Introduction
The Antarctic continent presents one of the harshest environments on this planet. It is cold and dry areas present a climate that it is not uniform, and, in that way, several main climatic zones can be recognized (Continental High Plateau, Continental Low Plateau, Continental High Latitude Coast, Continental Low Latitude Coast, Antarctic Peninsula, Antarctic Islands, Sub-Antarctic Islands). The existing environmental conditions in Antarctica - low temperature, water availability and precipitation; continuous freeze-thaw cycles; high UV incidence and intense winds - comprise a complex set of environmental conditions that make life difficult for animals as well as plants (Bokhorst et al. Reference Bokhorst, Huiskes, Convey and Aerts2007). Life in Antarctica is dominated by microbes, as they show an adequate level of adaptation to the environment that helps them to prevail in the extreme conditions (Ruisi et al. Reference Ruisi, Barreca, Selbmann, Zucconi and Onofri2007).
Microorganisms that colonize these environments are able to develop even at 0°C and can be classified as strict psychrophiles if their optimum growth temperature is 15°C or below, showing a maximal temperature for growth at about 20°C, or as psychrotolerants (psychrotrophics), which have the ability to grow at low temperatures, having optimal and maximal growth temperatures above 15°C and 20°C, respectively (Fernàndez et al. Reference Fernández, Martorell, Blaser, Ruberto, de Figueroa and Mac Cormack2017). They play a key role in the nutrient cycle and organic material mineralization in cold environments such as the Arctic and Antarctica. These capabilities are mainly related to the production of several extracellular enzymes known as cold-adapted or cold-active enzymes. As explained by Gerday et al. (Reference Gerday, Aittaleb, Bentahir, Chessa, Claverie, Collins, D’Amico, Dumont, Garsoux and Georlette2000), these enzymes present a higher catalytic efficiency than those showing optimal temperatures between 25°C and 50°C (mesophiles) at temperatures lower than 20°C. Hence, these enzymes are a promising tool for industrial processes that demand elevated enzymatic activity at low temperature. Examples of these enzymes are: cellulase, amylase, inulinase, protease, isomerase and lipase, which have been used in the food, soap and biofuel industries (Margesin & Feller Reference Margesin and Feller2010).
For these reasons, the search and study of cold-adapted microorganisms has increased considerably in the last decade. Several Antarctic biotopes were investigated in the search of cold-adapted fungi (Ruisi et al. Reference Ruisi, Barreca, Selbmann, Zucconi and Onofri2007). The major fungal phyla, Ascomycota, Basidiomycota, Zygomycota, Chytridiomycota and Glomeromycota, are well represented in the Antarctic continent (Godinho et al. Reference Godinho, Furbino, Santiago, Pellizzari, Yokoya, Pupo, Cantrell, Rosa and Rosa2013).
Based on this background, the objective of the present study was the isolation, identification and study of the exoenzyme production of psychrophilic/psychotropic fungi obtained from an Antarctic area located on King George Island. The study was focused on certain enzymatic activities in a collection of 51 isolates.
Materials and methods
Soil sampling and fungal isolation
Soil samples were collected during the 2013–2014 summer (December 2013–March 2014) in a radius of 5 km from the Argentinean Scientific Research Station, Carlini, on Potter Cove, King George Island (62°14′18″S, 58°40′00″W) (Fig. 1).
The sampling area on King George Island, South Shetland Islands, Potter Cove (62°14′18″S, 58°40′00″W) with sites marked. Sampling sites: 1. Nesting penguins in Barton Peninsula (Antarctic Specially Protected Area (ASPA) 171), 2. Carlini Station facilities, 3. Tres Hermanos Hill, 4. Elephant Refuge (ASPA 132), 5. Stranger Point (ASPA 132).
Samples were collected from several locations around the cove, including an ornithogenic site near the beach, a human refuge, two human-impacted areas (outside the station kitchen and near the fuel storage tanks), and a large and pristine vegetated area (Tres Hermanos Hill and nearby coastal soils) covered by Deschampsia antarctica Desv., lichens and mosses. Samples (c. 10 g) were aseptically taken from soil at 0–10 cm depth. After collection, the samples were stored in sealed sterile bags or sterile flasks and transported to the station, where they were kept at 4°C until processed for incubation and isolation.
Isolation was performed on solid media, using a diluted yeast morphology medium (DYM) (composition in g l-1: yeast extract 0.03, malt extract 0.03, peptone 0.05, dextrose 0.1, agar 15; pH adjusted to 4.5 by addition of HCl 1 N (to allow the growth of fungi instead of bacteria)). The isolation protocol followed two parallel procedures. First, a portion of each sample was excised under aseptic conditions, using a sterile spoon or spatula, and directly spread onto Petri plates containing DYM. Simultaneously, another portion of the same sample was re-suspended in a minimal volume of saline solution supplemented with 1% Tween 20 and then homogenized in a vortex mixer for 15 min. After that, 100 μl of the resulting homogenate was spread onto Petri plates with DYM.
The plates were incubated at 15°C for 7–21 days under natural lighting conditions. Actively growing fungi colonies were taken from the plates and subcultured onto fresh potato dextrose agar (PDA) plates as individual isolates.
All fungal isolates were maintained on PDA medium at 4°C in the Microbiological Resources Center Culture Collection (MIRCEN) of the PROIMI-CONICET Institute and in the Culture Collection in the Argentinean Antarctic Institute (IAA).
Molecular identification of selected isolates
Genomic DNA extraction was performed using a commercial kit (FastDNA™ Spin Kit, MP Biomedicals). The divergent domain at the 5’ end of the LSU rDNA gene (around 600 base pairs) was symmetrically amplified with primers NL-1 (5’-GCATATCAATAAGCGGAGGAAA AG) and NL-4 (5’-GGTCCGTGTTTCAAGACGG) according to standard methods, as described by Kurtzman et al. (Reference Kurtzman, Fell and Boekhout2011). This set of primers was chosen after trying to amplify the ITS region using ITS1 and ITS4 primers, which resulted in the production of several bands in some of the isolates (data not shown), as a consequence of unexpectedly high single nucleotide polymorphisms, a phenomenon previously reported by Simon & Weiß (Reference Simon and Weiss2008). The PCR products were purified and sequenced in MACROGEN (Korea). Sequences were analysed and edited, when necessary, using DNA Dragon software (Hepperle Reference Hepperle2011). DNA sequences were submitted to GenBank under accession numbers listed in Table I. Strain identifications were performed by comparison with the GenBank and UNITE databases. An identity criterion of ≥99% was employed to identify strains at the species level. Taxonomy was checked against Kurtzman et al. (Reference Kurtzman, Fell and Boekhout2011). Sequences showing 97–98% identity were tentatively identified to the genus level. Sequences showing less than 97% identity were considered unidentified.
Molecular identification of the isolated fungi.
* Presumptive identification corresponds to the database identification with higher percentage of identity and coverage (data not shown).
Screening of lytic enzymes production
Seven hydrolytic activities were tested in triplicate on fungal isolates growing on solid media at 15°C: amylase, cellulase, lipase, esterase, protease, laccase and xylanase. In all cases exoenzymatic activity was quantified as the diameter in mm of the halo (either colouration or decolouration) around the colony, from the edge of the colony to the edge of the halo.
For amylase, cellulase and xylanase activity, fungal isolates were grown in a YMD medium (1:10) with starch (2 g l-1), carboxymethylcellulose (5 g l-1), and xylane (5 g l-1), respectively, instead of glucose. To measure the activity after incubation, for amylase and xylanase, the plates were flooded with 1 ml of iodine solution, and positive activity was defined as a clear halo around the colony on a purple background (Carrasco et al. Reference Carrasco, Rozas, Barahona, Alcaíno, Cifuentes and Baeza2012). For cellulase, the plates were flooded with Congo red solution (1 mg ml-1), which was poured off after 15 min. The plates were then flooded with 1 M NaCl for 15 min. Positive cellulase activity was defined as a clear halo around the colony on a red background (Carrasco et al. Reference Carrasco, Rozas, Barahona, Alcaíno, Cifuentes and Baeza2012).
For protease and lipase activity, fungal isolates were grown in YMD medium (1:10) supplemented with skimmed milk (10 g l-1) and olive oil (4% v/v), and rhodamine B 0.01%, respectively. Positive protease activity was defined as a clear halo around the colony on a white opaque background (Rovati et al. Reference Rovati, Delgado, Figueroa and Fariña2010) whereas positive lipase activity was indicated as fluorescent halos around the colonies under UV exposure (Gopinath et al. Reference Gopinath, Hilda and Anbu2005).
For esterase activity, fungal isolates were grown in a medium composed of: bacto peptone, (10 g l-1); NaCl, (5 g l-1); CaCl2•2H2O, (4 g l-1) and Tween 80, (10 g l-1). Esterase activity was evidenced as a white precipitate around the colony (Carrasco et al. Reference Carrasco, Rozas, Barahona, Alcaíno, Cifuentes and Baeza2012). Finally, for laccase activity, the fungal isolates were grown in medium composed of: glucose, (2 g l-1); bacto peptone (0.1 g l-1); yeast extract, (0.01 g l-1) and ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) (1 g l-1). Laccase activity was indicated as green halos around the colonies after incubation (Maza et al. Reference Maza, Pajot, Amoroso and Yasem2014).
Growth temperature range
The effect of temperature on the growth of the isolates was investigated on PDA plates. Fungi (pre-grown on PDA plates) were used to inoculate two replicates per strain at each temperature (given below) on 24 multi-well plates filled with 1 ml of PDA agar. Plates were incubated at 5°C, 15°C, 25°C and 35°C. Growth was monitored every seven days up to 21 days, to avoid missing any slow-growing fungi at a specific temperature. Growth was expressed as the fungal colony diameter in mm (to assess optimal growth temperature, colony diameter (at each temperature) was recorded after 7 days of incubation). To classify fungi as psychrophilic or psychrotrophic, the presence or absence of growth after 21 days was considered. For microorganisms able to grow at low temperature, two classifications were used: psychrophilic (any fungi not able to grow at or above 25°C) and psychrotrophic (any fungi able to grow at or above 25°C).
Results
A total of 31 samples from pristine and anthropized sites were used. It is worth mentioning that some samples were taken from soils impacted by oil spills over recent years.
After 7–21 days of incubation, 51 fungal morphotypes were recovered as pure cultures and deposited at the MIRCEN and IAA culture collections (Table I). When analysing the origins of the samples (Table II), it was noticed that most isolates came from substrates associated with high organic matter content: 34% of the isolates (n = 18) were found in samples associated with lichen, moss and Deschampsia antarctica; 28% (n = 15) came from samples such as soil or mud with penguin faeces, feathers, wood from shipwrecks, decaying seals’ bodies; 28% (n = 15) came from soil or mud obtained near the station’s kitchen exit or near the fuel tanks, and were consequently considered as anthropogenic-impacted. Finally, only 8% (n = 4) came from soils related to neither the station nor areas with high organic matter content.
Enzyme activity of the fungal isolates.
* The diameter of the fungi colony at each temperature at 7 days of incubation was measured in mm and related to the optimal growth temperature. **The classification was made based on the growth at 21 days of incubation. ***Exoenzymatic activities were quantified as the radius (in mm) of the halo (of either colouration or decolouration) around the colony as explained in material and methods.
The 51 isolates were ascribed to 12 different genera. Nine genera belonged to the phylum Ascomycota (Cadophora, Helotiales, Monographella, Oidodendron, Penicillium, Phialocephala, Phialophora, Phoma and Pseudogymnoascus), one genus to the phylum Basidiomycota (Irpex) and other two genera to the phylum Mucoromycota (Mortierella and Mucor). Nine isolates could not be identified to genus level (Table I).
Only 25 isolates were identified to species level, and they belonged to five species Pseudogymnoascus pannorum (Link) Minnis & D.L. Lindner, Cadophora malorum (Kidd & Beaumont) W. Gams, Monographella lycopodina Jaklitsch, Siepe & Voglmayr, Mucor zonatus Milko and Mucor hiemalis Wehmer. The other isolates were only identified to genus level. More studies and other genes sequences are needed to reach the species level in those cases.
Pseudogymnoascus pannorum was the fungus most frequently found in this study, with 19 isolates. Monographella lycopodina (n = 2), Mucor zonatus (n = 1) and Mucor hiemalis (n = 1) came from the same sample, a mix of Deschampsia antarctica and lichen. Two isolates of Cadophora malorum came from impacted areas (around the kitchen exit and fuel tanks).
Results from the enzymatic screening are presented in Table II. Xylanase activity was the most abundant enzymatic activity, produced by 90% (n = 46) of the isolates. Cellulase activity was evidenced by 80% (n = 41) of the isolates whereas 78% of the isolates (n = 40) were proteolytic. Amylase was produced by 76% of the isolates (n = 39), Lipase by 71% (n = 36) and esterase, another lipolytic enzyme by only 53% (n = 27). Finally, laccase, a lignocellulolytic enzyme typical of basidiomycete fungi, was detected only in 37% of the isolates (n = 19).
Temperature response showed 23.5% (n = 12) were classified as psychrophiles and 76.5% (n = 39) were classified as psychrotrophic. Among the last group, only five isolates were able to grow at 35°C. The optimum temperature for growth was 15°C for 65% (n = 33) of the isolates and 25°C for 31% (n = 16). Only one isolate (Mortierella sp. CAV1-348) grew better at 5°C and isolates Mucor zonatus CAV1-23 and Mucor hiemalis CAV1-237 presented the same growth in the range 5–25°C.
Discussion
King George Island is the biggest of the South Shetland Islands, north-west of the Antarctic Peninsula, has a mild macroclimate that is strongly buffered by the surrounding ocean (Krishnan et al. Reference Krishnan, Alias, Wong, Pang and Convey2011). Its climate is typical of the peri-Antarctic islands: wet, windy and cold. It has an average temperature of 1–3°C in summer and -7°C in winter (Kostadinova et al. Reference Kostadinova, Krumova, Tosi, Pashova and Angelova2009). Soil temperature in winter ranges between -5°C and -9°C buffered from lower air temperatures by the snow layer (Krishnan et al. Reference Krishnan, Alias, Wong, Pang and Convey2011). In summer, maximal soil temperatures range from 14–26°C (Martínez-Álvarez et al. Reference Martínez-Álvarez, Ruberto, Balbo and Mac Cormack2017). While these habitats clearly experience chronically low mean temperatures, various other aspects of temperature stress, including long-term (seasonal or annual) and short-term (daily or weekly) variation, also present challenges to soil microbiota.
Results presented here helped to identify fungi not previously reported on the continent, proving the value of culture-based studies to determine the ecology of filamentous fungi on the island. The presence of several research stations in this environmentally sensitive area represents a source of high environmental impact. The effect of the human impact areas as well as those with natural high organic content (nesting birds, soil covered with Deschampsia antarctica, lichen or moss) on the mycodiversity was reflected by the fact that 93% of the fungi (37% from human-impacted areas and 56% from naturally high organic soils) were isolated from these areas. This observation is in accordance with Ding et al. (Reference Ding, Li, Che, Li, Gu and Zhu2016), who reported that cultivable fungal distribution and abundance patterns in Antarctic soils is positively correlated with soil nutrients, content and moisture. As a rule, differences in the composition of Antarctic soils lead to distinctive and characteristic microbial distributions.
In this work, the genus Pseudogymnoascus (formerly Geomyces) was the most frequently isolated and abundant taxon, with 19 members of Pseudogymnoascus pannorum isolated. The genus Pseudogymnoascus is one of the most reported fungal genera in Antarctica (Gonçalves et al. Reference Gonçalves, Vaz, Rosa and Rosa2012, Ding et al. Reference Ding, Li, Che, Li, Gu and Zhu2016) comprising psychrotrophic/psychrophilic fungi common in cold environments, and ubiquitous to soils not only from Antarctica, but also Arctic and Alpine soils (Hayes et al. Reference Hayes2012). In Antarctica, this genus has been isolated from different habitats (Ding et al. Reference Ding, Li, Che, Li, Gu and Zhu2016). Pseudogymnoascus species were reported by Arenz et al. (Reference Arenz, Held, Jurgens, Farrell and Blanchette2006) to have the ability to colonize and utilize several carbon sources, and therefore play and important role in the decomposition and nutrient cycle in Antarctica. From our 19 isolates of Pseudogymnoascus pannorum, six could produce all the enzymes tested in this work. Fungi with a broad enzymatic competence possess high eco-nutritional versatility: the ability to survive in a vegetative state, and to overcome environmental changes that might normally be harmful, increasing their survival chances under unfavourable environmental conditions (Cooke & Rayner Reference Cooke and Rayner1984).
Like Pseudogymnoascus, members of the genus Penicillium have frequently been isolated from Antarctic soils (Onofri et al. Reference Onofri, Selbmann, De Hoog, Grube, Barreca, Ruisi and Zucconi2007). This genus is ubiquitous, with a worldwide distribution, but the frequency of occurrence and range of the individual species may be more limited (McRae et al. Reference McRae, Hocking and Seppelt1999). Moreover, it is hard to determine if particular species are actually endemic to Antarctica due to their extensive distribution across the planet and their cold tolerance. Penicillia are easily dispersed and several Antarctic isolates could certainly represent the result of involuntary human contamination of natural Antarctic environments being transported with human foods, clothes, vehicles and other man-made materials (Comerio & Mac Cormack Reference Comerio and Mac Cormack2004). In this sense, for the Penicillium genus (with the ascomycetous state Eupenicillium), only one novel species has been described from cold regions. However, although isolated from Antarctica, this species, Penicillium antarcticum, is not particularly psychrotolerant (McRae et al. Reference McRae, Hocking and Seppelt1999). So, no penicillia have been reported as geographically confined to the Arctic or Antarctica, and even Penicillium antarcticum occurs worldwide. Despite this, Gunde-Cimerman et al. (Reference Gunde-Cimerman, Sonjak, Zalar, Frisvad, Diderichsen and Plemenitaš2003) indicate that there may be several novel cold-tolerant species of Penicillium in Antarctic and Arctic areas. Based on the growth temperature experiments (Table II), one isolate was psychrophilic (Penicillium sp. CAV1-21) and the other three (Penicillium sp. CAV1-11, CAV1-142 and CAV1-331) were psychrotrophic.
The class Leutuomycetes is represented by Phialocephala a common plant-root endophyte that is widespread in sub-Antarctic ecosystems and is also present in continental Antarctica (Newsham et al. Reference Newsham, Upson and Read2009). In this work, five isolates ascribed to these genera, all from soil samples, exhibited most of the evaluated enzymatic activities. Two of the isolates (Phialocephala sp. CAV1-225 and CAV1-230) were able to grow, although poorly, at 35°C.
Amongst the Mucorales, Mortierellaceae and Mucoraceae are the most frequently recorded families in Antarctica (Ruisi et al. Reference Ruisi, Barreca, Selbmann, Zucconi and Onofri2007). Genera belonging to these families are known to produce many mitotic spores that have resistance-reproductive structures that enhance survival opportunities (Onofri et al. Reference Onofri, Selbmann, Zucconi and Pagano2004). They exhibit worldwide distribution and are regarded as psychrotolerant organisms (Onofri et al. Reference Onofri, Selbmann, Zucconi and Pagano2004). In this work, members of two genera (Mortierella and Mucor) belonging to these families were isolated. Mortierella has frequently been obtained from different habitats from Antarctica (Ding et al. Reference Ding, Li, Che, Li, Gu and Zhu2016). In the present study, members of the genus were isolated from moss and soil with an elevated organic content (Mortierella sp. CAV1-145 and CAV1-348). These isolates were classified as psychrophilic and showed two of the seven evaluated enzyme activities, cellulase and lipase. The other Mucoromycota species isolated were Mucor hiemalis CAV1-237 and Mucor zonatus CAV1-23, which both came from a sample containing moss and from a Deschampsia antarctica specimen. These isolates showed psychrotrophic behaviour and grew particularly well at 35°C. Fungi belonging to this genus were previously isolated in Antarctica (Gesheva & Negoita Reference Gesheva and Negoita2012). Nevertheless, this work is the first report of Mucor zonatus in the Antarctic continent.
The size of halos and colonies were used to compare enzymatic activities and colony growth under different conditions. Several investigators have described the sensitivity of this method for semi-quantitative determinations and it has been used successfully for identification of novel activities from environmental isolates of microorganisms (Li et al. Reference Li, Chi, Wang, Peng and Chi2009). Two isolates, which presented six of the seven evaluated enzymes, and characterized as psycrothrophic, were identified as Cadophora malorum (CAV1-226 and CAV1-232). Four other isolates (CAV1-332, CAV1-179, CAV1-229 and CAV1-337) were identified to genus level as Phialophora sp. (synonym for Cadophora sp.). These isolates were also classified as psychrotrophic. Fungi belonging to genus Cadophora were previously reported on a range of different substrates (Arenz et al. Reference Arenz, Held, Jurgens, Farrell and Blanchette2006). The presence of previously reported species of Cadophora in Antarctica and the prevalence of these fungi at many locations in the continent suggests that they are indigenous (Blanchette et al. Reference Blanchette, Held, Jurgens, McNew, Harrington, Duncan and Farrell2004).
Other genera reported in Antarctica were also identified in this work, as Phoma sp. CAV1-327, isolated from soil, and Oidiodendron sp. CAV1-302, isolated from lichens and mosses. Both isolates were classified as psychrotrophic. These genera were previously isolated from different substrates such as wood from shipwrecks or structures (Held & Blanchette Reference Held and Blanchette2017), soil (Kochkina et al. Reference Kochkina, Ozerskaya, Ivanushkina, Chigineva, Vasilenko, Spirina and Gilichinskii2014) moss (Hirose et al. Reference Hirose, Hobara, Matsuoka, Kato, Tanabe, Uchida and Osono2016) and macroalgae (Godinho et al. Reference Godinho, Furbino, Santiago, Pellizzari, Yokoya, Pupo, Cantrell, Rosa and Rosa2013). In the case of isolate Irpex sp. CAV1-342, this genus was previously identified in Antarctic soil from the volcano Mount Erebus (Connell & Staudigel Reference Connell and Staudigel2013).
Two of the isolates (CAV1-14 and CAV1-20) identified as Monographella lycopodina, represent the first time this genus has been isolated in Antarctica. Monographella taxa include important plant pathogens, particularly of grasses and cereals. In cold to temperate regions Monographella nivalis, also known as ’pink snow mould’, is an economically important disease of wheat and barley (Jewell & Hsiang Reference Jewell and Hsiang2013). In this work, they were isolated from cellulolytic substrates as Deschampsia antarctica and lichen, and, as expected, presented two enzymatic activities related to mycorrhiza, laccase and cellulase.
Most of the isolates were successfully classified at either the species or genus level based on the LSU sequences of standard isolates deposited in the GenBank and the UNITE database according to the BLAST results, except for the isolates CAV1-55, CAV1-184, CAV1-204, CAV1-212, CAV1-227, CAV1-313, CAV1-335, CAV1-351 and CAV1-113. Also, isolate CAV1-319 showed 99% identity with the Helotiales sp. I12F-02289 sequence isolated from bryophytes as culturable endophytic fungi in Antarctica (Zhang et al. Reference Zhang, Zhang, Liu, Wei, Li, Su and Yu2013) and was further identified at order level as Helotiales sp. These fungi might represent novel species.
The results obtained from enzyme production suggest that several of these fungi could be used for industrial purposes, for example in large-scale microbial fermentation for the production of cold-active enzymes. In recent years, the use of lipases and proteases in the soup and dairy industries has become a reality. Other lytic enzymes as amylase, pectinase and cellulase have also proven to be helpful in making juice products more palatable and of better appearance (Pandey et al. Reference Pandey, Nigam, Soccol, Soccol, Singh and Mohan2000). These are only a few examples of the usefulness of some of the enzymes evaluated in this work.
The results presented here suggest that most of the fungal isolates from maritime Antarctica were psychrotrophs, in accordance with those reported in related studies (Ruisi et al. Reference Ruisi, Barreca, Selbmann, Zucconi and Onofri2007). The predominance of psychrotrophic fungi in surface soils from cold environments has been previously noted and it is attributable to seasonal and diurnal increases in soil temperature due to solar radiation that favours the dominance of the eurythermal cold-adapted microorganisms over the stenothermal ones represented by the strict psychrophiles (Robinson Reference Robinson2001). This ability also suggests an interesting way to select cold-adapted microorganisms for biotechnological purposes, because psychrotrophic microorganisms can grow at low temperatures but do not have the limited growth at moderate temperatures that the strict psychrophiles have.
Conclusion
This study investigated the taxonomic diversity and enzyme production in solid media of 51 cultivable fungi isolated from samples taken on King George Island. In agreement with previous research, some characteristic fungal taxa found in this region belong to cold-adapted and cosmopolitan taxa. Furthermore, some other taxa were found that have not been previously observed in Antarctica (Monographella lycopodina and Mucor zonatus) as well as several not fully identified fungal isolates, which will be more thoroughly investigated. The isolation of mainly psychrothrophic fungi having at least one cold-active enzymatic activity raises their potential for use in biotechnological/industrial processes at low and moderate temperatures.
Acknowledgments
This work was supported by Instituto Antártico Argentino/Dirección Nacional del Antártico (IAA/DNA), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, Project PICTO 2010-0124), Universidad de Buenos Aires (UBA, Project 20020130100569BA) and Universidad Nacional de Tucumán (UNT). The authors would like to thank the reviewers for their time spent on reviewing this manuscript and their helpful comments towards improving the article.
Authors contribution statement
All authors conceived and planned the experiments. Martorell and Fernández carried out the experiments. Ruberto, Figueroa and Mac Cormack. contributed to the interpretation of the results. Martorell took the lead in writing the manuscript. All authors provided critical feedback and helped shape the research, analysis, and manuscript.
Conflicts of interest
The authors declare no conflicts of interest.
Details of data deposit
DNA sequences were submitted to GenBank under Accession Numbers listed in Table I.
