Polar Biol (2011) 34:1535–1542 DOI 10.1007/s00300-011-1012-3
ORIGINAL PAPER
Extracellular hydrolase enzyme production by soil fungi from King George Island, Antarctica Abiramy Krishnan • Siti Aisyah Alias Clemente Michael Vui Ling Wong • Ka-Lai Pang • Peter Convey
•
Received: 27 January 2011 / Revised: 28 March 2011 / Accepted: 30 March 2011 / Published online: 17 April 2011 Ó Springer-Verlag 2011
Abstract Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. In order to examine extracellular enzyme production in this chronically low-temperature environment, fungi were isolated from ornithogenic, pristine and human-impacted soils collected from the Fildes Peninsula, King George Island, Antarctica during the austral summer in February 2007. Twenty-eight isolates of psychrophilic and psychrotolerant fungi were obtained and screened at a culture temperature of 4°C for activity of extracellular hydrolase enzymes (amylase, cellulase, protease), using R2A agar plates supplemented with (a) starch for amylase activity, (b) carboxymethyl cellulose and trypan blue for cellulase activity or (c) skim milk for protease activity. Sixteen isolates showed activity for amylase, 23 for cellulase and 21 for protease. One isolate showed significant activity across all three enzyme types, and a further 10
A. Krishnan S. A. Alias (&) Institute of Biological Science, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia e-mail:
[email protected] C. M. V. L. Wong Biotechnology Research Institute, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia K.-L. Pang Institute of Marine Biology, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 20224, Taiwan, ROC P. Convey British Antarctic Survey, High Cross, Madingley Road, Cambridge CB3 0ET, UK
isolates showed significant activity for at least two of the enzymes. No clear associations were apparent between the fungal taxa isolated and the type of source soil, or in the balance of production of different extracellular enzymes between the different soil habitats sampled. Investment in extracellular enzyme production is clearly an important element of the survival strategy of these fungi in maritime Antarctic soils. Keywords Warcup soil plating method Extracellular enzymes Psychrophilic Psychrotolerant Screening
Introduction King George Island is the largest island within the South Shetland Islands archipelago, north-west of the Antarctic Peninsula in the maritime Antarctic. As is typical of this region of Antarctica, the island hosts a variety of terrestrial ecosystems and soil habitats, ranging through largely pristine soils and vegetation, vertebrate-influenced habitats such as penguin rookeries and seal haul-out and resting areas, to sites under varying levels of human impact (Smith 1984a; Walton 1984; Tin et al. 2009). Although microbial diversity is generally poorly documented across Antarctica, to date a total of 197 species of fungi have been reported from the Antarctic Peninsula region, with 76 species reported from King George Island alone (http://www. antarctica.ac.uk/bas_research/data/access/fungi; see also Bridge et al. 2008). Studies on Signy Island in the South Orkney Islands (Davey et al. 1992; Arnold et al. 2003; Bokhorst et al. 2007), which experiences a similar maritime and predominantly cloudy climate to that of the South Shetland
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Islands, indicate that soil temperature minima in winter are commonly buffered by overlying snow and remain above about -5 to -9°C even though short-term air temperature minima may be much lower. Similarly, short-term soil temperature maxima in the range 14–26°C are typically experienced. However, for the majority of the biologically active period during the austral summer, these soil habitats experience low positive mean temperatures. Analogous features, in terms of both scale of variability and longerterm mean temperatures (Peck et al. 2006), are reported in studies documenting microenvironmental characteristics in soils at locations further south in the maritime Antarctic (Bokhorst et al. 2007; Engelen et al. 2008), in the continental Antarctic (McGaughran et al. 2009) and in vegetation (Smith 1984b). Cold-inhabiting microbes can be categorised, based on their growth characteristics at different temperatures, as psychrophilic or psychrotolerant (e.g. Morita 1975; Margesin et al. 2007). Psychrophilic taxa show optimum growth at 15°C or lower and cannot grow above 20°C, while psychrotolerant taxa typically have a higher optimum temperature and a maximum temperature for growth greater than 20°C, while retaining the ability to grow at low temperatures. Psychrophilic and psychrotolerant fungi contribute significant biomass in the soil microbial community and are important in the decomposition cycle in cold ecosystems (Margesin et al. 2007; Xin and Zhou 2007). With their ability to grow at low temperature, psychrophilic and psychrotolerant fungi are also often encountered as food spoiling agents in frozen and chilled food, causing important economic impacts (Margesin and Schinner 1994). Microbes are well known for the production of extracellular enzymes and other secondary metabolites (Singh et al. 2006). A range of protease, amylase, cellulase, pectinase, xylanase, chitinase and keratinase enzymes have been reported from the Antarctic region (Ray et al. 1992; Fenice et al. 1997; Raza et al. 2000; Duncan et al. 2008), with the majority of metabolites isolated from bacteria (Morita et al. 1997; Vincent 2000; Cavicchioli et al. 2002; Nichols et al. 2002). A smaller number of studies have been conducted on other microbial groups including actinomycetes (Gushterova et al. 2005; Gesheva 2009), cyanobacteria (Broady et al. 1987), fungi (Ray et al. 1992; Duncan et al. 2008) and microalgae (Gesheva 2009). Extracellular hydrolysing enzymes play an important role in the degradation of insoluble macromolecules such as cellulose and starch, with the degradation products subsequently being absorbed by the cell (Gibb and Strohl 1987; Oh et al. 2000) or being made available to other organisms in the soil microbial foodweb. Studies of extracellular hydrolysing enzymes of Antarctic soil fungi remain scarce, and virtually none have been undertaken in the maritime Antarctic region. The current study was designed to confirm and quantify the
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production and activity of several hydrolase enzymes under thermal culture conditions comparable to the chronically low temperatures of the maritime Antarctic soil environment. Using a range of psychrophilic and psychrotolerant fungal isolates obtained from soils of the Fildes Peninsula, King George Island, we screened for low-temperature production and activity of extracellular amylase, cellulase and protease enzymes.
Materials and methods Soil sampling and fungal isolation Soil samples were collected during the 2006/7 austral summer (February 2007) near the Chilean scientific research station, Prof. Julio Escudero, which is located on the Fildes Peninsula, King George Island (62°120 5700 S, 058°570 3500 W) (Fig. 1). The samples were collected from a range of locations, including an ornithogenic site close to nesting birds (62°110 51.600 S, 058°590 28.800 W), two human-impacted areas (62°120 5700 S, 058°570 3500 W and 062°120 14.900 S, 058°580 00.800 W) and two largely pristine and naturally vegetated areas (062°120 12.2300 S, 058°580 27.6300 W and 062°100 03.200 S, 058°510 16.200 W) (Fig. 1). Five subsamples were taken from each location, each consisting of 10 g of soil obtained from 0 to 10 cm depth using a sterile spatula, and stored in sealed sterile whirl packs or Falcon tubes. After collection, samples were rapidly returned to the research station, where they were refrigerated at 4°C, and subsequently transported at this temperature to the National Antarctic Research Center, Kuala Lumpur, Malaysia, taking 2 weeks in transit, where they were immediately frozen (-20°C). The samples were then stored frozen until processed in the laboratory. The soil plating method of Warcup (1950) was used to culture fungi from the soil samples. This involves dispensing approximately 0.1 g of soil per sample onto 6 individual petri dishes, with Potato Dextrose Agar (PDA) supplemented with chloramphenicol (0.2 g/l) then being poured into the dishes. PDA was chosen used as it is known to be an effective substrate for the isolation of fungi (Azmi and Seppelt 1998). Three plates were then incubated at each of either 4 or 25°C for 10–15 days. No specific illumination regime was followed, but the cultures were exposed to ambient room lighting in the refrigeration unit. Active growing mycelia were then taken from the plates and subcultured onto fresh PDA plates as individual isolates. In order to permit a pragmatic differentiation of thermal growth characteristics, isolates growing at 4°C only were classified as psychrophilic, those at both temperatures as psychrotolerant and those at only 25°C as mesophilic. Fungal strains were initially identified using colony
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broth in a 4°C orbital shaker for 10 days. The fungi were freeze-dried prior to DNA extraction at the National Taiwan Ocean University. Genomic DNA was extracted using the DNeasy Plant DNA Extraction Kit (Qiagen) according to the manufacturer’s instructions. The intergenic spacer regions of the nuclear rRNA genes were amplified using primer pairs ITS4/ITS5 (White et al. 1990). PCR reactions were performed in a 50 lL volume containing ca. 20 ng DNA, 0.2 lM of each primer, 0.2 mM of each dNTP, 2.5 mM MgCl2 and 1.25 U of Taq Polymerase (Invitrogen). The amplification cycle consisted of an initial denaturation step of 95°C for 2 min followed by 35 cycles of (a) denaturation (95°C for 1 min), (b) annealing (54°C for 1 min) and (c) elongation (72°C for 1.5 min) and a final 10-min elongation step at 72°C. The PCR products were analysed by agarose gel electrophoresis and sent to Tri-I Biotech. Inc., Taiwan for sequencing. The sequences obtained were checked for ambiguity, assembled and submitted to the National Center for Biotechnology Information (NCBI) for a nucleotide BLAST search. While this generates a number of ‘nearest neighbour’ sequence identities from those present in the database, we recognise that this provides only a coarse suggestion of isolate identity, even where named species are linked, not least as robust taxonomic knowledge of Antarctic fungi (as with all microbial groups) and their relationship with non-Antarctic taxa is currently unavailable. Enzyme activity
Fig. 1 a The location of King George Island in the South Shetland Islands; box indicates Fildes Peninsula. b Sampling locations on the Fildes Peninsula, King George Island
morphology and spore characteristics. Fungal isolates are deposited in the fungal culture collection of the Institute of Biological Science, University of Malaya, located in the National Antarctic Research Center, Kuala Lumpur. Molecular identification of selected fungi The six fungal isolates found to be the most effective enzyme producers (see below) were then grown in PDA
This study focused on identifying production and activity of extracellular enzymes at low temperatures, as are typical of the chronically cold soil habitats of the maritime Antarctic. Extracellular hydrolysing enzyme activity was therefore screened as described by Margesin et al. (2003), using cultures of psychrophilic and psychrotolerant fungal strains grown at 4°C. The presence of amylase, cellulase and protease activity was tested on R2A agar (casein acid hydrolysate 0.5 g/l, yeast extract 0.5 g/l, proteose peptone 0.5 g/l, dextrose 0.5 g/l, soluble starch 0.5 g/l, dipotassium phosphate 0.3 g/l, magnesium sulphate 0.024 g/l, sodium pyruvate 0.3 g/l, agar 15 g/l) supplemented with starch (0.4% w/v), carboxymethylcellulose and trypan blue (0.4% and 0.01% w/v), or skim milk powder (0.4% w/v), respectively. All test fungi were grown on PDA plates for 1 week after inoculation. Agar plugs (6 mm) were cut from the growing edge of the fungal colonies on the PDA plates using cork borer number 3 and inoculated into a small 6-mm well made at the centre of each assay agar plate, which were then incubated at 4°C. The assay agar plates were prepared in triplicate. After 10 days, the plates were examined for the presence of a clear zone in the agar
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around the colony, indicating extracellular enzyme activity. Amylase and protease activities were confirmed by staining the plates with Lugol’s solution and Coomassie brilliant blue solution, respectively. Relative enzyme activity (RA) was calculated using the following formula: Relative enzyme activity Clear zone diameter colony diameter ¼ clear zone diameter Following Bradner et al. (1999) and Duncan et al. (2008) strains exhibiting an RA of [1.0 were classified as having ‘significant activity’.
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exhibited significant activity (as defined by Bradner et al. (1999) and used by Duncan et al. (2008)) for amylase, as did seven for cellulase and 12 for protease. Twelve isolates showed no activity for amylase, five for cellulase and seven for protease. Yeast sp. 24 was the only isolate showing no activity in any of the assays. G. pannorum (strain AK07KGI2001 R2-1) was the only isolate with significant activity across all three enzymes (Table 2). In total, 25% of the fungal isolates examined showed significant activity for amylase, 25% for cellulase and 43% for protease.
Discussion Results Identification of fungi Of a total of 41 fungal taxa isolated based on morphological assessment (8 mitosporic fungi, 24 ascomycetes, 4 yeasts, a zygomycete, a basidiomycete, 2 unidentified species and a sterile mycelium), 28 strains classified as psychrophilic or psychrotolerant (Table 1) were screened for enzyme activity. Of these, 11 strains were psychrotolerant and 17 were psychrophilic. Six isolates showing the most significant activity were subject to molecular identification (Table 1). The closest sequence matches for the ITS regions of the rRNA gene of Geomyces sp. 1 (AK07KGI1001R1-2), Deuteromycete sp. 25 (AK07KGI102R203), Geomyces sp. 2 (AK07KGI102R1-4), Geomyces sp. 2 (AK07KGI1001R2-1) and Geomyces sp. 1 (AK07KGI301R3-3) include a number of Geomyces spp. and their subsequent closest sequence matches with full species identity were the same, with 98–99% similarity to Geomyces pannorum (DQ189229, DQ189228, DQ189224). For Mrakia sp. (AK07KGI103 R2-1), the closest sequence matches were many unidentified species and Mrakia spp., while the closest sequence matches with full species identity were Mrakia nivalis (AF144484) and M. frigida (AF144482), both with 98% similarity. Sequences obtained have been deposited in Genbank, accession numbers JF720026-31. Enzyme screening Relative enzyme activities across the three enzyme types and fungal isolates examined are illustrated in Fig. 2. The activities of isolates for each enzyme type are listed in Table 2. Although in some cases numbers of isolates are low, and there was limited overlap between individual isolates from different sampling sites, there was no obvious association between the presence of any type of strain or enzyme and the different collection sites. Seven strains
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King George Island lies towards the north of the maritime Antarctic region, experiencing a relatively mild macroclimate that is strongly buffered by the surrounding ocean. Mean monthly air temperatures vary between winter minima of about -13°C and maxima of ?4°C (Rakusa-Suszczewski 2002), at the upper end of the range typical of the maritime Antarctic (cf. Walton 1984). Although mean air temperatures are well known to provide a poor reflection of microhabitat temperatures in vegetation or soil ecosystems; there appear to be no accessible published datasets documenting these characteristics from locations in the South Shetland Islands. Soil temperatures in the range 5–10°C recorded during collection of the samples described here (data not shown) are not unusual for northern maritime Antarctic soils during the austral summer (cf. Davey et al. 1992; Arnold et al. 2003). While these habitats clearly experience chronically low temperatures, various other aspects of temperature stress, including large and shortterm variation, also present stress challenges to the soil microbiota (see Peck et al. 2006 for discussion). Therefore, while our study focused on the psychrophilic and psychrotolerant fungal strains obtained, it is unsurprising that mesophilic fungi were also noted to be present. Within the identification limitations imposed by both morphological and molecular analyses, many of the fungal taxa obtained in the current study (Table 1) have been reported in other studies of Antarctic fungi. However, detailed characterisation of these strains in either taxonomic/molecular or ecophysiological/biochemical contexts are unavailable. Some representatives of several of these strains (Table 2) have previously been described as psychrophilic or psyschrotolerant, including Mortierella sp., Antarctomyces sp.1, Mrakia frigida, Geomyces pannorum, Thelebolus sp. and Penicillium sp.1 (Zucconi et al. 1996; Wicklow and Malloch 1971; Robinson 2001; Tosi et al. 2002; de Hoog et al. 2005; Vishniac 2006; Brunati et al. 2009; Gesheva 2009, 2010). Few enzyme studies have been carried out on fungi isolated from the maritime Antarctic. Fungal production of
Polar Biol (2011) 34:1535–1542 Table 1 Fungal strains used in the present study, based on morphological examination, along with their identification based on molecular techniques
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Taxa
Strain number
GenBank accession number
Geomyces pannorum
AK07KGI102 R1-4(5)
JF720028
Geomyces pannorum
AK07KGI102 R2-3(1)
JF720027
Geomyces pannorum
AK07KGI301 R3-3(2)
JF720031
Geomyces pannorum
AK07KGI1001 R1-2(1)
JF720026
Geomyces pannorum
AK07KGI1001 R1-1(5)
Geomyces pannorum
AK07KGI2001 R2-1(1)
Geomyces pannorum
AK07KGI2002 R2-2(1)
Geomyces pannorum
AK07KGI102 R3-3
Geomyces pannorum
AK07KGI105 R3-2(1)
Geomyces pannorum Mortierella sp.
AK07KGI105 R3-1(1) AK07KGI105 R3-1
Penicillium sp. 1
AK07KGI2002 R3-1(2)
Trichocladium sp. 6
AK07KGI2002 R2-2(2)
Psychrotolerant
JF720030
Psychrophilic Aureobasidium sp.
AK07KGI101 R3-1(2)
Mrakia frigida
AK07KGI103 R2-1(2)
Yeast sp. 16
AK07KGI102 R1-4(2)
Yeast sp. 24
AK07KGI2001 R2-1(2)
Antarctomyces sp. 1
AK07KGI102 R1-3(2)
Antarctomyces sp. 1
AK07KGI1001 R1-1(1)
Antarctomyces sp. 2
AK07KGI102 R2-3(5)
Antarctomyces sp. 4
AK07KGI102 R3-2(5)
Antarctomyces sp. 6
AK07KGI301 R2-2
Antarctomyces sp. 8
AK07KGI1001 R1-1(4)
Ascomycete sp. 1 Thelebolus sp.
AK07KGI1001 R1-1(2) AK07KGI1001 R3-2(1)
Mitosporic fungi sp. 18
AK07KGI102 R2-1(2)
Ascomycete sp. 17
AK07KGI301 R1-3(6)
Sterile mycelium sp. 2
AK07KGI1001 R1-2(2)
amylase has been reported by Fenice et al. (1997) from Victoria Land in the continental Antarctic. Fungal production of cellulase has been reported from soils on Ross Island (Duncan et al. 2008), Victoria Land (Fenice et al. 1997) and from Syowa Station (Yamamoto et al. 1991). Protease production has been reported from Ross Island (Duncan et al. 2008), the Windmill Islands (Bradner et al. 1999), Victoria Land (Fenice et al. 1997) and the Schirmacher Oasis (Ray et al. 1992). However, the levels of enzyme activity displayed by Antarctic microbes have not been a focus of research, and Duncan et al.’s (2008) study is the only one to provide an assessment of this, showing significant cellulase activity in a Geomyces sp. The range of fungal isolates found in the current study to show significant activity levels of this enzyme suggests that this capability is an important element of the survival strategy of fungi in the low-temperature environments of the maritime Antarctic.
JF720029
No sexual stage ascomycetes isolated in the current study showed significant extracellular amylase activity, while asexual forms were active, as also reported by El-Safey and Ammar (2003). For instance, Aspergillus sp. is well known to degrade starch, and amylase activity has previously been detected in cultures of G. pannorum from Victoria Land (Fenice et al. 1997), while here isolates Geomyces sp. 1 and sp. 2 (again most closely aligning with G. pannorum) also showed significant activity. Fenice et al. (1997) detected amylase activity in Thelebolus microsporus. However, in the current study, Thelebolus sp. did not show any activity for amylase, which may indicate either that different strains were examined in the two studies, or that production was influenced by the different incubation temperatures used—Fenice et al. (1997) using 25°C, while the present study used 4°C. Over 80% of the isolated fungi showed extracellular cellulase activity, with M. frigida showing the best activity.
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Fig. 2 Comparison of relative enzyme activity of amylase, cellulase and protease across the different fungal strains isolated
Table 2 Occurrence of fungal strains at the different sampling locations and their relative enzyme activities (RA) for amylase (A), cellulase (C) and protease (P) production Species
Isolate number
Human-impacted lake
Ornithogenic site
Pristine sites
A
C
P
A
A
Mortierella sp.
AK07KGI105 R3-1
0.0
0.0
0.0
Aureobasidium sp.
AK07KGI101 R3-1(2)
1.5
1.0
0.6
Yeast sp. 16
AK07KGI102 R1-4(2)
0.4
1.1
1.1
Antarctomyces sp. 1
AK07KGI102 R1-3(2)
0.0
0.4
0.0
Antarctomyces sp. 1
AK07KGI1001 R1-1(1)
Antarctomyces sp. 2
AK07KGI102 R2-3(5)
0.0
0.4
0.0
Antarctomyces sp. 4
AK07KGI102 R3-2(5)
0.0
0.3
0.0
Antarctomyces sp. 6
AK07KGI301 R2-2
Ascomycete sp. 17 Mitosporic fungi sp. 18
AK07KGI105 R3-2(6) AK07KGI102 R2-1(2)
1.3 0.9
0.8 1.2
1.6 0.2
Mrakia frigida
AK07KGI103 R2-1(2)
0.0
1.7
1.8
Geomyces pannorum
AK07KGI102 R2-3(1)
0.8
0.2
2.5
Geomyces pannorum
AK07KGI102 R1-4(5)
1.8
0.8
2.0
Geomyces pannorum
AK07KGI301 R3-3(2)
Geomyces pannorum
C
P
C
Station site P
0.0
1.1
0.0
0.0
0.3
0.0
AK07KGI1001 R1-1(5)
0.4
1.1
1.5
Geomyces pannorum
AK07KGI1001 R1-2(1)
0.9
1.2
2.2
Geomyces pannorum
AK07KGI2001 R2-1(1)
0.0
0.0
0.0
1.8
0.8
1.0
A
C
P
2.1
1.2
1.5
Geomyces pannorum
AK07KGI2001 R3-1
0.9
0.8
1.3
Geomyces pannorum
AK07KGI2002 R2-2(1)
0.8
0.8
0.9
Geomyces pannorum
AK07KGI102 R3-3
Thelebolus sp.
AK07KGI1001 R3-2(1)
0.0
0.0
0.2
Antarctomyces sp. 8
AK07KGI1001 R1-1(4)
0.0
0.4
0.0
Ascomycete sp. 1
AK07KGI1001 R1-1(2)
0.0
0.0
0.2
Sterile mycelia sp. 2 Trichocladium sp. 6
AK07KGI1001 R1-2(2) AK07KGI2002 R2-2(2)
0.0
0.0
0.3
Penicillium sp. 1
AK07KGI2002 R3-1(2)
Yeast sp. 24
AK07KGI2001 R2-1(2)
Geomyces pannorum
AK07KGI105 R3-2(1)
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1.7
1.4
0.6
0.8
1.2
1.1
0.2
0.3
0.9
0.7
0.4
0.5
0.0
0.0
0.0
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This isolate was obtained from a highly human-impacted area, which may give some indication of its origin (cf. Ishihara et al. 1989; Duncan et al. 2008), as cellulose is a very common substance in waste materials. The majority of sequence database records of this species are from Antarctic locations, although sequences have also been reported from Japan and the Russian Federation. Previous studies have similarly reported extracellular cellulase activity in Geomyces sp. isolated from soil and moss from Victoria Land, continental Antarctica (Fenice et al. 1997) and obtained specifically from wood, artefacts and organic materials from the Cape Evans historic hut (Duncan et al. 2008). A large majority (75%) of fungal isolates showed extracellular protease activity, with G. pannorum showing the greatest activity level. As with amylase activity, almost all sexual stage ascomycetes did not show protease activity, with the exception of Ascomycete sp. 1 and Thelebolus sp. Extracellular protease production has previously been reported from fungi in the Windmill Islands, continental Antarctica (Bradner et al. 1999), and by Humicola marvinii M.E. Palm & Weinst obtained from Antarctic fellfield soil from Signy Island (Weinstein et al. 1997). Protease activity has also been reported from probably psychrotolerant members of the genera Kluyveromyces, Endomycopsis, Cephalosporium, Aureobasidium, Saccharomycopsis, Rhodotorula and Candida (Ray et al. 1992). In the current study, protease activity was detected in Geomyces sp, unlike the data reported by Fenice et al. (1997), again possibly linked with differences in the strains or culture conditions used between the two studies. Overall, most psychrotolerant and psychrophilic fungi isolated from soils on King George Island showed significant detectable extracellular enzyme activity in culture at 4°C, reinforcing the likely importance of investment in this nutrient-obtaining strategy for occupants of this relatively harsh environment. Of the 28 fungal strains examined, 25% showed good activity for amylase, 25% for cellulase and 43% for protease. Activity levels varied across the taxa isolated, with some being particularly strong producers of one or two of the three enzyme classes examined. Only a single taxon (G. pannorum isolated from behind the station) showed high activity across all three classes. Acknowledgments We thank the Malaysian Antarctica Research Program and the University of Malaya for support and the provision of research facilities, the Academy of Sciences Malaysia, Postgraduate Research Fund PS175/2008 and PS253/2009C, and the Instituto Antarctico Chileno for logistics and support of the fieldwork. We thank M. Bo¨lter, A.J.S. Whalley and anonymous reviewers for helpful and constructive comments on earlier manuscript versions. Figure 1a was provided by P. Fretwell of the BAS Mapping and Geographic Information Centre. PC is a member of the BAS core ‘Polar Science for Planet Earth’ programme. This paper also
1541 contributes to the SCAR ‘Evolution and Biodiversity in Antarctica’ research programme.
References Arnold RJ, Convey P, Hughes KA, Wynn-Williams DD (2003) Seasonal periodicity of physical and edaphic factors, and microalgae in Antarctic fellfields. Polar Biol 26:396–403 Azmi OR, Seppelt RD (1998) Fungi of the Windmill Islands, continental Antarctica: effect of temperature, pH and culture media on the growth of selected microfungi. Polar Biol 18:128– 134 Bokhorst S, Huiskes A, Convey P, Aerts R (2007) Climate change effects on decomposition rates in ecosystems from the Maritime Antarctic and Falkland Islands. Global Chang Biol 13:2642– 2653 Bradner JR, Gillings M, Nevalainen KMH (1999) Qualitative assessment of hydrolytic activities in Antarctic microfungi grown at different temperatures on solid media. World J Microb Biot 15:131–132 Bridge PD, Spooner BM, Roberts PJ (2008) Non-lichenized fungi from the Antarctic region. Mycotaxon 106:485–490 Broady P, Given D, Greenfield L, Thompson K (1987) The biota and environment of fumaroles on Mount Melbourne, Northern Victoria Land. Polar Biol 7:97–113 Brunati M, Rojas JL, Sponga F, Ciciliato I, Losi D, Gottlich E (2009) Diversity and pharmaceutical screening of fungi from benthic mats of Antarctic lakes. Mar Genomics 2:43–50 Cavicchioli R, Siddiqui KS, Andrews D, Sowers KR (2002) Lowtemperature extremophiles and their applications. Curr Opin Biotech 13:253–261 Davey MC, Pickup J, Block W (1992) Temperature variation and its biological significance in fellfield habitats on a maritime Antarctic island. Antarct Sci 4:383–388 de Hoog GS, Gottlich E, Platas G, Genniloud O, Leotta G, van Brummelen J (2005) Evolution, taxonomy and ecology of the genus Thelebolus in Antarctica. Stud Mycol 51:33–76 Duncan SM, Minasaki R, Farrell R, Thwaites JM, Held BW, Arenz BE, Jurgens JA, Blanchette RA (2008) Screening fungi isolated from historic Discovery Hut on Ross Island, Antarctica for cellulose degradation. Antarct Sci 20:463–470 El-Safey EM, Ammar MS (2003) Purification and characterization of NH-a-amylase isolated from Aspergillus flavus var. columnaris. In: International conferences of enzymes in the environment, activity, ecology and applications. Praha, Czech Republic, p 127, July 14–17, 2003 Engelen A, Convey P, Hodgson DA, Worland MR, Ott S (2008) Soil properties of an Antarctic inland site: implications for ecosystem development. Polar Biol 31:1453–1460 Fenice M, Selbmann L, Zucconi L, Onofri S (1997) Production of extracellular enzymes by Antarctic fungal strains. Polar Biol 17:275–280 Gesheva V (2009) Distribution of psychrophilic microorganisms in soils of Terra Nova Bay and Edmonson Point, Victoria Land and their biosynthetic capabilities. Polar Biol 32:1287–1291 Gesheva V (2010) Production of antibiotics and enzymes by soil microorganisms from the Windmill Islands region, Wilkes Land, East Antarctica. Polar Biol 33:1351–1357 Gibb GD, Strohl WR (1987) Physiological regulation of protease activity in Streptomyces peucetius. Can J Microbiol 34:187–190 Gushterova A, Vasileva-Tonkova E, Dimova E, Nedkov P, Haertle T (2005) Keratinase production by newly isolated Antarctic actinomycete strains. World J Microbiol Biotechnol 21:831–834
123
1542 Ishihara M, Inagaki S, Shimuzu K (1989) Use of woody biomass for production of cellulase by Trichoderma virde. Mokuzai Gakkaishi 35:572–577 Margesin R, Schinner F (1994) Properties of cold-adapted microorganisms and their potential role in biotechnology. J Biotechnol 33:1–14 Margesin R, Gander S, Zacke G, Gounot AM, Schinner F (2003) Hydrocarbon degradation and enzyme activities of cold-adapted bacteria and yeasts. Extremophiles 7:451–458 Margesin R, Neuner G, Storey KB (2007) Cold-loving microbes, plants, and animals- fundamental and applied aspects. Naturwissenschaften 94:77–99 McGaughran A, Redding GP, Stevens MI, Convey P (2009) Temporal metabolic rate variation in a continental Antarctic springtail. J Insect Physiol 55:129–134 Morita RY (1975) Psychrophilic bacteria. Bacteriolog Rev 30:144–167 Morita Y, Nakamura T, Hasanb Q, Murakamia Y, Yokoyama K, Tamiyaa T (1997) Cold-active enzymes from cold-adapted bacteria. J Am Oil Chem Soc 74:441–443 Nichols DS, Sanderson K, Buia A, Kamp JV, Holloway P, Bowman JP, Smith M, Nichols CM, Nichols PD, McMeekin DA (2002) Bioprospecting and Biotechnology in Antarctica. In: JabourGreen J, Haward M (eds) The Antarctic: past, present and future, Antarctic CRC Research Report 28. Hobart, pp 85–103 Oh YS, Shih IL, Tzeng YM, Wang SL (2000) Protease produced by Pseudomonas aeroginosa K-187 and its application in the deproteinization of shrimp and crab shell wastes. Enzyme Microb Tech 27:3–10 Peck LS, Convey P, Barnes DKA (2006) Environmental constraints on life histories in Antarctic ecosystems: tempos, timings and predictability. Biol Rev 81:75–109 Rakusa-Suszczewski S (2002) King George Island–South Shetland Islands, maritime Antarctic. In: Beyer L, Bo¨lter M (eds) Geoecology of Antarctic ice-free coastal landscapes, ecological studies, vol 154. Springer, Berlin, pp 23–39 Ray MK, Uma Devi K, Seshu Kumar G, Shivaji S (1992) Extracellular protease from the Antarctic yeast Candida humicola. Appl Environ Microb 58:1918–1923 Raza S, Fransson L, Hult K (2000) Enantioselectivity in Candida antarctica lipase B: a molecular dynamics study. Protein Sci 10:329–338 Robinson CH (2001) Cold adaptation in Arctic and Antarctic fungi. New Phytol 151:341–353
123
Polar Biol (2011) 34:1535–1542 Singh SM, Puja G, Bhat DJ (2006) Psychrophilic fungi from Schirmacher Oasis, East Antarctica. Curr Sci India 90:1388–1392 Smith RIL (1984a) Terrestrial biology of the Antarctic and subAntarctic. In: Laws RM (ed) Antarctic ecology. Academic Press, London, pp 61–162 Smith RIL (1984b) Recording bryophyte microclimate in remote and severe environments. In: Glime JM (ed) Methods in bryology. Hattori Botanical Laboratory, Nichinan, pp 275–284 Tin T, Fleming Z, Hughes KA, Ainley D, Convey P, Moreno C, Pfeiffer S, Scott J, Snape I (2009) Impacts of local human activities on the Antarctic environment: a review. Antarctic Sci 21:3–33 Tosi S, Casado B, Gerdol R, Caretta G (2002) Fungi isolated from Antarctic mosses. Polar Biol 25:262–268 Vincent WF (2000) Evolutionary origins of Antarctic microbiota: invasion, selection and endemism. Antarctic Sci 12:374–385 Vishniac HS (2006) Yeast biodiversity in the Antarctic. In: Rosa CA, Garbor P (eds) Biodiversity and ecophysiology of yeasts. Springer, Berlin, pp 419–440 Walton DWH (1984) The terrestrial environment. In: Laws RM (ed) Antarctic ecology. Academic Press, London, pp 1–60 Warcup JH (1950) The soil plate method for isolation of fungi from soil. Nature 166:117–118 Weinstein RN, Palm ME, Johnstone K, Wynn-Williams DD (1997) Ecological and physiological characterisation of Humicola marvinii, a new psychrophilic fungus from fellfield soils in the maritime Antarctic. Mycologia 89:706–711 White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky J, White TJ (eds) PCR protocols: a guide to methods and application. Academic Press, San Diego, pp 315–322 Wicklow DT, Malloch D (1971) Studies in the genus Thelebolus: temperature optima for growth and ascocarp development. Mycologia 63:118–131 Xin MX, Zhou PJ (2007) Mrakia psychrophila sp. nov., a new species isolated from Antarctic soil. J Zhejiang Univ Sci B 8:260–265 Yamamoto H, Ohtani S, Tatsuyama K, Akiyama M (1991) Preliminary report on cellulolytic activity in the Antarctic region (extended Abstract). In: Proceedings of the NIPR symposium on polar biology, vol 4, pp 179–182 Zucconi L, Pagano S, Fenice M, Selbmann L, Tosi S, Onofri S (1996) Growth temperature preferences of fungal strains from Victoria Land, Antarctica. Polar Biol 16:53–61
