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Keywords:

  • psychrophilic yeasts;
  • glacial environments;
  • Alpine glaciers

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The presence of psychrophilic yeasts in supra- and subglacial sediments, ice and meltwater collected from two glaciers of the Italian Alps (Forni and Sforzellina – Ortles-Cevedale group) was investigated. After incubation at 4 °C, subglacial sediments contained from 1.3 × 103 to 9.6 × 103 CFU of yeasts g−1. The number of yeast cells in supraglacial sediments was c. 10–100-fold lower. A significant proportion of isolated yeasts exhibited one or more extracellular enzymatic activities (starch-degrading, lipolytic, esterolytic, proteolytic and pectinolytic activity) at 4 °C. Selected isolates were able to grow at 2 °C under laboratory-simulated in situ conditions. In all, 106 isolated yeasts were identified by MSP-PCR fingerprinting and 26S rRNA gene sequencing of the D1/D2 region as belonging to 10 species: Aureobasidium pullulans, Cryptococcus gilvescens (over 50% of the total), Cryptococcus terricolus, Mrakia gelida, Naganishia globosa, Rhodotorula glacialis, Rhodotorula psychrophenolica, Rhodotorula bacarum, Rhodotorula creatinivora and Rhodotorula laryngis. Four strains, all belonging to a new yeast species, yet to be described, were also isolated.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Glacial environments have long been merely regarded as life-preserving habitats. As a consequence, microorganisms occurring in these environments have been studied mainly due to their ability to survive under such extreme conditions (Abyzov, 1993; Ma et al., 1999, 2000; Christner et al., 2000; Poglazova et al., 2001; Reeve et al., 2002; Gunde-Cimerman et al., 2003; Mader et al., 2006). Nevertheless, some studies have shown that such habitats, including Arctic and Antarctic ice, permafrost or high mountain glaciers, can be colonized by obligate and psychrophilic microorganisms (Deming, 2002). Conventionally, obligate psychrophiles are defined as organisms with an optimal temperature for growth of 15 °C, a maximum temperature below 20 °C and a minimal temperature for growth at 0 °C or lower. Microorganisms that grow at 0 °C, but have optima of 20–30 °C are called facultative psychrophiles (psychrotolerant) (Cavicchioli & Tortsen, 2000; Raspor & Zupan, 2006). Facultative psychrophiles have evolved to tolerate cold, but they are not as physiologically specialized as obligate psychrophiles (Gounot, 1976).

The occurrence of microbial life in glacier habitats has been amply documented. Viable bacterial populations have been observed beneath polythermal glaciers in the Alaskan and Canadian High Arctic regions (Skidmore et al., 2000, 2005; Bhatia et al., 2006) and in the New Zealand alps (Foght et al., 2004). More recently, de García et al. (2007) described the occurrence of viable yeasts in glacial meltwater rivers originating from glaciers of Argentinean Patagonia. Butinar et al. (2007) reported the occurrence of viable yeasts in the different ice layers of Arctic glaciers located in the Svalbard Islands (Norway), and Gostinčar et al. (2006) described the local evolution of black yeast-like organisms of the species Aureobasidium pullulans in basal ice of Arctic glaciers.

Concerning studies on southern European Alpine glaciers, Sharp et al. (1999) observed viable bacterial populations in subglacial sediments beneath glaciers in the Swiss Alps, although they did not isolate yeasts or filamentous fungi. On the other hand, Margesin et al. (2002) described the occurrence of heterotrophic Gram-positive and Gram-negative bacteria, as well as yeasts, in Alpine glacier cryoconites, and Buzzini et al. (2005) reported the occurrence of viable yeast cells in meltwaters running off from the ice tongues of glaciers in Italian Alps. Overall, the literature so far published gives only a fragmentary picture of the diversity of psychrophilic yeast populations occurring in glacial environments of Alpine glaciers. The aim of the present study has been the isolation and characterization of psychrophilic yeasts from supra- and subglacial sediments, ice, and meltwater from two glaciers in the Italian Alps.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Characteristics of glaciers

Forni glacier (46°23′ N, 10°35′ E) (Fig. 1) is the largest Italian valley glacier (c. 12 km2 of surface area, elevation range between 2600 and 3670 m a.s.l.) characterized by an ice thickness of about 100 m (Merlanti et al., 1995). The maximum recent extension of Forni glacier was observed at the end of the Little Ice Age (between 1550 and 1850). After this period, an almost continuous terminus retreat (1925–2005 average retreat value=−20 m year−1) and strong glacier thinning (2000–2005 average thickness decrease in the ablation area=−3 m year−1) have been observed.

image

Figure 1.  Location of Forni (a) and Sforzellina (b) glaciers. Arrows indicate the sampling areas.

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Sforzellina glacier (46°20′ N, 10°30′ E) (Fig. 1) is a cirque-shaped glacier (c. 0.3 km2 of surface area, elevation range between 2850 and 3050 m a.s.l.) characterized by an ice thickness of about 30 m (Pavan et al., 2000). This glacier also shrunk significantly during the 20th century (1925–2005 average terminus retreat value=−5 m year−1, 2000–2005 average thickness decrease in the ablation area=−1.2 m year−1).

Sample collection

Samplings were carried out in the month of August of 2 consecutive years (2004 and 2005). The sampling areas were located at the terminus of the two glaciers described above, and an average number of five to six different sampling sites per glacier per year (each one placed several meters from each other at the glacier margin) were considered. For all sampling procedures, clean hand tools were surface sterilized using 70% ethanol immediately before use, and between each sample. Glass sample containers were previously sterilized in the laboratory by autoclaving them at 121 °C for 15 min. Sediment, water and ice temperature were recorded in situ.

Various aliquots (about 500 g) of unfrozen subglacial sediments were collected. Overlying ice was previously removed to expose fresh undisturbed sediments. After removing and discarding about 5 cm of surface sediment, the underlying layer was aseptically collected and placed into sterile glass containers. A total of about 5 kg of sediment per glacier was collected. The sediments were stored at 4 °C until analysis, carried out within 72–96 h. This storage temperature was chosen because the sediments were unfrozen in situ when collected. For comparative purposes, aliquots (about 500 g) of supraglacial unfrozen sediments were also collected.

Aliquots (500 mL) of meltwaters, originating from subglacial flow exiting underneath the terminus of the glaciers, were collected using sterile glass bottles. The bottles were immersed in the melt stream while still sealed and then opened while still under the water. A total of about 15 L of water per glacier was collected. Samples were stored at 4 °C until analysis, carried out within 72–96 h.

Aliquots (about 300 g) of ice carrots (ice cores) were collected, in proximity to the sediments. After removing and discarding about 5 cm of ice surface, cores (diameter=8 cm) were obtained by drilling glacial ice to depths of about 30 cm. Ice cores were placed into sterile containers with dry ice and thus placed at −18 °C in the laboratory within 24–30 h. A total of about 3 kg of ice per glacier was collected. Ice cores never reached temperatures above 0 °C.

Physical and chemical analyses

Ice samples (after melting) and meltwaters were analyzed for dry mass (DM), pH, total organic carbon, total organic nitrogen, and total organic phosphorus using standard methods (Hunt & Wilson, 1986). Sediment samples were analyzed for the same components according to Mudroch et al. (1996).

Microbiological analyses

As sampling had been carried out aseptically, both sediments and melting waters were studied in the laboratory without decontamination procedures. However, a laboratory decontamination protocol according to Rogers et al. (2004) was used for ice cores to exclude the presence of external microorganisms on the sample surfaces introduced during drilling procedures.

Enumeration of culturable yeasts was carried out according to Buzzini et al. (2005) using three different substrates: Rose Bengal agar (RB)+tetracycline; Dichloran 18% Glycerol agar (DG18); Dichloran Rose Bengal agar (DRB)+chloramphenicol. All media were from Difco.

Aliquots (50–100 mL) of liquid samples (meltwaters or water originated after melting of ice carrots) were filtered through 0.22-μm filters (Millipore). Each filter was then aseptically divided into two equal parts and laid onto different Petri dishes, which were incubated at 4 or 20 °C for 12 or 3 weeks, respectively. Solid samples (supra- and subglacial sediments) were diluted with sterile 0.1% sodium pyrophosphate. Serial dilutions were then plated onto Petri dishes containing the above media and then incubated at the two different temperatures (4 and 20 °C). All viable counts were carried out in triplicate and statistical evaluation of average values was carried out by anova.

Determination of extracellular enzymatic activities of yeast isolates

A set of 73 yeast strains, representative of isolates, were tested for the ability to secrete extracellular hydrolytic enzymes (amylase, protease, lipase, esterase, pectinase, cellulase and chitinase) according to previously described procedures (Buzzini & Martini, 2002; Brizzio et al., 2007; de García et al., 2007).

Assessment of growth at 2 °C of yeast isolates in subglacial sediments under laboratory-simulated in situ conditions

Sediments were sterilized using ethylene oxide. Before exposure to sterilization treatments, sediment aliquots were stored in jars at 25 °C and exposed to anhydrous calcium sulfate crystals as desiccators to obtain a relative humidity of less than 1%. Treatments with ethylene oxide were carried out in a 100 L sterilization apparatus (Bioster Spa, Italy) at 25 °C using the following operative conditions: prevacuum pressure −70 kPa, prevacuum time 30 min, ethylene oxide inlet time 60 min, cycle temperature 50 °C, cycle relative humidity 65%, delta cycle pressure 120 kPa, ethylene oxide exposure time 20 h, postvacuum pressure –70 kPa, postvacuum time 45 min. Sterilized sediments were then stored in sterile containers until examination. Before utilization, the treated sediments were rehydrated to their original moisture content with sterile distilled water.

Sterile subglacial sediments collected from Forni were inoculated with Cryptococcus gilvescens DBVPG 4793 (previously isolated from subglacial sediments of the same glacier), whereas those from Sforzellina were inoculated with Cr. gilvescens DBVPG 4800 (a strain previously isolated from subglacial sediments of that glacier). Aqueous suspensions of washed cells of the two isolates were used to inoculate Erlenmeyer flasks containing 250 g of sediments to achieve an initial yeast concentration of 10 cell g−1 sediment DM. After inoculum, flasks were incubated under static conditions at 2 °C for 10 weeks. Yeast growth was monitored every 2 weeks using viable cell counts on RB+tetracycline agar plates. Flasks containing uninoculated sediments were used as controls.

Isolation and preliminary phenotypic clustering of yeasts

Microbial growth on the plates was periodically checked, and any yeast colonies were transferred to RB without tetracycline and purified. Colonies were selected for isolation on the basis of morphology, taking care to isolate all types occurring at the different incubation temperatures. All yeast strains isolated are deposited in the Industrial Yeasts Collection DBVPG (http://www.agr.unipg.it/dbvpg). The isolates were preliminarily typed using a series of conventional phenotypic tests: macroscopic and microscopic morphology, the Diazonium Blue B (DBB) assay, glucose fermentation, carbon (succinate, inulin, glucitol) assimilation, nitrate utilization, and growth at different temperatures (4, 10, 15, 20, 25, 30 and 37 °C), as well as without vitamins (Yarrow, 1998).

Molecular identification of yeasts: DNA extraction

Total genomic DNA extraction was done according to Sampaio et al. (2001) with some modifications. Disruption of the cell wall was achieved by suspending three loopfuls of 48-h cultures (YEPG agar: yeast extract 10 g L−1, peptone 10 g L−1, glucose 20 g L−1, agar 15 g L−1) in 300 μL of sterile water and 200 μL (calculated as equivalent volume) of glass beads (diameter=425–600 μm) were added. After vortexing for 2 min, the tubes were incubated for 1 h at 65 °C, after which samples were vortexed again for 1 min.The suspension was then handled according to the yeast protocol of NucleoSpin® Tissue (Macherey-Nagel GmbH & Co. KG, Düren, Germany).

Molecular identification of yeasts: MSP-PCR fingerprinting

The synthetic oligonucleotides (GTG)5 and M13 (MWG Biotech AG, Ebersberg, Germany) were used as single primers for MSP-PCR fingerprinting (Libkind et al., 2003). All PCR reactions were performed in 25-μL reaction volumes containing 1 × PCR buffer, 2 mm MgCl2, 250 μM of each of the four dNTPs, 0.8 μM of primer and 1 U Taq DNA polymerase (Fermentas Inc., Hanover, MD), according to Meyer et al. (1993). DNA amplification was performed in a T personal Combi Thermal Cycler (Biometra® Gmbh, Goettingen, Germany) using the following PCR program: (1) initial denaturing step at 95 °C for 5 min; (2) 40 cycles of 40 s at 95 °C, 1 min at 50 °C and 1 min at 72 °C; (3) final extension step at 72 °C for 5 min. A negative control was also included in all PCR reactions. Amplification products were separated by electrophoresis in 1.4% w/v agarose gels containing ethidium bromide in 1X TAE (Tris–Acetate–EDTA) buffer at 120 V for 1.5 h. Gels were stained with ethidium bromide. A molecular size marker was used for reference (Mass Ruler DNA ladder, Mix, Fermentas Inc.). Electrophoretic bands were visualized under UV light. DNA banding patterns were analyzed using the image software package. In agreement with Sampaio et al. (2001) and Gadanho & Sampaio (2002), strains exhibiting identical DNA-banding patterns were grouped and considered to belong to the same species.

Molecular identification of yeasts: sequencing of the D1/D2 domain of 26S rRNA gene

Representative strains from each of the groups were subjected to sequencing of the D1/D2 domain of 26S rRNA gene. DNA was first amplified using the primers ITS5 (5′-GGA AGT AAA AGT CGT AAC AAG G) and LR6 (5′-CGC CAG TTC TGC TTA CC) (MWG Biotech AG, Ebersberg, Germany) (Libkind et al., 2003). A 600–650 base pairs region was sequenced by the forward primer NL1 (5′-GCA TAT CAA TAA GCG GAG GAA AAG) and the reverse primer NL4 (5′-GGT CCG TGTTTC AAG ACG G) (MWG Biotech AG) (Libkind et al., 2003). Sequences were obtained with an Applied Biosystems DNA Sequencer, mod. ABI PRISM 377 (Applied Biosystems, Foster City, CA) using standard protocols. Alignments were made using Vector NTI Suite 8 Contig Express (Informax, Invitrogen). Strains were identified by comparing the sequences obtained with the GenBank database (blastn freeware from http://www.ncbi.nlm.nih.gov/BLAST). The phylogenic trees of yeast isolates were obtained by neighbour joining of the D1/D2 domain of the 26S rRNA gene (clustalx freeware from http://bips.u-strasbg.fr/fr/Documentation/ClustalX/) (Hall, 2004).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Physical and chemical analyses

As expected, the chemical parameters for the two glaciers were similar (Tables 1 and 2), in keeping with their geographical proximity and shared geological and geomorphologic characteristics. In addition, no significant (P<0.01) differences were observed between the samples of different years. The pH of meltwater and ice (after melting) was similar to that of the sediments. Dry mass of supraglacial sediments was significantly (P<0.01) lower than that of subglacial sediments (Tables 1 and 2).

Table 1.   Physical and chemical properties of supra- and subglacial sediment collected in Alpine glaciers
 Supraglacial sedimentsSubglacial sediments
No. of samplesMean ± SDNo. of samplesMean ± SD
  1. All samples were carried out in triplicate.

  2. DM, dry mass; TOC, total organic carbon; TON, total organic nitrogen; TOP, total organic phosphorus.

Forni glacier
 DM (%)866.8 ± 4.5983.6 ± 4.5
 pH84.8 ± 0.495.0 ± 0.4
 TOC (g kg−1 DM)80.11 ± 0.0890.33 ± 0.28
 TON (g kg−1 DM)80.09 ± 0.0790.15 ± 0.13
 TOP (mg kg−1 DM)80.16 ± 0.0990.25 ± 0.22
Sforzellina glacier
 DM (%)740.3 ± 9.81181.2 ± 5.7
 pH75.1 ± 0.9115.1 ± 0.6
 TOC (g kg−1 DM)70.34 ± 0.18110.52 ± 0.37
 TON (g kg−1 DM)70.12 ± 0.10110.21 ± 0.20
 TOP (mg kg−1 DM)70.21 ± 0.08110.33 ± 0.28
Table 2.   Physical and chemical properties of meltwater and melted ice collected in Alpine glaciers
 IceMeltwaters
No. of samplesMean ± SDNo. of samplesMean ± SD
  1. All samples were carried out in triplicate.

  2. TOC, total organic carbon; TON, total organic nitrogen; TOP, total organic phosphorus.

Forni glacier
 pH75.8 ± 0.285.6 ± 0.2
 TOC (mg L−1)710.7 ± 0.888.8 ± 0.9
 TON (mg L−1)70.77 ± 0.2980.59 ± 0.06
 TOP (mg L−1)70.13 ± 0.0280.13 ± 0.03
Sforzellina glacier
 pH95.8 ± 0.196.3 ± 0.2
 TOC (mg L−1)97.2 ± 0.998.9 ± 1.9
 TON (mg L−1)90.35 ± 0.1390.60 ± 0.51
 TOP (mg L−1)90.25 ± 0.0990.32 ± 0.13

Total organic carbon (TOC) and total organic nitrogen (TON) of sediments were from 10- to a few 100-fold greater than that observed in ice and meltwater. Conversely, no significant (P<0.01) differences were observed in the total organic phosphorus (TOP) of sediments, ice or meltwater (Tables 1 and 2).

Sediment and water temperature (recorded in situ) ranged from 1 to 3 °C, whereas ice temperature was from 0 to –2 °C. No significant (P<0.01) differences among samples of different years were observed.

Microbial enumeration

Like the chemical parameters, the microbiological data for the two glaciers were similar (Tables 3 and 4). In addition, no significant (P<0.01) differences were observed among samples of different years. The use of a variety of different incubation media for viable counts of yeasts and filamentous fungi (RB+tetracycline, DG18, and DRB+chloramphenicol) gave no significant (P<0.01) differences in the viable counts. Culturable yeasts in subglacial sediments ranged from 102 to 103 CFU g−1 DM. However, the number of yeast cells in supraglacial sediments was 10–100-fold lower (Table 3). Meltwater and ice contained a number of culturable yeasts from 0.17 to 1.05 and from 0.09 to 0.56 CFU mL−1, respectively, with no significant (P<0.01) differences between samples incubated at 4 or 20 °C (Table 4). The concomitant occurrence of both bacteria and filamentous fungi was observed.

Table 3.   Viable counts of yeasts in supra- and subglacial sediment collected in Alpine glaciers
 Supraglacial sedimentsSubglacial sediments
No. of samplesMean ± SD (CFU g−1 DM)No. of samplesMean ± SD (CFU g−1 DM)
  1. All samples were carried out in triplicate.

  2. DM, dry mass.

  3. Different superscript letters indicate significant (P<0.01) differences between average values.

Forni glacier: enumeration at 4°C821 ± 11a9(9.6 ± 7.1) × 103c
Sforzellina glacier: enumeration at 4°C715 ± 7a11(1.3 ± 0.9) × 103c
Forni glacier: enumeration at 20°C821 ± 10a9(2.5 ± 1.4) × 102b
Sforzellina glacier: enumeration at 20°C712 ± 5a11(2.0 ± 1.9) × 102b
Table 4.   Viable counts of yeasts in meltwater and melted ice collected in Alpine glaciers
 Ice (CFU mL−1)Meltwaters (CFU mL−1)
No. of samplesMean ± SD (CFU mL−1)No. of samplesMean ± SD (CFU mL−1)
  1. All samples were carried out in triplicate.

  2. Different superscript letters indicate significant (P<0.01) differences between average values.

Forni glacier: enumeration at 4°C70.11 ± 0.09a81.05 ± 0.44b
Sforzellina glacier: enumeration at 4°C90.56 ± 0.41a, b91.00 ± 0.68b
Forni glacier: enumeration at 20°C70.09 ± 0.08a80.49 ± 0.38a, b
Sforzellina glacier: enumeration at 20°C90.11 ± 0.05a90.17 ± 0.06a

Extracellular enzymatic activity of yeast isolates

All 73 tested strains secreted at least one extracellular hydrolytic enzyme active at 4 °C. No chitinolytic or cellulolytic yeasts were observed. Over 38% simultaneously exhibited three or more extracellular enzymatic activities, whereas more than 80% exhibited at least two activities. More than 86% of the strains tested had extracellular lipolytic activity. In particular most strains belonging to the genera Cryptococcus and Rhodotorula were able to hydrolyze at least one of the two lipophilic substrates tested (Tween 80 and tributyrin) (Table 5). In addition, the ability to degrade starch and proteinaceous compounds was expressed by over 80% and 50% of strains, respectively, in particular those belonging to the genera Mrakia and Rhodotorula. Extracellular pectinolytic activity, however, was expressed by a smaller percentage of strains (Table 5).

Table 5.   Extracellular enzymatic activity of yeast isolates from glacial environments of Alpine glaciers
Genus/speciesNo. of tested strains% of positive strains
SDALEA (Tw80)LEA (Tb)PrAPcA
  1. All determinations were carried out in triplicate.

  2. SDA, starch-degrading activity; LEA (Tw80), lipolytic/esterasic activity observed on Tween 80; LEA (Tb), lipolytic/esterolytic activity observed on tributyrin; PrA, proteolytic activity; PcA, pectinolytic activity.

Cryptococcus gilvescens3886.810092.121.013.1
Mrakia gelida1310076.976.910038.5
Rhodotorula laryngis4010010000
Rhodotorula glacialis1070.010080.090.00
Rhodotorula psychrophenolica510010060.010060.0
Unidentified yeasts333.333.310010033.3
Total tested yeasts7380.893.186.352.019.2

Assessment of growth ability of yeast isolates in subglacial sediments under laboratory-simulated in situ conditions

Growth of the strains DBVPG 4793 and DBVPG 4800 in the sediment samples at 2 °C began after an appreciable lag phase (about 1 week). Growth of about three logarithmic cycles was then observed after 6 weeks, followed by a stationary phase (Fig. 2). No microbial growth was detected in uninoculated (control) sediments.

image

Figure 2.  Time course of cell growth of yeast isolates in subglacial sediments under laboratory-simulated in situ conditions. Cryptococcus gilvescens DBVPG 4793 (▪); Cryptococcus gilvescens DBVPG 4793 (▴); uninoculated sediments (control) (▵ and □). All viable counts were carried out in triplicate. Error bars indicate the SEM.

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Yeast isolation and identification

A total of 106 yeast strains were isolated. About 55% of isolates originated from subglacial sediments, reflecting the higher viable yeast counts in these habitats. Strain characterization by MSP-PCR fingerprinting showed 29 distinct profiles, 14 of which were shared by at least two strains which formed groups of identical DNA banding patterns. Sequences obtained in this study were deposited in the GenBank database (National Center for Biotechnology Information). In agreement with the subdivision of basidiomycetous yeasts in Hymenomycetes, Uredinomycetes and Ustilaginomycetes (Fell et al., 2000), the phylogenic placement of yeasts isolated in the present study (obtained by neighbour joining of the D1/D2 domain of the 26S rRNA gene), are reported in Figs 3, 4 and 5, respectively.

image

Figure 3.  Phylogenic placement of some yeast isolates within Hymenomycetous yeasts obtained by neighbour joining of the D1/D2 domain of the 26S rRNA gene. Numbers on branches represent bootstrap percentages from 100 full heuristic bootstrap replications. Species with GenBank numbers (in parentheses) represent sequences obtained from GenBank database (National Center for Biotechnology Information). Species labelled as DBVPG represent strains sequenced in this study. T, type strains.

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image

Figure 4.  Phylogenic placement of some yeast isolates within Uredinomycetous yeasts obtained by neighbour joining of the D1/D2 domain of the 26S rRNA gene. Numbers on branches represent bootstrap percentages from 100 full heuristic bootstrap replications. Species with numbers (in parentheses) represent sequences obtained from GenBank database (National Center for Biotechnology Information). Species labelled as DBVPG represent strains sequenced in this study. T, type strains.

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image

Figure 5.  Phylogenic placement of some yeast isolates within Ustilaginomycetous yeasts obtained by neighbour joining of the D1/D2 domain of the 26S rRNA gene. Numbers on branches represent bootstrap percentages from 100 full heuristic bootstrap replications. Species with GenBank numbers (in parentheses) represent sequences obtained from GenBank database (National Center for Biotechnology Information). Species labelled as DBVPG represent strains sequenced in this study. T, type strains.

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The 26S rRNA gene D1/D2 region sequence analysis of representative strains from 27 out of 29 MSP-PCR profiles allowed them to be assigned to the following species: Aureobasidium pullulans, Cryptococcus gilvescens, Cryptococcus terricolus, Rhodotorula creatinivora, Mrakia gelida, Naganishia globosa, Rhodotorula bacarum, Rhodotorula laryngis, Rhodotorula glacialis and Rhodotorula psychrophenolica. The most frequently isolated one was Cr. gilvescens (51.2% of total strains), and R. glacialis and M. gelida accounted for 15.1% and 13.2% of total strains, respectively (Fig. 6). Sixteen strains were identified as belonging to the species R. glacialis, and five to R. psychrophenolica. In addition, both 26S rRNA gene D1/D2 sequences of the strains DBVPG 4736 and DBVPG 4804 exhibited 100% similarity with the sequence AF181540, deposited in the GenBank database with the formal name of Cryptococcus saitoi. However, as this species has been recently described to be a synonym of N. globosa (Fell et al., 2000), both strains have been identified as belonging to this species.

image

Figure 6.  Frequency of isolation of the yeast species found in the present study. (a) Aureobasidium pullulans; (b) Rhodotorula creatinivora; (c) Rhodotorula bacarum; (d) Naganishia globosa; (e) Cryptococcus terricolus; (f) unidentified yeast species; (g) Rhodotorula laryngis; (h) Rhodotorula psychrophenolica; (i) Mrakia gelida; (j) Rhodotorula glacialis; (k) Cryptococcus gilvescens.

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The 26S rRNA gene D1/D2 sequences of four yeast strains, grouped within two distinct MSP-PCR patterns, exhibited 100% similarity with a sequence (deposited as AY040645) obtained by so far unidentified strains labeled as ‘Antarctic yeasts’. They differed substantially (2%, corresponding to seven substitutions) from the closest known species Leucosporidium antarcticum (strain CBS 5942, sequence number AF189906) (Fig. 4), and their formal taxonomic designation is still in progress.

Fifty-six strains identified as Cr. gilvescens exhibited an identical MSP-PCR fingerprinting pattern, whereas a few species (M gelida, R. laryngis, R. glacialis, and R. psychrophenolica) were characterized by heterogeneous MSP-PCR profiles. On the other hand, profiles including a few strains (Cr. terricolus and N. globosa) or even single strains (A. pullulans and R. bacarum) were also observed.

The origin of isolates belonging to the different species is reported in Table 6. On the whole, subglacial sediments and, to a lesser extent, meltwater exhibited a greater yeast diversity than supraglacial sediments and ice. Strains of Cr. gilvescens were isolated from samples of supra- and subglacial sediments, ice and meltwater, whereas strains belonging to the species M. gelida and R. glacialis were isolated from subglacial sediments and meltwater. In contrast, a few species were detected only sporadically: strains of A. pullulans and R. bacarum were associated only with meltwater and supraglacial sediments, respectively (Table 6).

Table 6.   Growth temperatures of yeast species isolated from glacial habitats of Alpine glaciers
SpeciesGrowth at
4°C10°C15°C20°C25°C30°C37°C
Aureobasidium pullulans++++++
Cryptococcus gilvescens+++++
Cryptococcus terricolus++++++
Mrakia gelida+++
Naganishiaglobosa+++++
Rhodotorula bacarum++++
Rhodotorula creatinivora+++++
Rhodotorula glacialis++++
Rhodotorula laryngis++++++
Rhodotorula psychrophenolica++++
Unidentified yeast+++

All yeast isolates were able to grow at 4, 10 and 15 °C, but none at 37 °C (Table 7). For all strains belonging to the species A. pullulans, Cr. terricolus and R. laryngis, 30 °C represented the upper limit of growth, whereas all isolates of Cr. gilvescens (with the sole exception of the strain DBVPG 4722), N. globosa, and R. creatinivora grew at 25 °C but not at 30 °C. The upper growth limit for all strains of R. bacarum, R. glacialis, and R. psychrophenolica was 20 °C, whereas all strains of M. gelida, as well as those belonging to the unidentified species labeled as ‘Antarctic yeasts’, grew at 15 °C, but not at 20 °C (Table 7).

Table 7.   Occurrence of yeast isolates in supra- and subglacial sediments, ice and meltwater collected in Alpine glaciers
SpeciesForni glacierSforzellina glacier
SedimentsIceMeltwatersSedimentsIceMeltwaters
SupraglacialSubglacialSupraglacialSubglacial
Aureobasidium pullulans   +    
Cryptococcus gilvescens++++++++
Cryptococcus terricolus +      
Mrakia gelida + + + +
Naganishia globosa    + + 
Rhodotorula creatinivora +      
Rhodotorula bacarum+       
Rhodotorula laryngis  +  ++ 
Rhodotorula glacialis + + + +
Rhodotorula psychrophenolica +     +
Unidentified yeasts     +  

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Although bacterial populations sharing subglacial habitats have been widely observed (Sharp et al., 1999; Skidmore et al., 2000, 2005; Foght et al., 2004; Bhatia et al., 2006), only a few authors (Margesin et al., 2002; Gostinčar et al., 2006; Butinar et al., 2007) have recently reported the occurrence of viable yeast and yeast-like populations in both high Arctic and Alpine glaciers. Viable counts carried out in subglacial sediments gave concentrations of yeast cells several orders of magnitude higher than those observed in supraglacial sediments, ice and meltwater. This evidence is apparently consistent with the hypothesis that the unfrozen subglacial environment of Alpine glaciers could harbour actively growing yeast populations, in close analogy with previous studies reporting that bacterial populations can grow in such habitats (Sharp et al., 1999; Skidmore et al., 2000; Foght et al., 2004). The findings that all strains belonging to the species Cr. gilvescens (which represented over 50% of isolates) exhibited an identical MSP-PCR fingerprinting pattern, as well as their ability to grow under laboratory-simulated in situ conditions at 2 °C, support the hypothesis that subglacial sediments could harbour yeast growth, and even allow their in situ selective enrichment. This conclusion is in agreement with Gostinčar et al. (2006) and Butinar et al. (2007), who described the evolution of yeast and yeast-like populations in basal Arctic ice. Both sets of authors postulated that only some of the clones entering in the glacier might be selectively enriched in subglacial habitats and therefore could form distinct and genetically homogeneous yeast populations.

Physical and chemical analysis of subglacial sediments collected beneath the Forni and Sforzellina glaciers revealed the presence of organic carbon, nitrogen and phosphorous that might be utilized for microbial activity at the glacier bed, as previously supposed by Sharp et al. (1999) and Foght et al. (2004). Accordingly, the heterotrophic metabolism of yeasts and their observed ability to degrade organic macromolecules through the secretion of extracellular hydrolytic cold-adapted enzymes may suggest their potential auxiliary role as biogeochemical nutrient recyclers in subglacial environments. Over 80% of isolated strains were facultative psychrophiles. This finding is in agreement with Foght et al. (2004), who found that bacteria sharing subglacial habitats are mostly facultative psychrophiles. Vincent (1988) also found this feature common to microbial populations occurring in cold environments.

As far as the ecological significance of the presence of yeasts in subglacial microbial communities is concerned, some questions still remain, particularly in consideration of the fact that most microbial species cannot be cultured under laboratory conditions. As a consequence, the results of this study might represent at best a partial picture of the yeast biodiversity occurring in glacial environments.

The yeast diversity from Forni and Sforzellina glaciers has been compared with that previously observed in other glacial environments. Butinar et al. (2007) found Cr. gilvescens only sporadically in high Arctic glaciers, where, in contrast, Cryptococcus liquefaciens (over 90% of isolates) and Rhodotorula mucilaginosa represented the dominant species. Among the other species isolated in the present study, only A. pullulans and Cr. saitoi (synonym of N. globosa) have been previously isolated from high Arctic glaciers (Gostinčar et al., 2006; Turk et al., 2006; Butinar et al., 2007).

Only three of the species observed in the present study have previously been found in Alpine habitats: R. creatinivora, R. glacialis, and R. psychrophenolica (Margesin et al., 2002, 2007; Bergauer et al., 2005), whereas R. creatinivora and R. laryngis have been previously isolated from meltwater running off from glaciers of the Andes (de García et al., 2007). However, six of the 10 species identified in this study have been observed previously in polar habitats. In particular, M. gelida and R. laryngis have previously been isolated from Antarctic habitats (Deegenaars & Watson, 1998; Pavlova et al., 2001). The isolation of M. gelida from Alpine glacier habitats is not surprising and agrees with the well documented occurrence of the closely related obligate psychrophilic species Mrakia frigida in Alpine, Artic and Antarctic habitats (Fell & Stalzell-Tallman, 1998; Margesin et al., 2005). Analogously, the unidentified yeast species found in this study exhibited 100% similarity with the 26S rRNA gene D1/D2 sequence of a yeast strain originating from Antarctica.

Finally, the species Cr. terricolus and R. bacarum have previously been isolated from Alpine or Norwegian soils (Pedersen, 1958; Bergauer et al., 2005), as well as from air, plants and the phylloplane (Fell & Statzell-Tallman, 1998; Fonseca & Inácio, 2006). This is the first study reporting their isolation from glacial environments.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors thank Dr E. Ferrari for her skilful technical assistance. This work was supported by a grant from MIUR (PRIN Project, 2006).

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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