to wall components.Beveridge demonstrated that cell walls concentrated cationssuch as Mg

++

, Na

+

, K

+

, Cu

++

and Fe

+++

, but not Ba

++

, Li

+

orAl

+++

(Beveridge & Murray, 1976). Since these initial studies,Beveridge and his students and collaborators have contributedgreatly to our understanding of the complex interactions betweenmicrobial cell surface polymers and metals in the environment.As fellow scientists working in this research area, we have developeda deep admiration of Beveridge’s scientific insight, technicalskills and collegial demeanor. Not surprisingly, Beveridge’sresearch has had a significant impact on our research, as wellas on the research of our collaborators and colleagues, and willlikely influence the work of future generations of scientistsworking in the field of geobiology. Some examples are cited below.

Beveridge’s research from the very beginning provided thetype of detailed description of the interaction between bacterialcell wall components and various metal ions that offeredunique perspectives on function. For example, through a

quantitative comparison of metal bound to chemically modifiedcell walls of

B. subtilis

, Beveridge determined that cell wallcarboxyl groups more than other functional groups played animportant role in metal uptake (Beveridge & Murray, 1980).These results revealed for the first time the importance of cellwall biopolymer chemistry on metal ion uptake by bacterialcells. The interplay of metal concentrations and other environ-mental variables undoubtedly influence cell wall chemistry andstructure. Future researchers investigating metal interactionswith bacteria in their natural habitats will likely find that as cellwall biopolymer chemistry changes in response to the conditionsof the surrounding environment, other functional groups mayassume a greater role in binding metals.

A potentially useful approach to characterize at the micro-meter- and nanometer-scale functional groups associated withmicrobial cell walls and extracellular polymers that bind metalsis synchrotron radiation-based scanning transmission X-raymicroscopy (STXM). Distinct from other high-resolutiontechniques, STXM allows samples to remain hydrated at ambientatmospheric pressure, thus minimizing introduction ofartifacts. One of us (TB) has used this approach to generatecarbon K-edge and Fe L

III

-edge maps and near-edge X-rayabsorption fine structure (NEXAFS) spectra that correlate theabundance of Fe with carbonate and carboxyl concentrationsat specific sites on a single cell of the bacterium

Shewanellaputrefaciens

using ferric-citrate as terminal electron acceptorand lactate as the electron donor (Fig. 1A–C). Since thisapproach can also distinguish different species of a metal andtheir relative abundance at different sites on a single cell(Fig. 1D,E), it has the potential to correlate functionalmoieties of cell wall and extracellular polymers such as amide,phenolic or ketone, aromatic groups (carbon K-edge), andphosphate groups (P K-edge) with a specific species of a metal(i.e. Fe(II) and Fe(III) using Fe L

III

-edge spectra).

*Present address: Biological Sciences Division, Pacific North-westNational Laboratory, Richland, WA 99352-1793, USA.

UO2++

266

G. G. GEESEY

et al.

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd

complements his own in electron microscopy. Collaborativeresearch was a trademark of Beveridge’s approach to sciencelong before it became a necessity in today’s research climate.Terry’s easy-going manner facilitated collaborative interactions,while his passion for science made the collaborations highlyproductive. We cannot think of many other scientists who haveused the collaborative research approach as successfully asBeveridge. For example, through collaboration, he combinedtransmission electron microscopy (TEM) with small angleX-ray scattering, atomic absorption spectroscopy, and energy-dispersive X-ray spectroscopy (EDX) to describe the inter-actions of Ga, U, and Pt with

P. fluorescens

(Krueger

et

al

.,1993). Through other collaborations, he combined TEMwith EDX, and inductively coupled plasma atomic-emissionspectroscopy to investigate the influence of cell physiology onthe binding and uptake of metals by cells. Using this suite ofanalytical techniques Beveridge and coworkers showed thatprotons pumped into the cell envelope of

B. subtilis

duringrespiration to establish a proton motive force compete withmetal cations for binding sites in the envelope (Urrutia Mera

et

al

., 1992). Actively respiring cells bound less metal on theirsurface than did moribund cells. Terry always gave the impres-sion, however, that he valued the personal interactions with hiscolleagues as much or more than the resulting scientificdiscoveries.

Among Beveridge’s most significant contributions toscience is his work on the involvement of microorganisms inmineral diagenesis and the role of metal cations in the preser-vation of microbial cells in the fossil record (Ferris

et

al

., 1986;Ferris

et

al

., 1988). In the early 1980s, he and his collaboratorsemployed high-resolution electron microscopy to follow themineralization of bacteria and their component parts(Beveridge

et

al

., 1983). They demonstrated that the loading

of cell walls of

B. subtilis

with metal ions in an artificialsediment environment leads to the formation of authigenicmineral phases such as crystalline metal sulfides, and thepreservation of bacterial cells in sedimentary rock. Beveridge,in collaboration with Ron Oremland at the US GeologicalSurvey, using a suite of complementary analytical techniquesthat included UV-VIS spectroscopy, Raman spectroscopy, andtransmission and scanning electron microscopy, showed thatnanospheres of elemental Se formed through anaerobicrespiration of Se oxyanion had different properties thannanospheres formed abiotically (Oremland

et

al

., 2004). Theywere also successful in resolving differences in the propertiesof nanospheres of Se formed by different Se-respiring organisms.These findings will likely lead to the use of microorganisms inthe fabrication of new materials requiring the novel propertiesof microbiologically derived Se. More recently, Beveridge andcollaborators demonstrated the formation of intracellulargranules of ferric and ferrous iron formed by

S. putrefaciens

CN32 during anaerobic respiration on solid phase ferricoxide (Glasauer

et

al

., 2007). This was the first time mixed-valence metal particles had been detected

in situ

at highresolution.

It is important to note that Beveridge and collaboratorshave also shown that fine-grained precipitates associated withthe membranes of many Gram-negative bacteria are notnecessarily formed

de novo

at these sites (Glasauer

et

al

., 2001).Large quantities of nanoparticles of ferrihydrite, goethite andhematite, formed through abiotic reactions elsewhere, can betransported to and become bound to cell walls of

Shewanellaputrefaciens

CN32. They further demonstrated that nanocrystalsof these minerals can penetrate the outer membrane andpeptidoglycan layers of the cell envelope. Given these results,one gains a heightened appreciation of the rigor of the earlierstudies performed by Beveridge that enabled him to resolve

denovo

mineral formation on bacterial cell walls. As the field ofbiogeochemistry matures, the ‘Beveridge paradigm’ thatmicrobial cells are important sites of metal cation binding andmineral formation will only gain significance.

The observations made by Beveridge and colleagues duringtheir early investigations of metal interactions with naturalmicrobial biofilms in aquatic environments further heightenedour appreciation for his scientific insight. In work carried outwith Konhauser and others, Beveridge recognized that biofilmstructures are important sites of mineral precipitation in lotic(flowing water) environments (Konhauser et al., 1994). Theyprovided evidence that epilithic microbial biofilms dominatethe reactivity of the rock-water interface and appear to controlmineral formation in these dynamic systems. Although we hadsimilar evidence from electron micrographs prepared fromsamples of microbial biofilms that formed in mountain streamsnearly two decades earlier, unlike Beveridge, we failed torecognize at the time a role for microbial biofilms in this bio-geochemical context. Following Beveridge’s lead, we now paycloser attention to the mineral precipitates that accumulate in

Fig. 3 Transmission electron micrograph of a thin section of a cell ofShewanella oneidensis MR-1 associated with an iron oxide surface viaextracellular polymeric substances after incubation for 11 days in anaerobicliquid medium containing 30 mM HEPES buffer, 18 mM lactate as electron donorand 5 mM Si-stabilized ferrihydrite as electron acceptor. Bar denotes 0.5 µm.

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Bremer P, Geesey GG (1991) Laboratory based model of microbially induced corrosion of copper. Applied and Environmental Microbiology 57, 1956–1962.

Brown DA, Kamneni DC, Sawicki JA, Beveridge TJ (1994) Minerals associated with biofilms occurring on exposed rock in a granitic underground research laboratory. Applied and Environmental Microbiology 60, 3182–3191.

Bruno J, Depablo J, Duro L, Figuerola E (1995) Experimental study and modeling of the U(VI)-Fe(OH)3 surface precipitation/coprecipitation equilibria. Geochimica et Cosmochimica Acta 59, 4113–4123.

Duff MC, Coughlin JU, Hunter DB (2002) Uranium co-precipitation with iron oxide minerals. Geochimica et Cosmochimica Acta 66, 3533–3547.

Ferris FG, Beveridge TJ, Fyfe WS (1986) Iron-silica cyrstallite nucleation by bacteria in a geothermal sediment. Nature 320, 609–611.

Ferris FG, Fyfe WS, Beveridge TJ (1988) Metallic iron binding by Bacillus subtilis-implications for the fossilization of microorganisms. Geology 16, 149–152.

Garland JM, Archibald AR, Baddiley J (1975) An electron microscopic study of the localization of teichoic acid and its contribution to staining reactions in walls of Staphylococcus faecalis 8191. Journal of Bacteriology 89, 73–86.

Geesey GG, Mittelman MW, Iwaoka T, Griffiths PR (1986) Role of bacterial exopolymers in the deterioration of metallic copper surfaces. Materials Performance 25, 37–40.

Glasauer S, Langley S, Beveridge TJ (2001) Sorption of Fe (hydr) oxides to the surface of Shewanella putrefaciens: cell-bound fine-grained minerals are not always formed de novo. Applied and Environmental Microbiology 67, 5544–5550.

Glasauer S, Langley S, Boyanov M, Lai B, Kemner K, Beveridge TJ (2007) Mixed valence cytoplasmic iron granules are linked to anaerobic respiration. Applied and Environmental Microbiology 73, 993–996.

Gorby YA, Yanina S, Mclean JS, Rosso KM, Moyles D, Dohnalkova A, Beveridge TJ, Chang IS, Kim BH, Kamneni KS, Culley DE, Reed SB, Romine MF, Saffarini DA, Hill EA, Shi L, Elias DA, Kennedy DW, Pinchuk G, Watanabe K, Ishii S, Logan B, Nealson KH, Fredrickson JK (2006) Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proceedings of the National Academy of Sciences USA 103, 11358–11363.

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Hoyle B, Beveridge TJ (1983) Binding of metal ions to the outer membrane of Escherichia coli. Applied and Environmental Microbiology 46, 749–752.

Humphrey BA, Vincent JM (1966) Strontium as a substituted structural element in cell walls of Rhizobium. Nature 212, 212–213.

Irvin RT, Chatterjee AK, Sanderson KE, Costerton JW (1975) Comparison of the cell envelope structure of a lipopolysaccharide-defective (heptose-deficient) strain and a smooth strain of Salmonella typhimurium. Journal of Bacteriology 124, 930–941.

Jang LK, Harpt N, Grasmick D, Vuong LN, Geesey GG (1990) A two phase model for determining the stability constants for interactions between copper and alginic acid. Journal of Physical Chemistry 94, 482–488.

Jang LK, Nguyen D, Geesey GG (1999) An equilibrium model for adsorption of multiple divalent metals by alginate gel under acidic conditions. Water Research 33, 2826–2832.

Jolley JG, Geesey GG, Hankins MR, Wright RB, Wichlacz PL (1989) In situ, real time FT-IR/CIR/ATR study of the biocorrosion of copper by gum arabic, alginic acid, bacterial culture supernatant and Pseudomonas atlantica exopolymer. Applied Spectroscopy 43, 1062–1067.

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Konhauser K, Schultze-Lam S, Ferris FG, Fyfe WS, Longstaffe FJ, Beveridge TJ (1994) Mineral precipitation by epilithic biofilms in the Speed River, Ontario, Canada. Applied and Environmental Microbiology 60, 549–553.

Krueger S, Olson GJ, Johnsonbaugh D, Beveridge TJ (1993) Characterization of the binding of gallium, platinum, and uranium to Pseudomonas fluorescens by small-angle X-ray scattering and transmission electron microscopy. Applied and Environmental Microbiology 59, 4056–4064.

Kuyucak N, Volesky B (1988) Biosorbents for recovery of metals from industrial solutions. Biotechnology Letters 10, 137–142.

Lambert PA, Hancock IC, Baddiley J (1975) The interaction of magnesium ions with teichoic acid. Biochemical Journal 149, 519–524.

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Marshall M, Beliaev JAS, Dohnalkova AC, Kennedy DW, Shi L, Wang Z, Boyanov MI, Lai B, Kemner KM, Mclean JS, Reed SB, Culley DE, Bailey VL, Simonson CJ, Saffarini DA, Romine MF, Zachara JM, Fredrickson JK (2006) c-type cytochrome-dependent formation of U(IV) nanoparticles by Shewanella oneidensis. PLoS Biology 4, e268.

Marsili E, Baron DB, Shikhare ID, Coursolle DJ, Gralnick A, Bond DR (2008) Shewanella secretes flavins that mediate extracellular electron transfer. Proceedings of the National Academy of Sciences USA 105, 3968–3973.

Mullen MD, Wolf DC, Ferris FG, Beveridge TJ, Flemming CA, Bailey GW (1989) Bacterial sorption of heavy metals. Applied and Environmental Microbiology 55, 3143–3149.

Neal AL, Dohnalkova A, McCready D, Peyton BM, Geesey GG (2001) Iron sulfides and sulfur species produced at hematite surfaces in the presence of sulfate-reducing bacteria. Geochimica et Cosmochimica Acta 65, 223–235.

Oremland RS, Herbel MJ, Blum JS, Langley S, Beveridge TJ, Ajayan PM, Sutto T, Ellis AV, Curran S (2004) Structural and spectral features of selenium nanospheres produced by Se-respiring bacteria. Applied and Environmental Microbiology 70, 52–60.

Payne TE, Davis JA, Waite TD (1994) Uranium retention by weathered schists – the role of iron minerals. Radiochimica Acta 66–67, 297–303.

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Sani RK, Peyton BM, Amonette JR, Geesey GG (2004) Reduction