The microbe–mineral environment and gypsum neogenesis ina weathered polar evaporiteC. S. COCKELL,1 G. R. OSINSKI,2 N. R. BANERJEE,3 K. T. HOWARD,4 I . GILMOUR5 AND

J. S. WATSON5

1Geomicrobiology Research Group, Centre for Earth, Planetary, Space and Astronomical Research (CEPSAR), Open University,

Milton Keynes, UK2Department of Earth Sciences ⁄ Physics and Astronomy, University of Western Ontario, London, ON, Canada3Department of Earth Sciences, University of Western Ontario, London, ON, Canada4Impacts and Astromaterials Research Centre, Department of Mineralogy, Natural History Museum, London, UK5Centre for Earth, Planetary, Space and Astronomical Research (CEPSAR), Open University, Milton Keynes, UK

ABSTRACT

Evaporitic deposits are a globally widespread habitat for micro-organisms. The microbe–mineral environment in

weathered and remobilized gypsum from exposed mid-Ordovician marine evaporite beds in the polar desert of

Devon Island, Nunavut, Canadian High Arctic was examined. The gypsum is characterized by internal green

zones of cyanobacterial colonization (dominated by Gloeocapsa ⁄ Aphanothece and Chroococcidiopsis spp. mor-

photypes) and abundant black zones, visible from the surface, that contain pigmented cyanobacteria and fungi.

Bioessential elements in the gypsum are primarily provided by allochthonous material from the present-day polar

desert. The disruption, uplift and rotation of the evaporite beds by the Haughton meteorite impact 39 Ma have

facilitated gypsum weathering and its accessibility as a habitat. No cultured cyanobacteria, bacteria and fungi

were halophilic consistent with the expectation that halophily is not required to persist in gypsum habitats.

Heterotrophic bacteria from the evaporite were slightly or moderately halotolerant, as were heterotrophs

isolated from soil near the gypsum outcrop showing that halotolerance is common in arctic bacteria in this

location. Psychrotolerant Arthrobacter species were isolated. No psychrophilic organisms were isolated. Two

Arthrobacter isolates from the evaporite were used to mediate gypsum neogenesis in the laboratory,

demonstrating a potential role for microbial biomineralization processes in polar environments.

Received 6 October 2009; accepted 23 March 2010

Corresponding author: Charles S. Cockell. Tel.: +44 1908 652588; fax: +44 1908 848022; e-mail: c.s.cockell@

open.ac.uk

INTRODUCTION

Microbial communities that inhabit evaporitic deposits have

been described from a diversity of global locations. The best

studied communities associated with evaporitic deposits are

those of the marine intertidal zone and salterns (e.g. Cau-

mette, 1993; Caumette et al., 1994; Rothschild et al., 1994),

where organisms inhabit the salt crusts produced from the

evaporation of seawater. Within some evaporitic deposits that

are sufficiently stable, a well-defined zonation of the interior

community occurs. Oren et al. (1995) described the zonation

of communities inhabiting gypsum deposits in a saltern pond

in Eilat, Israel, with distinctive differentiation occurring

between layers of oxygenic phototrophs, dominated by cyano-

bacteria such as Synechococcus and Phormidium spp., and

purple sulphur bacteria beneath them. This distinct zonation

was also demonstrated by fatty acid (Ionescu et al., 2007) and

molecular biological analysis (Sørensen et al., 2005). Synecho-

coccus are reported to be a dominant phototroph taxa in

the evaporites in Guerrero Negro, Baja, California, which is

composed of 22% gypsum (Rothschild et al., 1994).

Some organisms within evaporitic deposits or the salty

waters associated with them are not merely passive inhabit-

ants, but play an active role in biogeochemical processes, for

example fixing nitrogen (Boison et al., 2004) or precipitating

minerals. Douglas and Yang report the biogenic formation of

rosickyite, a thermodynamically unstable form of sulphur in

gypsum crusts in Death Valley, USA (Douglas & Yang, 2002;

� 2010 Blackwell Publishing Ltd 293

Geobiology (2010), 8, 293–308 DOI: 10.1111/j.1472-4669.2010.00240.x

Douglas, 2004). The extent to which the organisms within

salt deposits take part in processes such as microbial mineral

neogenesis is still poorly understood. As these processes could

potentially leave mineral biosignatures or preserve organic

remains, evaporites have been considered promising targets

for investigating life on the early Earth or possibly elsewhere

(Mancinelli et al., 2004; Edwards et al., 2005; Stivaletta

et al., 2005), particularly on Mars where large quantities of

sulphate deposits, including gypsum, are known to occur

(Clark et al., 1982; Rieder et al., 1997; Gendrin et al., 2005;

Langevin et al., 2005; Squyres et al., 2006).

Communities in evaporitic deposits have been described in

extreme environments, where the habitat can provide protec-

tion from the environment, in a similar way to which endoliths

are protected from extreme conditions by living in the interior

of rocks. For example, cyanobacteria, principally Chroococcidi-

opsis morphotypes, inhabit evaporitic deposits in the Atacama

Desert (Chile) (Wierzchos et al., 2006). The organisms inha-

bit halite mounds within the hyperarid core of the desert,

achieving metabolic activity during brief periods of moisture

availability, potentially assisted by halite deliquescence (Davil-

la et al., 2008). Chroococcidiopsis phylotypes have been

described from gypsum soil crusts in the Atacama Desert,

Mojave Desert (USA), Al-Jafr Basin (Jordan) (Dong et al.,

2007) and a gypsum outcrop in the Harz Mountains

(Germany) (Boison et al., 2004).

Polar environments also host organisms capable of inhabit-

ing salt crusts. Hughes & Lawley (2003) described the coloni-

zation of thin gypsum crusts on sandstone boulders on

Alexander Island, Antarctica by endolithic organisms, includ-

ing cyanobacteria, bacteria and fungi. Although primary pro-

ductivity was low, they showed that the gypsum provides

protection from UV radiation and desiccation. Parnell et al.

(2004) described the cyanobacterial colonization of selenite

(calcium sulphate) crystals in the Haughton impact structure,

Devon Island, Canadian High Arctic. These crystals were

formed by post-impact hydrothermal activity at the margins

of the crater and the organisms inhabit the space between the

selenite sheets.

Organisms that inhabit gypsum deposits would not neces-

sarily be expected to be halophilic as the solubility of gypsum

is low in water [2.4 g L)1 (0.014 M) at 25 �C], compared to

halite [360 g L)1 (6.16 M) at 25 �C]. However, Garcia-Pichel

et al. (2001) studied soil crusts of the Colorado Plateau, USA

and found that gypsum crusts were the most phylogenetically

divergent of the crusts studied, which they suggested might

be caused by the requirement for halotolerance. Thus, the

physiological requirements for living within gypsum require

further study.

In this work, we investigated the microbe–mineral environ-

ment in weathered and remobilized gypsum associated with

Ordovician evaporitic outcrops in a polar desert on Devon

Island in the Canadian High Arctic. We tested hypotheses

concerning the ability of organisms to take part in mineral

neogenesis and their physiological requirements.

MATERIALS AND METHODS

Study sites

Gypsum outcrops of the mid-Ordovician Bay Fiord Forma-

tion occur throughout the 39 Ma, 23-km diameter Haugh-

ton impact structure (Bischoff & Oskierski, 1988; Osinski

et al., 2005a). Samples of evaporites from a gypsum outcrop

at 75�23.85¢ N, 89�32.37¢ W (Fig. 1) were collected for

geomicrobiological analysis. The outcrop comprised a block

Fig. 1 Location of gypsum outcrop studied in this

article in relation to major lithological units in the

Haughton impact structure, Nunavut, Canadian

High Arctic (map after Osinski et al., 2005b).

294 C. S. COCKELL et al.

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of rotated ⁄ disrupted gypsum constituting part of the cen-

tral uplift draped by impact melt breccia in the east of the

Haughton impact structure in the Haughton River valley

(Fig. 2A,B). Samples were collected into aseptic bags and

maintained at ambient temperatures until return to the

laboratory.

Light microscopy

Light microscopy was used to examine the zonation of organ-

isms within the gypsum crust. Material from several represen-

tative parts of colonized gypsum was examined by crushing

the material gently into a fine powder. Some of this powder

was distributed onto a microscope slide and doubly distilled

H2O was added to suspend the material. The material was

examined using bright field microscopy on a Leica DMRP

fluorescence microscope (Leica Microsystems, Wetzlar,

Germany). Autofluorescence of cyanobacteria was examined

using a Leica N2.1 cube, which uses an excitation filter with a

bandpass of 515–560 nm and a long-band emission filter

(>590 nm).

To visualize prokaryotes, gypsum samples were stained by

soaking them in SYBR Green I DNA binding dye according

to the manufacturer’s instructions (Invitrogen, Paisley, UK).

Stained cells were observed fluorescing green using an excita-

tion waveband of 450–490 nm (Leica filter cube I3) and a

long-band emission cutoff filter of >515 nm. Images of

stained cells were superposed on bright field micrographs of

the same field of view to show relationships between stained

prokaryotes and cyanobacteria. Control tests on autoclaved

material showed that the stain did not non-specifically bind to

gypsum or other minerals present in the habitat.

Scanning electron microscopy and energy dispersive

spectroscopy

The location and characteristics of micro-organisms within

the gypsum and their mineral environment were examined by

scanning electron microscopy (SEM). Selected blocks (�0.3–

1 cm3) of gypsum encrustations from green colonized zones

were mounted on aluminium stubs with two-sided carbon

tape. Samples were carbon coated (15–20 nm thickness) and

were examined in secondary electron mode at a 20 kV acceler-

ating voltage and 7–15 mm working distance using a Quanta

3D dual beam FIBSEM (FEI, Hillsboro, OR, USA). Energy

dispersive spectroscopy (EDS), with a count time of 50–100 s

and data analysis with the software, Inca (Oxford Scientific

Instruments, Oxford, UK) were used to study qualitatively

the elemental composition of the samples.

To obtain high resolution images of crystals formed in gyp-

sum neogenesis experiments, a field emission gun (FEG)

SEM (ZEISS Supra 55-VP; Zeiss Microimaging, Gottingen,

Germany) was used. Samples were carbon coated (15–20 nm

thickness) and were examined in secondary electron mode at a

2–15 kV accelerating voltage and 7–10 mm working distance.

Analysis of the crystal composition was accomplished by EDS

as described above.

Transmission electron microscopy of gypsum sections

Transmission electron microscopy (TEM) was used to exam-

ine the relationship of organisms to the surface of the gypsum

and the presence of extracellular polysaccharide (EPS) around

organisms. Small fragments of gypsum from the green colo-

nized zone were dropped into 2.5% glutaraldehyde in 0.1 M

A B

C D

Fig. 2 Characteristics of the gypsum habitat. (A)

Gypsum outcrop at 75�2385¢ N, 89�3237¢ W emb-

edded within the melt-rocks of the Haughton

impact structure. Note rotation and disruption of

bedding planes (white dashed lines) caused by

impact (scale bar 1 m). (B) Close-up of black square

in (A) showing detail of bedding planes (scale bar

1 m). (C) Surface of outcrop showing precipitation

of gypsum along the vertical bedding planes and

fractures in which organisms preferentially grow

(scale bar 1 cm). Note visible black colouration,

which is biological. (D) Green zone of phototrophs

within reprecipitated gypsum (arrow) (scale bar

1 cm).

Microbe–mineral environment and gypsum neogenesis 295

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sodium cacodylate pH 7.4. The gypsum fragments were

post-fixed in 2% osmium tetroxide in sodium cacodylate,

dehydrated through an ascending series of acetone concentra-

tions, infiltrated in Epon (Hexion, Columbus, OH, USA)

minus accelerator at 60 �C with six changes. The samples were

finally embedded in complete Epon and polymerized at 60 �Cfor 48 h.

Ultrathin sections of �70 nm were cut using a microtome

diamond knife and collected on copper slot grids with a

formvar film. The sections were counter-stained with uranyl

acetate and Reynolds lead citrate before examination in a JEM

1400 transmission electron microscope (Jeol, Tokyo, Japan)

operating at 80 kV. Digital images were acquired using an

AMT XR60 digital camera (AMT Imaging, Danvers, MA,

USA).

X-ray diffraction

X-ray diffraction was used to study the parent material in the

outcrop and the reprecipitated gypsum. An INEL X-ray

diffractometer (XRD) (Inel, Strasbourg, France) with a

curved position sensitive detector was used for XRD analyses

at the Natural History Museum, London. A Ge monochro-

mator allowed selection of only CuKa radiation. Samples were

ground to a 35 lm powder and mounted on single-crystal

quartz substrates that were rotated continuously during

analysis. Post-monochromator slits were used to restrict the

beam to 0.24 · 5.00 mm and patterns were collected with

the beam at an incident angle of 3.6� to the flat-topped

sample. Analyses were conducted over 30 min. Silicon and

silver behenate were used as external standards in this

study. Calibration and data collection were performed using

Diffgrab�. WinXPow� was used for phase identification.

X-ray fluorescence

X-ray fluorescence (XRF) was used to examine the elemental

composition of the parent and reprecipitated gypsum and thus

the potential supply of nutrients to the enclosed biota. Sam-

ples of source evaporite taken from beneath the colonized

zones and reprecipitated gypsum deposits from green colo-

nized zones were sampled for XRF analysis. The samples were

crushed into an homogenous powder in a mortar and pestle

and pressed into discs using a Methyl methacrylate binder.

Analysis was carried out using an XRF Philips PW1480 Spec-

trometer (PANalytical, Almelo, The Netherlands) with semi-

quantitative software.

Raman spectroscopy

Raman spectroscopy was used to investigate the mineralogy of

the parent material and reprecipitated gypsum, to examine

whether the parent material contained anhydrite and to cross-

correlate spectra to XRD and XRF data. Raman was carried

out using a Horiba Jobin Yvon LabRAM (Horiba Jobin Yvon,

Stanmore, UK) and samples were excited using a 514.5-nm

(green) laser at 0.7 mW. For each material, three separate

samples of �10 g were powdered in a mortar and pestle and

pooled for analysis to obtain spectra representative of the

mean composition of the material. The footprint area of analy-

sis was a 2-lm-diameter spot. Spectra were the mean of five

separate spectra, each acquired over 20 s. Data were gathered

in the program LABSPEC (Horiba Jobin Yvon).

Penetration of light into the samples

The penetration of light into the reprecipitated gypsum was

examined to determine the light environment for photo-

trophs and the protection that they receive from ultraviolet

(UV) radiation. Light transmission spectra (250–750 nm)

were measured with an optical microspectrophotometer

(Craic Technologies, San Dimas, CA, USA), linked to a Leica

DMR microscope. The light source was a 75-W xenon-arc

lamp and the light was focussed onto an area of 10 · 10 lm.

Two representative samples of thin consolidated gypsum crust

were examined of 0.9-mm thickness each. Spectra at 10 differ-

ent spots were acquired and a mean spectrum was calculated.

The penetration of light at 300 nm (midway through the

UVB radiation region), 360 nm (midway through the UVA

radiation region) and at 680 nm (taken as the chlorophyll a

absorption maximum) was calculated.

Sulphur isotopic analysis

Sulphur isotopic analysis was carried out to determine if

gypsum reprecipitation was microbially mediated. Samples for

isotopic analysis were prepared by collecting �2 g from four

independent gypsum samples and crushing them into an

homogenous powder using a mortar and pestle. Two sets of

samples were prepared for analysis in this way. Samples of unal-

tered source evaporite, reprecipitated gypsum near the colo-

nized zones (uncolonized by phototrophs) and heavily

colonized (from the green colonized zones) gypsum were

prepared. Sulphur isotope analysis was undertaken by

EA-IRMS using a Europa Scientific 20-20 isotope ratio mass

spectrometer linked to a Sercon elemental analyser (Sercon,

Crewe, UK). Tin capsules containing reference or sample

material plus vanadium pentoxide catalyst were loaded into an

automatic sampler and dropped into a furnace held at

1080 �C and combusted in the presence of oxygen. The

combusted gases were treated in a helium stream over com-

bustion catalysts (tungstic oxide ⁄ zirconium oxide). Water was

removed using a Nafion� membrane (DuPont, Willmington,

DE, USA). The reference material used for 34S isotope analysis

was IA-R036 (barium sulphate, d34SV-CDT = +20.74&).

Samples of IA-R036, IA-R025 (barium sulphate, d34SV-CDT =

+8.53&) and IA-R026 (silver sulphide, d34SV-CDT =

+3.96&) were used for calibration and correction of the

296 C. S. COCKELL et al.

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18O contribution to the SO ion beam. IA-R036, IA-R025 and

IA-R026 are in-house standards calibrated and traceable to

NBS-127 (barium sulphate, d34SCDT = +20.3&) and IAEA-

S-1 (silver sulphide, d34SV-CDT = )0.3&). For quality control

purposes, test samples of IA-R036 and IAEA-SO-5 (barium

sulphate, d34SV-CDT = +0.50&) were measured. The data are

reported in the usual delta notation relative to V-CDT. The

error was ±0.5 per mil.

Thermochemolysis–GC–MS analysis

Thermochemolysis–GC–MS analysis was used to study the

major lipids present in the gypsum. Approximately 350 mg of

crushed colonized gypsum was dissolved in deionized water

(8 mL), sonicated and centrifuged. This procedure was

repeated five times. The residue was then freeze-dried. To

analyse microbial lipids in the gypsum, an aliquot of the

freeze-dried residue was placed in a quartz pyrolysis tube

plugged at either end with quartz wool; 10 lL of 25% tetram-

ethylammonium hydroxide in methanol was added to the

sample and the methanol was allowed to evaporate for 12 h.

The sample was heated using a CDS 5000 Pyroprobe fitted

with a 1500 valve interface held at 250 �C (CDS Analytical,

Oxford, PA, USA) and coupled to a GC-MS. GC-MS analysis

was carried out using an Agilent Technologies 6890 gas

chromatograph coupled to a 5973 mass spectrometer. Separa-

tion was performed on a S.G.E. (UK) BPX-5 column (30 m

length, 0.25 mm internal diameter and 0.25 lm film

thickness). Helium at a column flow rate of 1.1 mL min)1

was used as the carrier gas. Injection was at a 5:1 split and the

injector temperature was 270 �C. The GC oven temperature

was held for 1 min at 50 �C and then programmed at

5 �C min)1 to 310 �C. The final temperature was held for

9 min.

Microbial physiology

Enrichments and isolations were carried out to investigate the

physiology of organisms within the material and to determine

their degree of halotolerance or halophily. Cyanobacteria were

cultured by scattering fragments of crushed gypsum (5 g in

total) from green and black colonized zones onto BG-11

(Rippka et al., 1979) agar plates (2% Bacteriological Agar No.

1; Oxoid, Basingstoke, UK) with 50 lg mL)1 cycloheximide

to prevent fungal growth. Selection for halotolerant ⁄ halo-

philic species was accomplished by scattering fragments of

material onto BG-11 plates with amended NaCl concentra-

tions of 0.05, 0.1, 0.5, 1, 2 and 4 M. Plates were incubated at

4 and 21 �C under natural day ⁄ night light cycles for 2 months

until colonies had formed.

BG-11 was found to provide a suitable medium for the

cultivation of many fungal species from the gypsum when

employed without cycloheximide. Halotolerance ⁄ halophily of

fungal species was tested in an identical manner to the cyano-

bacteria. Plates were incubated at 4 and 21 �C for 5 weeks.

Selected isolates of fungi were also grown on 2% Malt agar.

Heterotrophic bacteria were isolated by scattering frag-

ments of crushed gypsum (5 g in total) from colonized zones

onto 1 ⁄ 10 Tryptone Soya Agar (TSA) plates made with

0.06 g L)1 TSA broth (Oxoid), 0.2 g CaSO4.2H2O and 2%

Bacteriological Agar No. 1 (Oxoid). Plates were incubated at

4 and 21 �C for 5 weeks.

To test for halophily (the requirement for salt to allow

growth), any organisms that grew on the NaCl-amended

BG-11 and 1 ⁄ 10 TSA plates were subcultured onto NaCl-free

plates and colony formation was monitored after 3 weeks. To

determine the maximum halotolerance of heterotrophic bac-

teria, isolates obtained on the NaCl-amended plates were

grown on plates with 0.1, 0.5, 1, 2 and 4 M NaCl and colony

formation was monitored after 3 weeks.

To investigate for halotolerance of bacteria in the surround-

ing arctic environment, soil samples were collected �50 m

east of the outcrop. Three samples were obtained by collecting

a soil core of �5 cm depth and pooling the samples. The soil

was collected into aseptic bags and maintained at ambient

temperature. Upon return to the laboratory 2 g of this soil

was added to 100 mL of TSA broth and shaken vigorously for

�2 min. Half mL aliquots of this solution (10 and 100X dilu-

tions) were spread onto TSA plates. After 2 weeks, sixty-four

distinct colonies (selected based on varying colour or mor-

phology) were removed from the plates and spread onto TSA

plates without NaCl and plates amended with 0.1, 1, 2 and

4 M NaCl to test for halotolerance. Colony formation was

monitored after 3 weeks.

All media and other reagents were obtained from Sigma

(Sigma-Aldrich, Gillingham, UK).

Identification of isolates

Cyanobacteria were identified by morphological criteria using

bright field microscopy on a Leica DMRP fluorescence micro-

scope (Leica Microsystems) at the Open University (Milton

Keynes, UK). Fungi were identified based on morphological

criteria at CABI (Centre for Agricultural Bioscience Interna-

tional; Egham, UK).

Selected heterotrophic bacterial isolates were identified by

16S rRNA gene partial sequencing. Microbial isolates were

identified by direct PCR from colonies. DNA template was

prepared by dispersing a bacterial colony in 50 lL of 10 mM

TE buffer, pH 7.5, and freeze-thawing at )20 �C. PCRs

were performed with the primers pA (5¢-AGA GTT TGA

TCC TGG CTC AG-3¢) and pH (5¢-AAG GAG GTG ATC

CAG CCG CA-3¢). The reaction mixture contained: 5 lL

of template DNA; 200 lM dNTP; 1 lM primers; 1.25 U of

Taq DNA polymerase; 1X PCR Buffer [20 mM Tris–HCl

(pH 8.4), 50 mM KCl] and 2.5 mM MgCl (Invitrogen).

Amplification consisted of incubation at 94 �C for 15 min

(which was used to assist cell disruption). This was followed

Microbe–mineral environment and gypsum neogenesis 297

� 2010 Blackwell Publishing Ltd

by 35 cycles of 1 min at 94 �C, 1 min at 55 �C and a 1-min

extension at 72 �C, with a final extension of 5 min at

72 �C. The PCR products were run on a 0.8% (w ⁄ v) agar

gel and extracted using a GenElute Gel Extraction kit

(Sigma-Aldrich, Poole, UK) according to the manufacturer’s

instructions. DNA sequencing of the PCR products was car-

ried out directly by DNA Vision (Liege, Belgium).

Sequences (up to 750 bp) were phylogenetically classified

and their nearest 16S rRNA gene sequences identified in the

Ribosomal Database Project (RDP) using the Classifier and

Sequence Match tools.

A phylogenetic tree of Arthrobacter isolates was

constructed. A region of 507 nucleotides (Escherichia coli

numbering 210–716 covering 16S rRNA variable regions

V2–V4) was used for tree construction with putative close

phylogenetic relatives obtained in RDP. A tree was first con-

structed using the neighbour-joining method (Saitou & Nei,

1987) and Kimura 2-parameter for distance correction

(Kimura, 1980) using the PHYLIP v. 3.67 inference package

(DNADIST and NEIGHBOR). SEQBOOT (1000 bootstraps) and

CONSENSE were used to provide confidence estimates for the

tree topology for a consensus tree. This tree was used as an

input tree to obtain a maximum-likelihood tree with distance

estimates using DNAML. Trees were visualized with NJPLOT

and TREEVIEW.

Nucleotide sequence accession numbers

The partial 16S rRNA sequences of the bacterial isolates

(Table 2) were deposited in GenBank (accession numbers

FJ706080–FJ706092).

Bacterial gypsum neogenesis by Arthrobacter

To test the hypothesis that Arthrobacter species could take

part in gypsum neogenesis, Arthrobacter isolates were grown

on CaSO4.2H2O-saturated 1 ⁄ 10 TSA agar plates. Two iso-

lates that formed bright white colonies were selected on the

basis that white colonies had the greatest chance of signify-

ing gypsum precipitation. The isolates were grown on 1 ⁄ 10

TSA agar plates for 2 weeks with no CaSO4 supplement to

ensure that there was no residual gypsum. A CaSO4.2H2O

saturated 1 ⁄ 10 TSA broth was prepared by vigorous shaking

of 10 g of CaSO4.2H2O in 500 mL 1 ⁄ 10 TSA which had

been previously sterilized and cooled. The medium was left

to settle overnight at 21 �C and was then filtered through a

0.2-lm filter. The isolates were inoculated into 50 mL of

the medium in sterilized and dried glass bottles and the

bottles were incubated at 21 �C for 4 weeks. At the end of

the incubation period, 2 mL of medium was filtered onto

0.2-lm black nitrocellulose filters to dryness and the filter

was examined by FEG-SEM as described above. All experi-

ments were prepared in replicate. Replicate uninoculated

controls were prepared.

RESULTS

Characteristics of colonized gypsum

Gypsum outcrops are found widely distributed across the

Haughton impact structure (Osinski et al., 2005a). They are

often disrupted and rotated and have been in this state since

an asteroid or comet impact �39 Ma (Sherlock et al., 2005).

The outcrop studied here exhibited conspicuous impact-rota-

tion of the bedding planes (Fig. 2A,B); the bedding prior to

impact was subhorizontal with a 3–5� westerly dip. The repre-

cipitated colonized gypsum was localized along the edges of

the near-vertical bedding planes (Fig. 2C) through which

precipitation in the form of snow-melt or rain can percolate.

The surface of the outcrop exhibited other irregular gypsum

reprecipitation textures (Fig. 2D). With the unaided eye,

phototrophs displayed separation into three distinct zones.

Black regions on the surface and within the gypsum corre-

spond to highly pigmented organisms (Fig. 2C,D). Beneath

the surface at a depth of between�2 and 5 mm the organisms

formed green zones of growth, although the depth of the

zones was irregular depending on the crystallinity and density

of the gypsum precipitate (Fig. 2D). Regions of pink pigmen-

tation were much more rarely observed within the gypsum,

which were usually associated with green zones. Unlike previ-

ously studied gypsum evaporites (e.g., Oren et al., 1995),

these zones were not developed into a well-defined vertically

stratified community.

Light microscopy

Within the black-zoned regions of the gypsum were abundant

pigmented cyanobacteria of size �5–10 lm (Fig. 3D). The

cells sometimes formed small colonies, but were mainly iso-

lated coccoid forms. Within the black zones, black fungal con-

idia were observed associated with patches of clear fungal

filaments.

Within green regions of the evaporite (Fig. 2D), the

primary morphotype corresponded to Gloeocapsa ⁄ Aphanot-

hece spp. (Fig. 3A). The cells were small (�1–2 lm) and grew

in a colonial growth habit surrounded by a conspicuous polys-

accharide sheath. Very rarely cyanobacterial filaments were

also observed, which were often encrusted with presumptive

gypsum crystals (Fig. 3C). Also abundant were organisms dis-

playing Chroococcidiopsis-like morphotypes (Fig. 3B). The

cells had the appearance of single cells, but many of them were

heavily pigmented colonies. Some displayed tetrad growth

patterns. All of these morphotypes displayed autofluores-

cence.

SYBR Green I staining revealed the presence of abundant

prokaryotes (Fig. 3E,F). The organisms were often, but

not exclusively, associated with cyanobacteria and dead cell

matter. They displayed both coccoid and rod morpho-

logies.

298 C. S. COCKELL et al.

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Mineralogy of the habitat

Scanning electron microscopy–EDS showed that the unal-

tered evaporite comprised a consolidated matrix of calcium

sulphate (Fig. 4A). The material contained other minor com-

ponents, which EDS showed to be comprised the major ele-

ments Mg, Si, Al, Fe and K (spectrum 1). The reprecipitated

gypsum (Fig. 4B) had a greater porosity than the parent mate-

rial, which provides space for microbial colonization. Under

SEM, organisms were observed as large amorphous masses,

consistent with the colonial growth forms (Fig. 4B) observed

by bright field microscopy (Fig. 3). The reprecipitated gyp-

sum contained grains of alumino-silicate materials containing

Mg, Si, Al, Fe and K (spectrum 2), some of which were on the

surface of the gypsum, suggesting an allochthonous source.

In some newly precipitated regions, the gypsum was pure

(spectrum 3) consistent with the white appearance of the gyp-

sum by visual inspection. The integrated spectrum shown

(spectrum 4) was representative of the reprecipitated gypsum.

By XRD, the original evaporite material and the newly

precipitated material on its surface exhibited peaks corre-

sponding to gypsum (CaSO4.2H2O). No anhydrite was

detected and no other major minerals were detected (data not

shown). Minor alumino-silicate phases are inferred on the

basis of the SEM–EDS spectra.

X-ray fluorescence data are shown in Table 1. Newly pre-

cipitated gypsum was depleted in non-gypsum elements,

except magnesium and barium, in which it was enriched.

Transmission electron microscopy of gypsum sections

Transmission electron microscopy revealed the presence of

numerous cell clusters associated with the gypsum surface.

Large gypsum crystals fell out of the sections, presumably

because of a weak connection with the resin, leaving cells pre-

viously on the gypsum surface associated with white empty

zones (Fig. 5). Most cells displayed abundant EPS production

(Fig. 5A). Many cells also had small gypsum crystals, which

did survive sectioning, associated with their surfaces (Fig. 5B).

Raman spectroscopy

The major peaks associated with the newly precipitated gyp-

sum and the underlying unweathered material corresponded

A B

C D

E F

Fig. 3 Micro-organisms within the gypsum. (A–C)

Micrographs of cyanobacterial morphotypes inhab-

iting the green zone in the gypsum (scale bar

10 lm), (D) micrograph of dominant morphotype

within the back zoned regions of the gypsum (scale

bar 20 lm), (E,F) SYBR Green I staining of prokary-

otes associated with gypsum crystals and cyanobac-

teria. Presumptive heterotrophs associated with

phototrophs are highlighted by an arrow. Photo-

trophs are highlighted with a dashed arrow. The

SYBR Green I image is superposed with the back-

ground of the bright field image to show location of

gypsum crystals and phototrophs. In image (E), the

cyanobacteria are the same morphotype as in (D).

The dark pigmentation prevents visualization by

SYBR Green I in contrast to (F) where the cyanobac-

terium [morphotype as in (B)] has stained [scale bar

in (E) and (F) is 10 lm].

Microbe–mineral environment and gypsum neogenesis 299

� 2010 Blackwell Publishing Ltd

to gypsum (identical with previously reported peaks at 416,

495, 621, 671, 1006, 1136 cm)1; Sharma et al., 2009)

(Fig. 6). The major peak at 1006 cm)1 is attributed to the

symmetric stretching vibrational mode of the sulphate ion.

No peaks corresponding to other minerals were observed.

Both the reprecipitated gypsum and the unweathered parent

material showed characteristic peaks attributed to O-H

stretching bands of water at 3406 and 3498 cm)1 showing

that the parent material is predominantly gypsum, not anhy-

drite. The parent material had a pronounced, but broad, peak

at 1600 cm)1 and a shoulder at �1375 cm)1. These features

most closely correspond to the organic G (aromatic carbons)

and D bands, respectively (Schopf et al., 2002; Jehlicka et al.,

2003; Guedes et al., 2005), showing the presence of organic

material in the Ordovician parent evaporite material.

Sulphur isotopic analysis

The mean d34S for the three samples was: source evaporite,

29.38&; reprecipitated uncolonized gypsum 29.91&; colo-

nized gypsum 30.25&.

Penetration of light into the gypsum

Light was effectively attenuated through gypsum crusts.

Through a 0.9-mm-thick gypsum crust, the mean penetration

through 10 different locations at 300 nm was 0.23% (±0.12),

at 360 nm it was 0.34% (±0.18) and at 680 nm it was 0.5%

(±0.24).

Thermochemolysis–GC–MS analysis of microbial

community

Thermochemolysis of organic material within the reprecipitat-

ed gypsum liberates a number of different compounds. The

most abundant compounds are fatty acids (as methyl esters)

which have an even predominance and range from C10 to C26.

10-methyl octanoic acid (also known as tuberculostearic acid)

is present (Fig. 7) which is a cell wall lipid produced by

Actinobacteria; the C18 homologue is also present

(O’Donnell et al., 1982; Brennan, 1988; Macnaughton &

O’Donnell, 1994). The presence of relatively high molecular

weight (>C18) fatty acids is also a common component of

actinobacterial cell walls.

Microbial isolations and physiology

On BG11 plates, cyanobacterial isolates were obtained on

plates amended with NaCl up to 0.1 M concentration, but not

on plates with 0.5 M NaCl concentration or higher. All organ-

isms obtained in NaCl-amended plates grew on unamended

plates showing that they were halotolerant, but not halophilic.

No cyanobacteria grew at 4 �C.

Diverse fungal species grew on BG-11 plates. One species

formed conspicuous and large quantities of black conidia,

which are morphologically similar to the black conidia

observed in field samples. Morphological criteria showed this

0Full scale 4416 cts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0Full scale 255 cts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0Full scale 2311 cts

1 2 3 4 5 6 7 8 0Full scale 4193 cts

1 2 3 4 5 6 7 8

1

A

2 B 3

4

Fig. 4 SEM–EDS analysis of minerals in the habitat surrounding a typical

cyanobacterial colonist. (A) A control area of unaltered Ordovician evaporitic

material under the habitat (scale bar 50 lm). EDS spectrum 1 corresponds to

unaltered material. Note presence of trace alumino-silicate material in evapo-

rite. (B) An example region of colonized gypsum (green zone in Fig. 2D; scale

bar 50 lm). Arrow is dried colonial form of cyanobacterium in rock. Spectrum 2

is of representative grains of presumptive allochthonous alumino-silicates, spec-

trum 3 is reprecipitated gypsum (note more pure form of gypsum than control

spectrum 1), spectrum 4 is the integrated spectrum of minerals around the

organisms.

Table 1 XRF data of reprecipitated gypsum from green colonized zones and

parent evaporite. Data are mean of three samples (SD < 5% for each analyte)

Analyte Reprecipitated gypsum (%) Parent evaporite (%)

Ca 25.23 26.52

SO4 68.74 70.54

BaO 3.9 –

SiO2 0.8 1.4

MgO 0.5 0.3

SrO 0.4 0.4

Al2O3 0.2 0.4

K2O 0.06 0.2

Fe2O3 0.07 0.2

Cl 0.02 0.03

300 C. S. COCKELL et al.

� 2010 Blackwell Publishing Ltd

species to most closely resemble Stachybotrys chartarum. A

common fungus observed on the plates formed septate dema-

tiaceous mycelia. It failed to sporulate and colonies reached

only 1.3 cm diameter after 50 days on 2% Malt agar plates. It

was morphologically similar to other rock-inhabiting fungi

belonging to the Herpotrichiellaceae. Fungi with black coni-

dia were obtained on plates amended with NaCl up to 1 M

concentration, but none were obtained at 2 or 4 M. No fungi

grew at 4 �C.

All heterotrophic bacteria isolated on plates saturated with

CaSO4 also grew on non-CaSO4-amended plates showing

that none required saturated CaSO4 concentrations to grow.

No heterotrophic bacteria were isolated on 4 M NaCl-

amended plates. One isolate (N5; Table 2) was isolated on

2 M NaCl-amended plates. Four isolates were obtained on

1 M NaCl-amended plates (N6–N9; Table 2). At 0.5 M NaCl,

similar morphology colonies were obtained as observed at 1 M

NaCl. At 0.1 M NaCl, plates were covered in many small red

colonies (N12; Table 2) in addition to those morphotypes

observed at higher concentrations. An actinobacterial mor-

photype was also obtained (N13; Table 2). At 0.05 M NaCl, a

variety of white and cream-coloured colonies grew in addition

to organisms observed at higher concentrations of NaCl.

None of 16 heterotrophic organisms selected from NaCl-

amended plates (N5–N9, N12, N13 and nine colonies from

the 0.05 M plates) were halophilic (all were able to grow on

unamended plates). Of the 16 heterotrophic bacteria obtained

on NaCl-amended plates and studied for their maximum salt

tolerance, all were able to grow at 0.1 M NaCl. Thirteen could

grow at 0.5 M NaCl (81%), 10 could grow at 1 M NaCl (62%),

one could grow at 2 M NaCl (N5; Table 2) and none grew at

4 M NaCl. The isolate that grew at 2 M NaCl could not grow

at 3.4 M NaCl, showing that it was moderately, but not extre-

mely, halotolerant. Four heterotrophic bacterial isolates were

obtained on plates incubated at 4 �C (N1–N4; Table 2).

These isolates were able to grow at 21 �C and grew faster than

at 4 �C, showing that they were psychrotolerant. No psychro-

philic bacteria were isolated.

Many of the isolates we obtained (Table 2) belonged to the

Arthrobacter. The partial 16S rRNA sequence phylogenetic

tree of the Arthrobacter isolates is shown in Fig. 8.

Of sixty-four heterotrophic isolates obtained from arctic

soil, all could grow at 0.1 M, 51 (81%) could grow at 0.5 M

NaCl, 29 (45%) could grow on 1 M NaCl and one isolate grew

on 2 M NaCl. None could grow at 4 M NaCl.

Bacterial gypsum neogenesis by Arthrobacter

Two Arthrobacter isolates that formed white colonies on

CaSO4-saturated TSA plates were examined for gypsum

crystal formation. In CaSO4-saturated 1 ⁄ 10 TSA solution,

gypsum crystals were observed in biological and control flasks.

However, in inoculated bottles (e.g. isolate N10; Fig. 9A,B),

gypsum crystals were observed to form along the periphery of

A B

Fig. 5 Transmission electron micrographs of

(unknown) prokaryotes associated with gypsum

surfaces. (A) Abundant extracellular polysaccharide

(EPS) (arrow) produced around two cells. The

empty area below corresponds to the location of

gypsum (scale bar 500 nm). (B) Cell showing gyp-

sum crystals (arrow) attached to the cell membrane

(cf. Fig. 3C) (scale bar 500 nm).

Fig. 6 Raman spectra of parent evaporite beds

(upper scan) and reprecipitated gypsum (lower

scan). Major peaks correspond to gypsum. Both

materials show characteristic O-H stretching bands

in water (shown to the right) at 3406 and

3498 cm)1. The parent material also shows a broad

peak at 1600 cm)1 (arrow).

Microbe–mineral environment and gypsum neogenesis 301

� 2010 Blackwell Publishing Ltd

carbonaceous material or to form columnar crystals on

organic surfaces (Fig. 9B). The carbonaceous material was

usually too large to be single cells and was probably EPS or

colonies of cells surrounded by EPS. All results were observed

to be replicated for both isolates. Plate-like crystals with pris-

matic edges (Fig. 9C) and clusters of needle-like crystals

(Fig. 9D) were also observed in the inoculated flasks, but not

in control flasks. In both control and inoculated flasks, many

crystals were composed of small plate-like crystals (compare

Fig. 9A to E). Many crystals in both control and inoculated

flasks exhibited irregular morphologies (e.g. Fig. 9F).

DISCUSSION

Gypsum colonization

Microbial communities inhabiting salt deposits have been

described from many environments. Here, we have investi-

gated the colonization of exposed reprecipitated gypsum on

Ordovician evaporites on Devon Island, Nunavut, Canadian

High Arctic. Unlike inter-tidal and saltern evaporites (e.g.

Rothschild et al., 1994; Oren et al., 1995), these modern

habitats are created by the remobilization of ancient evaporite

beds, rather than the evaporation of existing ponds, lakes or

marine waters. In marine evaporites, seawater provides many

required nutrients. In the material studied here, discrete min-

eral grains were observed on the surfaces of reprecipitated

gypsum by SEM–EDS. They were enriched in bioessential

cations such as magnesium, iron and potassium, showing that

the organisms in the polar desert must obtain most of their

nutrients from allochthonous input. The polar desert is dusty

and the frequent high winds pick up a diverse mineral suite

including fluvio-glacial minerals and minerals mixed and

exposed in the impact breccia melt hills, such as dolomites and

shocked crystalline basement gneiss and mafic clasts (Osinski

et al., 2005a). Neither XRD nor Raman suggests the presence

of other major minerals in the parent gypsum, although SEM

and XRF show that it does contain low abundances of magne-

sium, iron and potassium.

Phototrophs probably provide the main source of carbon

for the abundant bacterial populations associated with them.

This is consistent with observations of heterotrophic bacteria

associated with phototrophs in halite deposits in the Atacama

Desert (Chile) (Wierzchos et al., 2006), the gypsum-contain-

ing halite deposits of Baja, California (USA) (Rothschild

et al., 1994) and the gypsum outcrops of Bad Sachsa, Harz

Mountains (Germany) (Boison et al., 2004). Heterotrophs

were also observed associated with dead cells, indicating that

community turnover provides carbon. Raman spectroscopy

shows that the unweathered gypsum contains organics, which

Fig. 7 Thermochemolysis–GC–MS total ion chroma-

togram (TIC) and partial mass chromatogram (m ⁄ z74) of freeze-dried residue isolated from the

gypsum. Actinobacterial-associated tuberculostea-

ric acid (10Me18:0) is shown.

302 C. S. COCKELL et al.

� 2010 Blackwell Publishing Ltd

is lost in the reprecipitated gypsum. This material has either

leached away or it may also provide a source of organics for

heterotrophic micro-organisms. Another potential source of

organic material is the weathered products of dolomite (e.g.

Allen Bay Formation) in this region of Devon Island, which

contains bitumen and hydrocarbon fluid inclusions (Parnell

et al., 2007) and may be one component of the allochthonous

alumino-silicate material in the reprecipitated gypsum.

Micro-organisms do not play a major role in biogeochemi-

cal cycling of the gypsum itself, suggested by the largely

unchanged sulphur isotopic composition. This is consistent

with the aerobic environment in the evaporite which will

generally preclude sulphate reduction, although the small

enrichment in 34S might suggest localized anaerobic zones of

sulphate reduction or other mechanisms leading to loss of

light sulphur.

Physiological requirements

We were unable to isolate halophilic cyanobacteria, fungi or

heterotrophic bacteria in the gypsum. This is consistent with

theoretical expectations on the water activity (aw) in gypsum,

which provides a better measure of the biological availability

of water than solubility. Water potential is the effective water

content expressed as its mole fraction given by the van’t Hoff

equation:

w ¼ �m/CRT ; ð1Þ

where m is the number of particles in solution per mole-

cule (2 for gypsum), / is the activity coefficient (which is

assumed to be 1 for dilute solutions such as gypsum in

water), C is the concentration in mol kg)1, R is the

gas constant (8.31 J K)1 mol)1) and T is temperature in

Kelvin.

Water activity is computed from the inverse Kelvin equa-

tion:

aw ¼ expMwwRT

� �; ð2Þ

where Mw is the molecular mass of water (0.018 kg mol)1).

Substituting eqn (2) into eqn (1) gives

aw ¼ expð�MwmwCÞ: ð3Þ

The solubility of gypsum at 18 �C is 2.6 g L)1 and its molecu-

lar mass is 172.17 kg mol)1 giving a saturated solution a con-

centration of 0.015 molal (solubility drops to 2.23 g L)1 at

0 �C, which would be experienced in the Arctic, which

increases slightly the water activity) (Marion & Farren, 1997,

1999). Thus, the aw of a saturated gypsum solution at

18 �C = 0.9995.

Table 2 Identity of bacterial isolates obtained in this work

Isolate

number

Genbank

accession

number Description ⁄ Phylum Closest uncultured match (similarity) Closest cultured match (similarity)

N1 FJ706080 Psychrotolerant strain 1

Actinobacteria

DQ125753 Uncultured bacterium

clone AKAU3866 (95%)

EU029648 Arthrobacter sp. MY14015

from Minyong glacier (95%)

N2 FJ706081 Psychrotolerant strain 2

Actinobacteria

AM268326 Microbacterium

phyllosphaerae (98%)

EU468057 Uncultured bacterium clone

CE2_b03_2 (98%)

N3 FJ706082 Psychrotolerant strain 3

Actinobacteria

EF651060 Uncultured Arthrobacter

sp. clone AUVE_03D09 (91%)

EU157717 Arthrobacter sp. 31 (91%)

N4 FJ706083 Psychrotolerant strain 4

Actinobacteria

DQ125724 Uncultured bacterium

clone AKAU3822 (98%)

EF601820 Arthrobacter sp. WX4 (98%)

N5 FJ706084 Isolate from 2 M NaCl plate

Actinobacteria

FJ429561 Uncultured Streptomyces

sp. clone 2537 (100%)

FJ406045 Streptomyces albidoflavus

strain AS 4.1291 (100%)

N6 FJ706085 Isolate 1 from 1 M NaCl plate

Actinobacteria

EU536500 Uncultured bacterium

clone nbt214 h04 (99%)

AM990781 Kocuria sp. MOLA 5 (99%)

N7 FJ706086 Isolate from 1 M NaCl plate

Actinobacteria

AM167988 Uncultured Arthrobacter

sp.(98%)

AJ315069 Arthrobacter tumbae (99%)

N8 FJ706087 Isolate from 1 M NaCl plate

Actinobacteria

EF614070 Uncultured bacterium

clone NGD49 (96%)

EF471900 Arthrobacter sp. GW10-3

(96%)

N9 FJ706088 Isolate from 1 M NaCl plate

Actinobacteria

EU979035 Uncultured

actinobacterium clone g26 (92%)

DQ830985 Aestuariimicrobium

kwangyangensis strain R47 (93%)

N10 FJ706089 Gypsum neogenesis isolate 1

Actinobacteria

EF614054 Uncultured bacterium

clone NGD34 (99%)

AM933512 Arthrobacter sp. FB21

(99%)

N11 FJ706090 Gypsum neogenesis isolate 2

Actinobacteria

EU153020 Uncultured bacterium

clone G22-20 (99%)

AF479354 Glacial ice bacterium

G50-TB7 (Arthrobacter) (99%)

N12 FJ706091 Common red colony

a-proteobacteria

EF071489 Uncultured

Acetobacteraceae (98%)

AF170742 Paracraurococcus sp.

QSSC5-3 (Antarctic quartz rocks)

(97%)

N13 FJ706092 Common actinobacterium EU132912 Uncultured soil bacterium

(96%)

AY253863 Kribella flavida (97%)

Microbe–mineral environment and gypsum neogenesis 303

� 2010 Blackwell Publishing Ltd

This high water activity is equivalent to a NaCl concentra-

tion of 0.01 M and would not be expected to select for halo-

tolerance or halophily (Grant, 2004) and is not sufficiently

high to select even for slight halotolerance (0.2–0.85 M NaCl;

as defined by DasSarma & Arora, 2001).

Despite this observation, many of the heterotrophic bacte-

rial isolates were moderately halotolerant (tolerant of salt

concentration >0.85 M and <3.4 M), as were heterotrophs

from the surrounding soil, showing that halotolerance is a

widespread attribute in arctic bacteria. Statistical comparisons

between the soil and gypsum isolates are not reliable because

it is not possible to tell which isolates in the gypsum crust are

active and which ones are blown in from the surrounding

environment and are inactive; furthermore some soils in the

region contain gypsum (Bowden & Parnell, 2007). Studies

on halotolerance are generally confined to hypersaline envi-

ronments, so we do not know how widespread halotolerance

is in soil bacteria generally. In polar environments, a contribu-

tory selection pressure could plausibly be freeze-thaw cycles

that might form briny micro-environments within soils.

Although living in gypsum does not require halophily, the

habitat does select for specific organisms. The diversity of

Gloeocapsa morphotypes observed in black epilithic biofilms

on arctic rocks (Fig. 3D; Cockell et al., 2002) is not observed

in the gypsum at such high abundance and filamentous cyano-

bacteria are rare. Parnell et al. (2004) similarly observed a low

diversity of cyanobacteria belonging to Gloeocapsa in selenite

from the same region, although Nostoc sp. was commonly

observed in that material. Factors other than the requirement

for halophily are responsible for species selection. For exam-

ple, the gypsum has a lower concentration of bioessential

nutrients compared to rock surfaces; a different nutrient regi-

men might act as a selection pressure. The gypsum-encrusted

prokaryotes and cyanobacterial filaments observed by trans-

mission electron and bright field microscopy, respectively, and

the gypsum neogenesis shown in Arthrobacter isolates suggest

that growing in the presence of gypsum precipitation is a

challenge for organisms. Despite the high water activity in a

saturated gypsum solution, the material is highly porous.

These factors probably make the gypsum a desiccating envi-

ronment, which is consistent with the copious EPS produced

by many organisms observed by TEM. In principle, the anhy-

drite from which these gypsum deposits formed would also

make the habitat desiccating. Although trace quantities of this

mineral cannot be ruled out, the XRD and Raman analysis

show that it cannot be a significant contributor to desiccation

in the outcrop studied here.

No psychrophilic isolates were obtained. Tang et al. (1997)

showed that many arctic cyanobacteria had optimum meso-

philic growth temperatures, which suggests successful coloni-

zation through slow persistent growth rather than optimum

adaptation to low temperatures. Our data show that many

arctic bacteria and fungi also possess mesophilic growth

optima.

Within the gypsum, the organisms are protected from UV

radiation. In the High Arctic, the minimum zenith angle

experienced in July when the temperatures are highest and

rain water is available is 51�, so that the maximum instanta-

neous UV radiation flux is lower than in temperate or tropical

evaporitic habitats, but the 24-h photoperiod makes the

potential cumulative dose as high (Cockell et al., 2001).

Parnell et al. (2004) reported a high level of UV penetration

to organisms within selenite sheets with a reduction of only

25% through a 1-mm-thick sheet. This is caused by the poor

UV radiation absorption of calcium sulphate and the trans-

parent nature of selenite crystals. The organisms within the

habitat described here gain advantage from the precipitation

Fig. 8 Phylogenetic tree of gypsum Arthrobacter isolates and phylogenetic

relatives. A maximum-likelihood tree was generated using a neighbour-joining

tree (1000 bootstraps) as the input tree and using a mask of 507 nucleotide

positions (Escherichia coli numbering 210–716 covering V2–V4). Aquifex sp.

(accession number AB304892) was used as an outgroup. The scale bar

represents the number of changes per nucleotide position. The black arrows

correspond to Arthrobacter isolates obtained in this study. The two white-

headed arrows are the isolates used to demonstrate gypsum neogenesis.

304 C. S. COCKELL et al.

� 2010 Blackwell Publishing Ltd

of small gypsum crystals which can effectively scatter UV radi-

ation (Cockell & Raven, 2004). Our data are consistent with

Hughes & Lawley (2003) who observed the effective attenua-

tion of UV radiation in a 1.2-mm-thick Antarctic gypsum

crust (0.005% of the UVB and 0.05% of UVA). Concomi-

tantly, however, the organisms’ exposure to photosyntheti-

cally active radiation will be lower than it would be in selenite

(Parnell et al., 2004) as this region of the spectrum is also

scattered.

Gypsum neogenesis by Arthrobacter

Many of the isolates that were obtained belonged to the

Arthrobacter. Arthrobacter, Gram-positive, pleomorphic,

non-spore forming members of the Actinobacteria, are known

to be an extremophilic genus of bacteria adapted to survival in

dry, cold and oligotrophic environments (Cacciari & Lippi,

1986; Reddy et al., 2002). They have been isolated from the

surface of rocks (Gounot, 1967; Heyrman et al., 2005) and

from permafrost (Vishnivetskaya et al., 2006) and are known

to sequester metals from rocks (Brantley et al., 2001).

Arthrobacter have previously been isolated from meadow soils

on Devon Island (Nelson & Parkinson, 1978a,b). Our obser-

vations that the dominant cultured psychrotolerant and halo-

tolerant organisms in this study belonged to the Arthrobacter

are consistent with these reports. The identification of the

lipid, tuberculostearic acid, which is associated with the

Actinobacteria (Brennan, 1988; Macnaughton & O’Donnell,

1994), is consistent with the importance of Actinobacteria in

this habitat. This lipid is formed by Arthrobacter spp. (O’Don-

nell et al., 1982). The recent report that Gram-positive

Actinobacteria constitute the major component of bacterial

communities in carbonates in this region of the Arctic (Lacelle

et al., 2009) is consistent with these findings.

In view of these observations, we hypothesized that in the

polar environment Arthrobacter might be one group playing a

role in mediating mineralization of the gypsum habitat.

Microbially mediated mineralization processes are known to

occur in the arctic (Konhauser et al., 2005). The data

obtained here show that Arthrobacter species can enhance

gypsum neogenesis.

In the inoculated experiments, crystals were observed to

have formed on carbonaceous materials. They were composed

of small plate-like crystals consistent with gypsum crystals on

cell surfaces observed by TEM and bright field microscopy.

The morphology of the crystals was indistinguishable from

similar crystals observed in controls. Small crystals were

observed to have formed in both abiotic and biotic experi-

ments. Precipitation within the controls suggests either

nucleation of crystals around abiotic particles or growth of

crystals around gypsum particles <0.2 lm that passed through

the filter. This would have occurred in the inoculated bottles,

A B

C D

E F

Fig. 9 Evidence for gypsum neogenesis. (A) Forma-

tion of gypsum crystals on an organic template in

culture N10 (scale bar 10 lm). Arrows in close-up,

(B) (scale bar 4 lm), are gypsum (EDS analysis).

Smooth material underlying crystals is carbona-

ceous (EDS analysis showed only carbon and

oxygen as constituents), (C) (scale bar 10 lm)

and (D) (scale bar 50 lm) show plate-like and

needle-like crystals, respectively, which are com-

mon crystal morphologies in inoculated experi-

ments. (E) and (F) show platy and irregular crystal

morphologies, respectively, which are seen in both

inoculated and abiotic experiments (compare E to

A) (scale bar 10 lm).

Microbe–mineral environment and gypsum neogenesis 305

� 2010 Blackwell Publishing Ltd

so that some of the crystals observed in the biological experi-

ments are likely to be abiotic.

The biologically mediated gypsum neogenesis that was

observed could plausibly be triggered by two processes: (i) the

nucleation of gypsum around bacteria whereby the bacteria

merely act as particles for gypsum growth as has been observed

with cyanobacteria (Braithwaite & Whitton, 1987), or (ii) the

more specific formation of gypsum crystals on cell membranes

or EPS. A plausible mechanism might be the binding of Ca2+

to negatively charged bacterial cell surfaces (Beveridge & Fyfe,

1985; Ferris, 1990; Mitchell & Ferris, 2006) or negatively

charged moieties on EPS (Tourney & Ngwenya, 2008), caus-

ing localized supersaturation and precipitation of gypsum. A

similar mechanism was proposed for the precipitation of gyp-

sum on the cell wall of the cyanobacterium Synechococcus sp. in

Green Lake, New York (Thompson & Ferris, 1990). This

process may also cause the permanent entrapment of micro-

organisms within the reprecipitated gypsum. The fact that we

observed gypsum crystal formation on organic matter in the

biotic experiment suggests that metabolic activity is not

required to cause gypsum neogenesis.

Implications for impact crater geobiology

Finally, we note the association of the gypsum with an impact

crater. Impact events are known to have an important influ-

ence on the geomicrobiological environment. They can

increase the abundance of lithic habitats for micro-organisms

by fracturing rocks and in some instances increasing their

translucence for phototrophs (Cockell et al., 2002, 2005).

The communities observed in the gypsum deposits are associ-

ated with evaporite beds disrupted by an asteroid or comet

impact 39 Ma and now emplaced in impact breccia deposits.

The impact-induced rotation and disruption of the beds, such

as in the unit examined here, have weakened the bedding

planes, thus improving water penetration, facilitating gypsum

mobilization and subsequent colonization.

Impact hydrothermal systems, such as the one established

by the Haughton impact event (Osinski et al., 2001), remobi-

lize and precipitate salts. At Haughton, a habitat was created

in the recrystallized hydrothermally precipitated selenite (Par-

nell et al., 2004), which is colonized by cyanobaacteria, analo-

gously to the habitat described in this study.

The crater cavity created by impact events can lead to the

formation of salty, evaporitic habitats if the crater cavity ponds

water. For example, a hypersaline lake exists within the bowl

of the 50 000-year-old 1-km diameter Tswaing impact crater

in South Africa (Schoeman & Ashton, 1982; Ashton & Scho-

eman, 1983). In addition to surface habitats, the confinement

of seawater from the shallow marine Chesapeake Bay impact

�35 Ma has trapped briny habitats in the deep subsurface

(Sanford, 2005; Cockell et al., 2009; Sanford et al., 2009).

Thus, the data presented here are another instance of the

diverse ways in which impact events can influence the distribu-

tion and accessibility of salt deposits as habitats for a micro-

biota.

ACKNOWLEDGMENTS

We would like to thank Pascal Lee and the Haughton-Mars

Project for work in the field site and the Canadian Space

Agency (Canadian Analogue Research Network) for funding.

We thank Brady Carter of Decagon Devices, Pullman, WA for

discussions on water activity and Gordon Imlach, Open

University, for assistance with the FEG-SEM. Preparation of

the TEM samples was carried out by Heather Davies at the

Open University. We thank two anonymous referees who

provided helpful review comments.

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