Collection of Samples Isolation and Identification of Fungi from High Arctic Glaciers

A study involving isolation of xerotolerant/ halotolerant fungi from an Arctic coastal environment was performed in Kongsfjorden. The fjord is located at 79°N, 12°E and is one of the larger fjords on the western coast of Spitsbergen, in the Svalbard Archipelago. It is 26 km long and 8 km wide and stretches from ESE to WNW from the Greenland Sea. The majority of the drainage basin is covered by glaciers, which calve pieces of glacier ice into the fjord throughout the year. The annual mean temperature is around -5 °C, although the water is warmer and less salty than the open sea during the summer. On average, the fjord water temperature is >0 °C by the end of May and 3.8 °C at the end of August. The mean salinity ranges from 34.00 to 35.00 PSU. Lowering of salinity can occur in summer and near the surface [Ito and Koduh, 1997].

The glaciers studied were Conwaybreen, Kongsvegen, austre Lovenbreen and austre Br0ggerbreen. They have polythermal characteristics, and therefore they mainly consist of ice at subfreezing temperatures [Copland and Sharp, 2001]. Melting in the temperate cores of the glaciers and seasonal inputs of meltwater from the glacier surfaces provide liquid water at their base [Skidmore et al., 2000]. The unfrozen sediments beneath the glaciers are entrained into the basal ice where the meltwaters refreeze beneath the cold-based marginal regions of the glacier. The ice flow then transports them to the glacier margins, where they can be easily accessed and aseptically sampled [Butinar et al., 2007; Skidmore et al., 2000]. Although all of the glaciers studied are polythermal, austre Br0ggerbreen is almost entirely cold-based and thus no prolonged interactions between the meltwaters and the glacier bed occur [Hodson et al., 2005].

Samples from the supra- and subglacial environments were collected aseptically during the two melt seasons (in 2001 and 2003), as previously described [Butinar et al., 2007; Gunde-Cimerman et al., 2003]. Samples were collected from Conwaybreen, Kongsvegen and austre Lovenbreen coastal glaciers and inland glacier austre Br0ggerbreen. Twenty-six subglacial samples included sediment-rich, overlying clear basal ice and subglacial meltwater. Additionally, two samples of subsurface ice from cryokarst formations were collected. The supraglacial samples comprised four samples of snow/ ice mixtures and nine samples of seasonal meltwaters on the glacier surfaces.

Physico-chemical parameters (pH, Na+, Mg2+ and K+ concentrations, and total phosphorus content) were determined for five basal ice samples (originating from Kongsvegen), and a sample of subglacial meltwater, as described by Gunde-Cimerman et al. [2003].

Isolation conditions were designed to accommodate xerotolerant/ halotolerant fungi by using media with high concentrations of salt or sugar, and thus low aw, such as previously used for the isolation of xerotolerant/ xerophilic and halotolerant/ halophilic fungi from temperate hypersaline environments [Gunde-Cimerman et al., 2000] (Table 1). These media should give a selective advantage to cultivable microorganisms adapted to ice, thereby possibly enabling the isolation of higher fungal colony forming units (CFU) numbers than previously reported [Gunde-Cimerman et al., 2000, 2003].

Melanized yeast-like fungi were identified by their morphology, physiology, and by multi-loci sequencing to the species level [Zalar et al., 1999; data not published]. Isolates of filamentous fungi were identified to the species level by morphology, physiology, and in most cases also by secondary metabolite profiles using HPLC-DAD [Smedsgaard, 1997; Sonjak et al., 2005, 2006]. The identification methods for non-melanized yeasts followed those described by Yarrow [1998], Fonseca [1992] and Sampaio [1999], and the molecular characterisation included analyses of the electrophoretic band patterns following minisatellite-primed PCR (MSP-PCR) [Gadanho et al., 2003], and their determining the

D1/D2 domain sequences of the 26S rDNA and/ or ITS sequences [Kurtzman and Robnett, 1998; Fell et al., 2000; Fonseca et al., 2000].

Table 1. Enumeration and selective isolation media, and respective water activity (aw)

values, used in this study

Table 1. Enumeration and selective isolation media, and respective water activity (aw)

values, used in this study

Medium

aw

Reference

Dichloran rose bengal chloramphenicol agar (DRBC)

~1

[King et al., 1979]

Dichloran 18% glycerol agar (DG18)

0.946

[Hocking and Pitt,

1980]

Malt yeast 10% glucose and 12% NaCl agar (MY10-12)

0.916

[Samson et al.,

2004]

Malt yeast 20% glucose agar (MY20G)

0.941

[Gunde-Cimerman

et al., 2000; 2003]

Malt yeast 35% glucose agar (MY35G)

0.915

[Gunde-Cimerman

et al., 2000; 2003]

Malt yeast 50% glucose agar (MY50G)

0.890

[Samson et al.,

2004]

Malt extract agar (MEA)

~1

[Samson et al.,

2004]

Malt extract 5% NaCl agar (MEA5NaCl)

0.951

[Gunde-Cimerman

et al., 2000; 2003]

Malt extract 10% NaCl agar (MEA10NaCl)

0.924

[Gunde-Cimerman

et al., 2000; 2003]

Malt extract 15% NaCl agar (MEA15NaCl)

0.881

[Gunde-Cimerman

et al., 2000; 2003]

Malt extract 17% NaCl agar (MEA17NaCl)

0.861

[Gunde-Cimerman

et al., 2000; 2003]

Malt extract 24% NaCl agar (MEA24NaCl)

0.828

[Gunde-Cimerman

et al., 2000; 2003]

Malt extract 30% NaCl agar (MEA30NaCl)

0.782

[Gunde-Cimerman

et al., 2000; 2003]

All isolates are maintained in a genetically stable way in the Culture Collection of the National Institute of Chemistry (MZKI) (Slovenia) and in the EXF Culture Collection of the Department of Biology, Biotechnical Faculty, University of Ljubljana (Slovenia). Most penicillia are preserved as well in the fungal collection (IBT) at the Centre for Microbial Biotechnology (BioCentrum-DTU), Denmark, while yeast-like melanized fungi are preserved in CBS, Utrecht, The Netherlands.

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