ORI GIN AL PA PER

Geomicrobiology of Iron Layers in the Sedimentof Lake Superior

M. Dittrich1• L. Moreau1

• J. Gordon1•

S. Quazi1 • C. Palermo1• R. Fulthorpe1

•

S. Katsev2• J. Bollmann3

• A. Chesnyuk1

Received: 18 August 2014 / Accepted: 27 February 2015� Springer Science+Business Media Dordrecht 2015

Abstract Previous studies of deeply oxygenated (8–12 cm) sediments in Lake Superior

revealed prominent iron (Fe)-rich layers, up to 2 cm thick. Whereas metal enrichments are

common near the oxic–anoxic boundary in aquatic sediments, several geomicrobiological

aspects of their formation remain unknown. In this study, we analyzed geochemical and

microbiological signatures associated with the Fe-rich layer in Lake Superior at two locations

(230 and 310-m water depth) in the Eastern Basin of the lake. We measured oxygen and metal

contents in the sediment solid phase and imaged the Fe-rich layer with transmission and

scanning electron microscope (TEM and SEM). Bacterial communities were characterized

using tag-encoded FLX small subunit ribosomal gene amplicon pyrosequencing. The SEM

images reveal filamentous structures encrusted with spheres of ca. 20 nm in diameter. TEM

and energy dispersive X-ray spectroscopy observations of thin sections indicate that bacteria

cells served as nucleation surfaces for Fe-oxide formation. The Fe-rich layers, in contrast to

the layers directly above them, were dominated by members of the NC10 phylum, known for

anaerobic methane oxidation, and also harbored a high proportion of Nitrospira species that

were most similar to members of a Fe-rich seep. Taken together the results suggests that

microorganisms, whose cell surfaces are serving as templates for iron oxide precipitates that

might also be mediating the iron oxidation, support formation of the Fe-rich layers.

Keywords Iron-rich layer � Pyrosequencing � Nitrospira � Sediments � Lake Superior

Electronic supplementary material The online version of this article (doi:10.1007/s10498-015-9258-y)contains supplementary material, which is available to authorized users.

& M. Dittrichmdittrich@utsc.utoronto.ca

1 Department of Physical and Environmental Sciences, University of Toronto Scarborough, Toronto,ON, Canada

2 Large Lakes Observatory and Department of Physics, University of Minnesota Duluth, Duluth,MN, USA

3 Department of Earth Sciences, University of Toronto, Toronto, ON, Canada

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Aquat GeochemDOI 10.1007/s10498-015-9258-y

1 Introduction

Sediments of several freshwater bodies across the planet contain oxidized iron (Fe) and

manganese (Mn) in the form of nodules or thin metal-rich layers, which have recently

become a topic of emerging interest and speculation (Och et al. 2012; Torres et al. 2014).

Metal-rich layers are well known in marine sediments, where their formation has been

linked either to direct sedimentation from the water column or to diagenetic processes in

the sediment, such as mineralization of organic matter, reductive mineral dissolution,

diffusion of dissolved metals, and mineral re-precipitation (Granina et al. 2004; Kasten

et al. 1998; Richardson 1974). At the oxic–anoxic interface, Fe and Mn are particularly

important due to their redox chemistry: Their reduced forms are highly soluble, while the

solubility of the oxidized species is very low (Stumm and Morgan 1995). The oxidized

phases of Fe and Mn are used as electron acceptors in organic matter mineralization after

the more potent electron acceptors, oxygen and nitrate, are depleted (Berner 1980; Froelich

et al. 1979). The reduced Fe and Mn can be preserved in sediment solids mainly as pyrite,

iron sulfides, or carbonates (Schaller and Wehrli 1997).

Whereas the redistribution of Fe and Mn in marine sediments during early diagenesis has

been extensively recorded and modeled for many years (Robbins and Callender 1975;

Froelich et al. 1979; Aller 1980; Kasten et al. 1998), detailed studies of Fe- and Mn-rich

layers in freshwater sediments are still scarce (Och et al. 2012; Torres et al. 2014; Granina

et al. 2004; Zakharova et al. 2010). Particularly rare are investigations that go beyond simple

geochemistry and include geomicrobiological aspects of the formation of metal-rich layers.

Past research focused mainly on the geochemical processes, such as reduction–oxidation

reactions and redox-sensitive metal redistribution, as well as formation of multiple layers

under non-steady-state conditions. The composition of the associated microbial communities

and their distribution in the sediment are rarely studied, despite the recognized importance of

microorganisms (Posth et al. 2014). In this study, we investigate the composition of the

microbial community associated with Fe-rich sediment layers and visualize the association

of Fe-mineral precipitates and microbes. Our study uses the sediment recovered from Lake

Superior, a large and relatively unproductive lake where sediment is characterized by low

organic matter content and deep penetration of oxygen (Li et al. 2012).

In oligotrophic freshwater lakes, Fe-rich layers occur at neutral pH and puzzlingly, often in

both anoxic and oxic sediment depths. In Lake Constance, a mesotrophic lake in Europe, a Fe-

rich layer was found in sediments 4 cm below the sediment–water interface, in the zone of

oxygen and nitrogen reduction where Fe oxidation can occur (Hauck et al. 2001). An attempt to

identify the group of microorganisms associated with iron oxidation, such as those that may

couple metal oxidation to nitrate reduction, was not successful (Hauck et al. 2001). The distri-

bution of all metabolic groups, such as chemotrophic, mixotrophic, and lithotrophic denitrifiers,

as well as denitrifiers capable of aerobic iron oxidation throughout the upper sediment layers was

relatively uniform (Hauck et al. 2001), leading to the conclusion that no specific microbial

metabolism could be linked to Fe-rich layers in Lake Constance. In oligotrophic Lake Vermilion

(USA), ferromanganese nodules were found near the sediment surface, similar to nodules found

in other lakes around the globe (see references in Sommers et al. 2002). The nodules consisted of

laminated layers enriched in Mn and Fe, and differed in size and morphology. The hypothesis of

a biogenic origin of Fe–Mn nodules has been supported by electron microscopy images of

microbial cells from cultured bacteria able to precipitate Fe and Mn oxides, which in turn served

as substrates for sulfate reducers (Sommers et al. 2002). However, the geochemical data on pore

water and sediment particles were not available in that study.

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Similar Fe-rich layers and nodules were found in Lake Baikal (Russia), along with high

concentrations of manganese (Baturin et al. 2009). The Fe enrichments occurred as either

irregular nodules or crusts 1 to 4 cm thick (Baturin et al. 2009; Granina et al. 2011). Some

Fe-rich layers were found in the oxidized upper sedimentary layer, whereas others were

found in the reduced sediment below (Baturin et al. 2009). Chemical analyses revealed that

these nodules and crusts consisted of 18.3–84.4 % Fe2O3, 0.57–27.3 % MnO2,

7.94–47.38 % SiO2, and 2.1–14.00 % Al2O3 (Baturin et al. 2009). Several other elements

were found in these formations in trace amounts (Muller et al. 2002), but the main com-

ponents were clearly oxidized iron and manganese, as well as silicon from diatom frustules.

Baturin et al. 2009 proposed that ferromanganese nodules and Fe- and Mn-rich layers

found in the anoxic sediments could be attributed to abiotic processes in Lake Baikal. At

the oxic sediment surface, Fe(II) is oxidized, whereas in anoxic sediment below, iron

precipitates dissolve. The Fe-rich layers are subsequently covered by sediment deposited

above them (Baturin et al. 2009), forming multiple Fe-rich layers in sediments. However,

this explanation does not account for the existence of a well-defined layer, as opposed to

continuous burial of oxidized iron. Taking into consideration possible secondary diagenetic

pathways, Och et al. (2012) hypothesized that Fe and Mn layers could have resulted from

recycling of Fe and Mn oxides, which consist of reductive dissolution of Fe and Mn oxides

below the O2 penetration depth, followed by Fe and Mn oxides precipitation and accu-

mulation. Indeed, in a recent study in Lake Baikal, Torres et al. (2014) found that Fe and

Mn layers can be formed by dissolution of particulate Fe and Mn coupled to the anaerobic

oxidation of methane. Dissolved Fe(II) diffuses upward to reduce particulate Mn(IV) and

thus forming a sharp mineral boundary. It has been also shown that particulate Mn can be

dissolved; hence, an anaerobic nitrification of NH4? has been observed at locations with

Mn(IV).

Most of the studies conducted to date in both marine and freshwater sediments have

focused on abiotic processes in Fe- and Mn-rich layer formation. Arguments exist, how-

ever, that support both abiotic and biotic roles for their origin. Most recent studies in Lake

Baikal show high abundances of Fe and Mn oxidizers close to the metal-enriched layers

(Granina et al. 2010; Zakharova et al. 2010). The biogenic origin of ferromanganese

nodules in freshwater systems is not a new idea. For example, Butkevich (1928) demon-

strated the development of layering in brown water-rich top sediments from the Pechora

Lake and White Seas, and isolated Fe-depositing microorganisms from metal-rich layers.

Direct precipitation of Fe- and Mn-oxides by bacteria has also been postulated (Zakharova

et al. 2010).

Similar to Lake Baikal, Lake Superior has a large surface area, a high abundance of Fe-

rich minerals in its watershed, permanently cold bottom waters, and relatively low bio-

logical productivity (Sterner 2010). Sediments found across the Lake Superior basin

contain prominent Fe-rich crusts, sometimes as multiple layers (Li et al. 2012). Well-

defined Mn and Fe layers in the oxidized upper sediment have been reported at multiple

locations (Sly and Thomas 1974; Li et al. 2012). Though typically located near the depth of

oxygen penetration or nitrogen penetration (Li and Katsev 2014), these layers are also

sometimes found in the presently anoxic or in the presently oxic zones (Richardson and

Nealson 1998). The organic carbon mineralization in Lake Superior is dominated by

aerobic respiration: At the offshore low-sedimentation locations, more than 95 % of carbon

is mineralized using oxygen as electron acceptor (Carlton et al. 1989; Li et al. 2012),

denitrification accounts for less than 2 % (Li and Katsev 2014), while metal reduction and

sulfate reduction account for even smaller fractions (Li and Katsev 2014; Li 2014). At the

Eastern Basin locations investigated here, oxygen penetrates 10–12 cm into the sediment

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(Li et al. 2012), and in low-sedimentation offshore areas exceeds 6–12 cm (Li et al. 2012;

Li and Katsev 2014). Nitrate is found to penetrate even deeper (Carlton et al. 1989; Li and

Katsev 2014). The overlap of the geochemical profiles of nitrate and ferrous iron provides

evidence for the coupling of iron oxidation to nitrate reduction (Li and Katsev 2014), a

phenomenon known from previous studies (Kasten et al. 2003; Wilson et al. 1985; Berger

1983; Froelich et al. 1979).

The involvement of microorganisms in the oxidation of metals and their roles in the

coupling of Fe and N geochemical cycles remain speculative. Bacteria are primarily in-

volved in reducing Fe (Straub and Buchholz-Cleven, 1998). Their involvement in Fe

oxidation was demonstrated for chemotrophic, denitrifying bacteria (Hauck et al. 2001)

isolated from freshwater ditches, streams and ponds (Straub and Buchholz-Cleven 1998),

as well as Lake Vermilion (Sommers et al. 2002) and Lake Constance. In the latter case,

bacterial strains such as Acidovorax sp. strain BoFeN1 have been shown to oxidize iron

through the reduction of nitrate (Pantke et al. 2012). Verification of the anaerobic oxidation

of ferrous iron by denitrifying bacteria would imply the closing of the iron cycle within the

anoxic zone of the sediment, with implications for both modern geochemical budgets and

the processes that might have shaped the sediment geochemistry during the Earth’s geo-

logical history (e.g., Crowe et al. 2008). Thus, more information is needed on the microbial

populations, functions, and activities.

In this study, we tested the hypothesis that Fe-oxidizing microbes participate in the

formation of Fe-rich layers in Lake Superior. We used electron microscopy to image

sediment sections and analyzed the microbial populations within the iron-rich layers. These

microbial communities have been compared to the communities in overlying layers, in-

cluding the layers enriched in manganese, to understand the origin and diagenesis of the

Fe-rich layers.

2 Materials and Methods

2.1 Sample Collection

Sediment cores were collected from Lake Superior with an Ocean Instruments multi-corer in

September 2010 and July 2012 aboard the R/V Blue Heron. Samples were collected at two

locations in the Eastern Basin of Lake Superior: Eastern (EM, 229 m) Sep 2010, 47� 33.370

86� 35.680 and Eastern Deep (ED, 310 m) Sep 2010, 47� 31.530 87� 07.490 (Fig. 1). Sediment

cores of 94 mm inner diameter ranged 30–35 cm in length. The cores were kept at the in situ

temperature of 4 �C and sectioned in anaerobic conditions in a glove bag to prevent

oxidation. Oxygen vertical distributions were determined within 40 min of sampling in

subsampled cores with an internal diameter of 5 cm and were from the same multicorer in

2010. A Unisense (Clark-type) microelectrode (Revsbech 1989) calibrated in air-saturated

and N2-purged water saturated with bi-dithionite was used for O2 measurements.

2.2 Sediment Chemical Analysis

Sedimentary iron content was determined using a modified approach of that described by

Tessier et al. (1979), Chester and Hughes (1967), (Mehra and Jackson 1960), as well as in

Poulton and Canfield (2005) (see details in Supplementary Material). Here, we present the

results on reducible Fe and Mn oxides sediment depth profiles.

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2.3 Electron Microscopy and Electron Dispersive X-Ray Spectroscopy

2.3.1 Transmission Electron Microscopy

Transmission electron microscopy (TEM) was employed to study sediment layers for

evidence of microbial cells in Fe-rich layers. Samples were homogenized and then incu-

bated in 2 % glutaraldehyde solution in Sorenson’s phosphate buffer (0.1 M, pH 7.4) to

allow for protein fixation and then washed with ethanol and propylene oxide. Samples were

then embedded in Spurr low-viscosity epoxy, cut into 70 nm sections onto copper grids

and stained with a uranyl acetate and lead citrate mixture. These samples were then

analyzed by transmission electron micrograph using the Hitachi H7500 with the energy of

10 kV at the Centre for the Neurobiology of Stress (CNS) at the University of Toronto,

Scarborough Campus. The Hitachi S-5200 in conjunction with the OXFORD INCA energy

dispersive X-ray spectrometer (SEM–EDS) from the Center for Nanostructure Imaging

(CNI) at the University of Toronto was also used in elemental mapping of samples.

2.3.2 Scanning Electron Microscopy

Scanning electron microscope (SEM) photographs and electron dispersive X-ray (EDX)

mapping were carried out to determine whether bacteria were visible on the iron crust.

Samples were prepared with a gold coating to provide electrical conductivity and achieve

higher image resolution. The images were then taken with a Zeiss Supra VP55 SEM, at the

Geology Department of the University of Toronto. Images were taken at the energy of

10 kV and magnification levels ranging from 5.56–192.74 kx.

Fig. 1 Map of Lake Superior, on the inlet is an image of Fe crust in the sediment sample from the stationEM in 2010

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2.4 Microbial Community Composition

2.4.1 Enumeration of Bacteria Cell Abundances

Sediment samples from EM station, 6–7 cm, and 9–12 cm, enriched in Fe, collected in 2012

and as well as sediments sampled from the 8–10 cm (Fe-rich layer) and 10–12 cm depths of

cores ED stations collected in 2010, were preserved in Life Guard1 2868-10 soil preservation,

were thawed at room temperature, and were shaken vigorously to break up large particles. The

total cell count of prokaryotes was determined using 4060-diamidin-2-phenylindole (DAPI), a

fluorescent blue dye, which binds to DNA. Cells were separated from the sediment particles

using a procedure modified from Epstein and Rossel (1995). For each sample depth, 0.5 g

preserved sample was combined with 2.0 ml filter-sterilized water, vortexed, and sonicated

for 90 s at 30 W using a Bronwill Scientific Biosonick III ultrasonic cell disrupter, at an

intensity setting of 40/100. The sample was then diluted 10009 and combined with DAPI to

produce a final concentration of 10 lg/ml. After 30 min of incubation in the dark, the solution

was filtered onto a 0.2-lm-pore-size polycarbonate membrane. The filters were placed on

slides in mounting oil and frozen. When counted, samples were thawed for at least 15 min and

viewed using a Nikon Eclipse E600 microscope 1009 magnification oil emersion objective

lens set. Slides were viewed using a DAPI filter of excitation wavelength 340–380 nm. At

12509 magnification, the number of cells per viewing field were counted and recorded, to a

minimum of 400 cells. Concentration factors were used to back calculate to the original

density of bacteria/g of dry sediment (DW) using porosity values.

2.4.2 Community Composition

2.4.2.1 DNA Extraction DNA was extracted and amplified from the sediment samples at

the following sites and depths: ED 7–8 cm, ED 10–12 cm, ED 18–20 cm, EM 7–8 cm, EM

10–12 cm, and EM 18–20 cm. DNA was extracted from the sediment following the pro-

tocol of a PowerSoil DNA isolation kit. The 16S ribosomal DNA (rDNA) region was

amplified in a polymerase chain reaction (PCR) using 341F-clamp forward primer and

907R reverse primer (Supplementary Material Table 1). Amplified DNA was then ana-

lyzed in denaturing gradient gel electrophoresis (DGGE), as described by Straub and

Buchholz-Cleven (1998). The gel was run from 20 h at 70 volts and was stained with

ethidium bromide for 30 min. Two ladders were used: one consisting of known microbes,

and the other of combined samples of 16S extracted from Lake Superior’s sediment.

Using GelCompar II, DGGE fingerprints were analyzed to compare communities at the

iron layers of sediment (10–12 and 18–20 cm), with those above. The comparison was

performed using Pearson’s correlation similarity coefficient, with 2 % optimization, and

UPGMA (unweighted pair group method with arithmetic mean) cluster analysis.

Using a PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc), approximately

350 lg of each sediment sample was used to extract bacterial genomic DNA. To con-

centrate DNA yield prior to purification, sediment samples were dried in a Savant

SpeedVac centrifugal evaporator (Thermo Fisher Scientific, USA). The DNA samples

were purified to remove amplification inhibitors using Healthcare illustra MicroSpin S-300

HR Columns (General Electric, USA) and following the manufacturer’s protocol. DNA

concentrations were determined using a NanoDrop ND-1000 v.3.7.0 UV–Vis spec-

trophotometer (NanoDrop Technologies, USA) according to the manufacturer’s guidelines.

Extracted and purified DNA samples were stored at -80 �C.

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2.4.2.2 Pyrosequencing DNA samples from 8 to 10 cm and 10 to 12 cm of cores ED

and EM collected in 2010 were sent to the Research and Testing Lab (Texas) for

pyrosequencing of 16S rRNA gene amplicons generated using barcoded universal pri-

mers 27f-519r. Sequences obtained were initially processed in MOTHUR for trimming

and chimera checking (http://www.mothur.org/wiki/Sequence_processing). Sequences

were aligned, clustered, and dereplicated using tools in the RDP II pyrosequencing

pipeline. Sequences were clustered using various percent similarities as cutoffs to define

OTU’s. Representative sequences from clusters were compared to those in GenBank

using BLASTn and submitted to RDP II classifier in order to obtain a phylogenetic

assignment if possible.

DNA samples from Mn and Fe layers (6–7 cm and 9–12 cm sediment depths, respec-

tively) from EM station collected in 2012 were sent to the MrDNA Molecular Research LP

for pyrosequencing of 16S rRNA gene amplicons. In a modified version of amplicon

pyrosequencing (bTEFAP) initially described by Dowd et al. (2008), a single-step 30 cycle

PCR was performed using HotStarTaq Plus Master Mix Kit (Qiagen, USA) and the same

primers as above.

The PCR was performed under the following conditions: 94 �C for 3 min, then 28

cycles of 94 �C for 30 s; 53 �C for 40 s and 72 �C for 1 min; followed by a final elon-

gation step at 72 �C for 5 min. Amplicon products from different samples were combined

in equivalent concentrations and purified using Agencourt Ampure beads (Agencourt

Bioscience Corporation, USA). Roche 454 FLX titanium instruments and reagents were

used to obtain sequences the samples following the manufacturer’s protocol.

A proprietary analysis pipeline (www.mrdnalab.com, MR DNA, Shallowater, TX) was

used to process the Q25 sequence data derived from the sequencing process. Barcodes and

primers were removed; then, sequences containing fewer than 200 base pairs, sequences

with ambiguous base assignments, and sequences with homopolymer runs greater than six

base pairs were eliminated. Sequences were then deionized, and chimeras were removed.

After removing singleton sequences, the sequences were clustered into OTUs and defined

based on relatedness at 3 % divergence (97 % similarity). Taxonomic classifications of

OTUs were achieved using BLASTn against a curated database derived from Greengenes

(http://greengenes.lbl.gov; DeSantis et al. 2006), RDPII (http://rdp.cme.msu.edu) and

NCBI (www.ncbi.nlm.nih.gov). Sequences were initially processed chimera for trimming

and checking. Sequences were divided into those from the iron layer and those from the

layer directly above using a perl script. Sequences were aligned, clustered, and derepli-

cated using tools in the RDP II pyrosequencing pipeline. Sequences were clustered using

various percent similarities as cut offs to define community microbial relationships.

Representative sequences from clusters were compared to those in GenBank using

BLASTn and submitted to RDP II classifier in order to obtain a phylogenetic assignment if

possible.

3 Results

3.1 Iron and Manganese Layers

The sequential iron extractions from Lake Superior sediments show high amounts of iron

from the dithionite extraction within the 8–10 cm band for site ED, while the EM site has a

broader layer at a depth of 9–12 cm as shown in Fig. 2. The EM also displayed an overall

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higher concentration of iron (III) oxides than the ED site at every depth measured. The

reducible oxides of Fe exhibited a maximum content (45.73 mg/g DW) at 8–10 cm below

the sediment–water interface at the same depth as Mn at the ED station (Fig. 2a, c). The

Mn layer at the EM station occurs above the Fe-rich layer (Fig. 2b, d).

3.2 Scanning Electron Microscopy Imaging

Figure 3 shows the typical features of Lake Superior sediment Fe and Mn layers. SEM and

EDX analyses revealed a number of features (Fig. 3a), such as honeycomb (Fig. 3b), plate-

like (Fig. 3c, d), and filamentous structures (Fig. 3e, f). The plates contained silica and

aluminum and were covered with EPS-like substances. Small particles (inlet of Fig. 3d) on

the surface of the plates showed a strong Fe peak in the spectra collected by EDX.

Figure 3e, f shows a representative stalk that was observed to measure over 1.4 lm in

length, with an average diameter of 400 nm. The structures appear to be organic in origin

and encrusted with small inorganic precipitate particles across their surface. Electron

dispersive X-ray (EDX) mapping on these particles shows a strong iron signal, as depicted

in iron mapping in Fig. 4.

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Fig. 2 Depth profiles of oxygen measured by microsensors at sediment–water interface and concentrationsof iron (III) oxides (a, b) and manganese oxides (c, d) within sediment samples at both sites, ED (a–c) andEM (d–f) in 2010

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3.3 Transmission Electron Spectroscopy

TEM images revealed small fibril structures roughly 1 lm in length after staining with

uranyl acetate and lead citrate (Fig. 5). Our observations of unstained samples showed

little to none of these structures, indicating that they may be of organic nature. Heavily

encrusted ring structures were also identified with and without staining, and are similar in

diameter to the previously observed stalks from the SEM images. EDX of these ring

Fig. 3 SEM images of iron-layer samples a represents an overview over the sample, b represents a honey-home structures, c–d show a thin Al–Si plates covers with Fe particles and extracellular polymericsubstances. Filamentous structure similar to sheaths of iron-oxidizing bacteria Leptothrix ochracea(e) covered with spherical to irregular shapes particles attached to sheaths/thin filamentous (f)

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structures revealed them to be encrusted with iron particles, consistent with the previous

observations of stalks. Other elements identified within the encrustation include P, C, Si,

and O as shown in Fig. 6.

3.4 Microbial Community Composition

The prokaryotic cell density in the sediment samples decreases with distance from the

sediment surface, once below the uppermost 0.5 cm of the sediment (Fig. 1 Supplementary

Material). At both the ED and EM sites, the sediment in the first 0–0.5 cm contains a much

lower concentration of bacteria than that in the layer from 0.5 to 1 cm of sediment. At the

ED site, the bacterial concentration was only (7.97 ± 3.30)*108 cells/g dry weight in the

uppermost 0.5 cm of sediment, as opposed to (2.30 ± 2.11)*109 cells/g dry weight found

in the sediment directly below. At the EM site, there were (7.07 ± 4.1)*108 cells/g dry

weight in the uppermost 0.5 cm of sediment, versus (1.09 ± 2.9)*108 cells/g dry weight

living directly below. This strong increase in bacterial abundance at the ED station occurs

within uppermost 0.5 cm (Fig. 1 Supplementary Material). Although bacterial abundance

is a nearly twice as great at the ED site as the EM site, nearly identical trends in abundance

occur. The bacterial abundance increases strongly in the layer 0.5–1 cm beneath the sur-

face. The populations then show exponential decline, rapidly dropping to concentrations of

1.5–2*108 cells/g of dry sediment (ED) and 3–5*108 cells/g of dry sediment (EM). The

density then plateaus, remaining relatively constant within these ranges.

6507 sequence reads were obtained for sample ED 8–10 cm, 4131 for sample ED

10–12cm, 9455 reads for sample EM 8–10 cm, and 5969 reads for sample EM 10–12 cm.

The iron layers, ED 8–10 cm and EM 10–12 cm, both differed from the sediments directly

above them in that they contained far fewer gram-positive organisms, far more pro-

teobacteria, and a high proportion of Nitrospira species within the community as shown in

Supplementary Materials Table 1 and 2. Both sediment layers contain very high propor-

tions of unclassifiable sequences 121 of 189 clusters from the no Fe layer, and 163 of 234

clusters from the iron layers. This is reflected in the data shown in Supplementary Ma-

terials Table 1, where representative sequences of the dominant clusters (those containing

Fig. 4 SEM image and EDX iron mapping of the particle attached to Al–Si plate (a). The gray square onthe left image indicates the boundaries of the mapping shown on the (b)

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high percentages of the total number of sequences) shown with their closest GenBank

matches. We show here the data for representatives of clusters defined at 20 % similarity of

sequence. This represents a groups of bacteria defined as being roughly in the same family

or order. The same exercise performed with sequences representing clusters defined at 5 %

similarity gives much the same information. Both sediment layers contain very high

proportions of unclassifiable sequences—121 of 189 clusters from the no Fe layer, and 163

Fig. 5 TEM images from the iron-layer sediment samples obtained from site ED (a–d) and site EM (e,f) stained with uranyl acetate and lead citrate, a, c show an overview of the samples with cross sections ofsheaths encrusted by an amorphous to poorly crystalline precipitates appear as circular or elongatedstructures, which are little or heavily encrusted (b, d) showing different level of biomineralization; e,f represent EM site with small little encrusted structures, similar to viruses

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of 234 clusters from the iron layers. The most dominant cluster (20 %) in Fe-rich layer

contains unclassified bacteria showing the best matches to organisms from Lake Wash-

ington sediments and from groundwater (Table 1 Supplementary Material). The next most

common organism appears to be Nitrospira, with relatively high similarity to organisms

found in an iron II-rich seep (Table 1 Supplementary Material). Other relatively common

organisms (i.e., found in the top ten most abundant clusters) are related to those found in

limestone systems, groundwater systems, soils, or sediments.

4 Discussion

4.1 Origin of Iron and Manganese Layers

Sedimentary Fe and Mn transformations have been studied extensively in marine systems

(Kasten et al. 1998; Maerz et al. 2012) and less intensively in freshwater environments. It is

widely recognized that mineralization of organic matter, which drives the redox zonation in

sediment, leads to the reduction of Mn and Fe (oxy) hydroxide after the oxygen and nitrate

supplies are depleted. The reduced Mn and Fe diffusing from the deeper sediment are

oxidized by oxygen and/or nitrate. As a result, Mn and Fe layers can form at the oxic–anoxic

boundary in the sediment. The position of metal-rich layers within the sediment can be

strongly affected by non-steady-state conditions, which may shift the layers vertically: up on

the reduction time scale or down on the burial time scale (e.g., Katsev et al. 2006). While the

abiotic controls of the involved reactions have been investigated in detail, studies of the

microbial impacts on Fe- and Mn-rich layers in freshwater sediments are still scarce.

Whereas previous studies focused on isolation of microorganisms and mineral pre-

cipitation, our microscopic investigations show microbial cells embedded in Fe oxides,

providing direct evidence of microbial involvement in Fe-rich layer formation (Posth et al.

2014). Our microscopic and spectroscopic images suggest that Fe oxides precipitated on

the surface of cells, so that cells became completely encrusted in Fe particles. The

Fig. 6 TEM-EDX line scans of a sheaths demonstrated a homogenous distribution of mostly Fe, significantamounts of Si, and minor P. Number at the left corner reflects the amplitude of the EDAX-signal

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microscopic images do not offer details on the mechanism of Fe precipitation, but they do

demonstrate that cell surfaces are able to act as templates for the growth of Fe particles.

The observations stress the importance of microbial-induced Fe oxides formations in

sediments; in addition, any abiotic pathway and or the Fe-oxide precipitation known to be

carried out by microaerophilic or anoxygenic phototrophic Fe oxidizers (Posth et al. 2014).

The images of the encrusted cells from Lake Superior sediments look similar to cells found

in Fe-rich hydrothermal vents (Fortin et al. 1998).

4.2 Microbial Communities in Sediments of Lake Superior

Lake Superior has a low bacterial abundance in comparison with other large lakes. For

example, the microbial cell density measured in different freshwater sediments is typically

between 1.6*109 cells/g DW and 1.7*1010 cells/g DW (Straub and Buchholz-Cleven

1998). Lake Superior’s maximum bacterial density estimate of 2.30 ± 2.11*109 cells/g

DW was only found at the ED site between 0.5 and 1.0 cm depths. Most of the other cell

densities found in the sediment cores from Lake Superior were on the order of 108. In

upper sediment layers, this disparity in bacterial density may be related to sampling in mid-

September, after the growing season has peaked. June bacterial density measurements

observed in a series of temperate freshwater reservoir sediments of different trophic levels

were invariably higher than those taken in the fall (Wobus et al. 2003). However, the low

bacterial density measurements are unsurprising given the lake’s low productivity and low

sedimentary concentration of organic matter (3–5 wt %; Li 2014). In oligotrophic Lakes,

there is less organic matter to act as a nutrient and energy source for bacterial populations.

Surprisingly, Wobus et al. (2003) found no statistically significant differences in total

bacterial population densities between eutrophic and oligotrophic reservoirs. The bacterial

densities measured through DAPI staining for all reservoirs ranged from 1*1010 to

10*1010 cells/g DW, regardless of trophic state (Wobus et al. 2003). However, the study

included only shallow water bodies, so organic matter content even in oligotrophic systems

would have been higher than those in the sediments of Lake Superior.

In their profile of a 23-cm sediment core from Lake Erken, Sweden, Haglund et al.

(2003) found that bacterial abundances decreased with depth, after peaking 0.5 cm below

the sediment interface. The cell density in Lake Erken’s sediment drops from 4*1010 cells/g

DW at 0.5-cm depth to 1.8*1010 cells/g DW at the bottom of sediment. This decrease in

bacterial abundance with depth is a common trend (Haglund et al. 2003). Deep sediment

does not contain a higher percentage of dead cells; the number of viable bacteria at any

depth is approximately 57–63 %, which suggests that neither grazing nor viruses are

primarily responsible for the decrease in abundance (Haglund et al. 2003). Since porosity

decreases with depth, and diffusion from the surface becomes more difficult with increased

distance, reduced organic matter and nutrient availability may be responsible for the de-

cline in Lake Superior’s sediment population with depth.

At most sediment depths, bacteria appear to be twice as abundant in the sediment at the

deep site (ED) than at the shallower one (EM). This may be due to different nutrient

concentration in the pore water at the two sites: the concentrations of ammonium and

phosphate were considerably higher at EM (Li and Katsev 2014; Li 2014). Higher levels of

oxygen and organic matter both allow increased cellular respiration and can support

bacterial communities at greater densities. At the ED site, oxygen levels were higher than

at the EM site, allowing increased respiration. Varying nutritional levels for microorgan-

isms, such as OM content, could also affect growth patterns. These parameters vary most

in the surface sediments, where the largest difference in bacterial populations between the

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two sites has been found (Fig. 1 Supplementary Material). The difference is less between

the EM and ED site’s bacterial abundance at the lower layers, regions with low metabolic

activities.

The pyrosequencing data show that the microbial community of the non-iron layer

above the iron crust is strongly dominated (46.5 % of reads) by gram-positive bacteria

represented by the Paenisporosarcina species. All other clusters containing relatively high

numbers of sequences are unclassifiable—but do show high similarity to sequences derived

from soils, groundwater areas, deep sediments and/or limestone environments. The Fe-rich

layer community is highly dissimilar; proteobacteria dominate rather than Firmicutes

(Supplementary Materials Table 2, Fig. 7). The dominant strains, representing 20 % of

sequences, are related to candidatus Methylomirabilis, a member of the NC10 phylum that

oxidizes methane via denitrification through an unusual pathway that produces oxygen

from nitrate (Wu et al. 2012; Ettwig et al. 2010). Also of interest is the Nitrospira cluster

representing [5 % of the community, for it the sequences are most similar to an unclas-

sified organism found within Lake Washington and to a microbe found within a Fe-rich

seep (Supplementary Materials Table 2). Iron precipitating microbes have been observed

0

5

10

15

% o

f Seq

uenc

es

ED 8-10 cmEM 8-10 cmEM 10-12 cmED 10-12 cm

0

10

20

40

50

60

% o

f Seq

uenc

es

ED 8-10 cmEM 8-10 cmEM 10-12 cmED 10-12 cm

(a)

(b)

Fig. 7 Percentage of sequences of each phylum for four sediment layers a represents 0–60 % of thesequences, while 0–15 % sequences are shown at (b)

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in Lake Washington, described as twisted tubules and irregular particulates (Shapiro et al.

1971). The Fe-seep microbes were isolated from a Fe-rich pond in which Fe oxidation was

occurring (Bruun et al. 2010). Sequence matches strongly support the presence of iron

oxidizers in these layers. We postulate these are the species that might precipitate Fe

oxides at the cell surfaces by serving as the templates as shown by TEM experiments.

Pyrosequencing of Fe-rich and above-lying sediment layers from the EM station was

carried out in 2012 and showed agreement with the previous results. Indeed, phyla of

proteobacteria, together with actinobacteria, chloroflexi and nitrospirae, dominated in both

above-lying and Fe-rich layers. Recently, neutrophilic proteobacteria were found in the

environment where microbes actively drive Fe and Mn cycles (Supplementary Materials

Table 2, Baskar et al. 2012), confirming the presence of the similar microbial communities

in Fe-rich habitats. The 2011 data show the presence of Methylomirabilis, a nitrate re-

ducing methane oxidizer that was thought to be NC10.

4.3 Cell-Mineral Interfaces: Biomineralization Phenomenon

In many studies, the identification of iron biomineralization has relied on the observation

of characteristic iron-encrusted structures such as stalks and sheaths (Hanert 2002; James

et al. 2002). Iron-encrusted, filamentous structures observed under SEM in this study

indicate the presence of an iron-oxidizing microorganism.

Studies on Gallionella ferruginea, iron-oxidizing chemolithotrophic bacteria that live in

low-oxygen conditions (Koschinsky et al. 2002) show similarities to organisms within

Lake Superior. G. ferruginea oxidizes dissolved iron, therefore removing it from the water

and producing an insoluble precipitate of ferric hydroxide onto the outside of long, stalk-

like structures.

Studies on BoFeN1, another iron-oxidizing microbe, also find similar encrustation pat-

terns of iron. However, Fe precipitates directly onto the outer membranes of these cells

(Kappler and Straub 2005). Experiments, such as those by Clarke et al. (1997) using TEM

techniques on other iron-biomineralizing microbes, reveal a common theme in the circular

encrustation of microbes with iron precipitates. These structures were observed within the

Lake Superior iron-layer samples, with inorganic precipitates on the outer surface of cells.

The presence of nodules on the outer surface of these structures, as well as the approximate

size correlates with the observed encrustation of the filamentous structures seen under SEM.

SEM-EDX mapping analysis of the observed nodules shows a high concentration of

iron within the nodule structure, leading to the conclusion that the observed particles are

iron precipitates (Fig. 5). Using TEM-EDX, the observed circular structures were analyzed

to determine the nature of the precipitates on their outer edge. Iron concentrations were

found to be higher on these structures than in the background sediment. Carbon was also

found on the ring structures at a much higher reading than the background-embedding

plastic. These results suggest that these structures are organic in nature and may be re-

sponsible for the concentration of iron within the iron layer.

5 Conclusions

We investigated oxidized Fe formations from sediments of Lake Superior. Microorganisms

identified in the Fe-rich layers included many that are capable of iron oxidation and iron

oxide precipitation at their cell surfaces. The identification of DNA sequences of organisms

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closely related to neutrophilic proteobacteria supports this hypothesis. Sequence matches

strongly support the presence of iron oxidizers in Fe-rich layers. We postulate that these

are the species that may precipitate Fe oxides besides the cell surfaces that serve as the

templates as shown by TEM experiments. The Fe-rich layers, in contrast to the layers

directly above them, were dominated by members of the NC10 phylum, known for

anaerobic methane oxidation, and also harbored a high proportion of Nitrospira species

that were most similar to members of a Fe-rich seep. Electron microscopy reveals

filamentous microbial structures, which were covered with Fe precipitates. Finally, TEM

sections demonstrated that iron oxides as well as silica and phosphorus are mineralized

directly on cell surfaces, supporting the contribution of the biomineralization process on

the formation and preservation of iron and manganese crusts.

Acknowledgments We acknowledge the support from the National Sciences and Engineering ResearchCouncil of Canada (Discovery grant to M. Dittrich), Canada Foundation for Innovation and Ontario Re-search (Fund for Leaders Opportunity Fund, Grant Nr 22404), and the US National Science Foundation(Grant OCE 0961720 to SK). M.D. was supported by start-up funding from the University of TorontoScarborough. We are grateful to Basirath Raoof for help with the laboratory experiments, B. Temkin(UTSC) and D. Holmyard (Mount Sinai Hospital, Toronto) for their assistance with SEM and TEMimaging and Jiying Li for sharing the data and help during the cruises 2010 and 2012. We thank BernhardWehrli and the anonymous reviewer whose comments and suggestions helped us to improve the manuscriptsignificantly.

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