Screening Fungi Isolated from Historic Discovery Hut on Ross Island, Antarctica for Cellulose Degradation SHONA M
Total Page：16
File Type：pdf, Size：1020Kb
Antarctic Science 20 (5), 463–470 (2008) & Antarctic Science Ltd 2008 Printed in the UK doi:10.1017/S0954102008001314 Screening fungi isolated from historic Discovery Hut on Ross Island, Antarctica for cellulose degradation SHONA M. DUNCAN1§*, RYUJI MINASAKI1#, ROBERTA L. FARRELL1, JOANNE M. THWAITES1, BENJAMIN W. HELD2, BRETT E. ARENZ2, JOEL A. JURGENS2 and ROBERT A. BLANCHETTE2 1Department of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton 3240, New Zealand 2Department of Plant Pathology, University of Minnesota, St Paul, MN 55108, USA §Present address, Department of Bioproducts & Biosystems Engineering, University of Minnesota, St Paul, MN 55108, USA #Present address: Eckmann Group, Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany *[email protected] Abstract: To survive in Antarctica, early explorers of Antarctica’s Heroic Age erected wooden buildings and brought in large quantities of supplies. The introduction of wood and other organic materials may have provided new nutrient sources for fungi that were indigenous to Antarctica or were brought in with the materials. From 30 samples taken from Discovery Hut, 156 filamentous fungi were isolated on selective media. Of these, 108 were screened for hydrolytic activity on carboxymethyl cellulose, of which 29 demonstrated activities. Endo-1, 4-b-glucanase activity was confirmed in the extracellular supernatant from seven isolates when grown at 48C, and also when they were grown at 158C. Cladosporium oxysporum and Geomyces sp. were shown to grow on a variety of synthetic cellulose substrates and to use cellulose as a nutrient source at temperate and cold temperatures. The research findings from the present study demonstrate that Antarctic filamentous fungi isolated from a variety of substrates (wood, straw, and food stuffs) are capable of cellulose degradation and can grow well at low temperatures. Received 23 September 2007, accepted 3 December 2007 Key words: cellulolytic, endo-1, 4-b-glucanase, microfungi, psychrotolerant Introduction Chromista) and 99.4% are composed of true fungi including In 1902, Discovery Hut was erected at Hut Point, Ross Island, yeasts and filamentous fungi from the phyla Chytidiomycota, Antarctica, by the National Antarctic Expedition led by Zygomycota, Ascomycota and Basidiomycota. Onofri et al. Robert F. Scott. This wooden building was the first structure stated that most Antarctic filamentous fungi are cold tolerant to be built on Ross Island and was to serve as a shelter, a mesophiles or psychrotrophs rather than psychrophiles. workshop and to store supplies. Due to the design of the hut Psychrotrophic fungi (organisms capable of growth at around it was too cold to house the men. When the expedition 08C as well as grow above 208C (Cavicchioli et al. 2002)) members left the continent, the hut and supplies were have been previously isolated from Antarctica and have been abandoned. The hut was also used extensively by four other defined as strains of filamentous mesophilic fungi adapted to expeditions in the Heroic Age, both as a key stepping stone to grow at temperatures of 18C (Kerry 1990a, Abyzoz 1993, the southern latitudes and as shelter for those returning from Azmi & Seppelt 1997). the south. After use by Shackleton’s 1914–1917 Imperial Much of the previous work on fungi found in association Trans-Antarctic expedition, the hut was abandoned until the with the Ross Island historic huts focused on the long-term late 1940s and visited only periodically until 1957 when survival of organisms in the food supplies and horse- restoration work began. In the past 20 years, the hut and the associated materials (Meyer et al. 1962, 1963, Nedwell surrounding area has attracted increasing numbers of tourists et al. 1994). Recently Held et al. (2005) reported on the from cruise ships and scientists from the nearby research presence of large fungal blooms within Scott’s Terra Nova facilities, Scott Base and McMurdo Station. Of the three Hut at Cape Evans. Investigations of biological and non- historic huts, on Ross Island, Discovery Hut has been the biological causes of deterioration in the historic huts of most affected by human impact due both to being close to the Antarctica produced evidence of decay fungi associated research facilities and to the increasing numbers of visitors. with exterior wood in contact with the ground, including There are many reports of fungi isolated from Antarctic soils, several previously undescribed Cadophora species, (based some described as endemic or indigenous to the continent on the recent taxonomic revision of some of the while many were reported to be introduced (Vishniac 1996). Phialophora-like fungi (Harrington & McNew 2003)). Onofri et al. (2004) reported that in Antarctica 0.6% of the Blanchette et al. (2004) suggested, on the basis of their known fungal species are water moulds (kingdom molecular evidence, that some of these fungi are 463 464 SHONA M. DUNCAN et al. indigenous species to Antarctica. Along with providing wood, foodstuffs (straw, meat, molasses, biscuits and oats) a nutrient source for the fungi, the hut creates a and organic materials were aseptically collected using microenvironment with conditions suitable for fungal tweezers and scalpels from inconspicuous locations growth during the summer. Fungi found in Discovery Hut throughout the hut. Swab samples were taken, using a sterile appeared well adapted to cold temperatures, and cotton swab saturated with sterile distilled water from the environmental monitoring within the hut indicated more conspicuous areas. Scrapings were taken from surfaces temperatures ranging from a maximum of 6.68Ctoa with visible fungal growth or an accumulation of organic minimum of -398C (Held et al. 2005). material was seen. All samples were taken under the New Many micro-organisms are known to degrade cellulose, Zealand Ministry of Agriculture and Fishery permit numbers a linear polymer of b-linked glucosyl units. The enzymes 1999006429 and 2000010576. Samples were placed in sterile responsible for hydrolysis of cellulose are extracellular and vials and kept between 0–58C while in Antarctica and during collectively known as cellulases. Endo-1, 4-b-glucanase transit to New Zealand. Samples were then stored under (EC 3.2.1.4), one of the components of the cellulase sterile conditions at 48C until isolations were made. enzyme complex catalyses randomly the hydrolysis of the b 1,4-glucosidic linkage. Cellulose decomposition in the Antarctic region has been Isolation of fungi studied by Smith (1981) and Walton (1985) at South Georgia, Pugh & Allsopp (1982) at Signy Island and Fungi from the samples taken in January 1999 and December Yamamoto et al. (1991) at Syowa station and Langhovde 1999 were isolated on the following media: hut, Antarctica. All concluded that cellulolytic fungi were YM agar (yeast extract 0.2%, malt extract 1.5%, agar present in the Antarctic region and that cellulose 1.8%), decomposition was occurring but at a slower rate when Medium 4 (yeast extract 0.2%, malt extract 1.5%, agar compared with more temperate environments. Antarctic 1.8%, chloramphenicol 0.2 g l-1, streptomycin sulphate fungi have been evaluated for extracellular enzyme activity 0.1 g l-1 ) for isolation of streptomycin resistant fungi including cellulases by Hurst et al. (1983), seven of eight (Harrington 1981), fungi isolated from this study (Acremonium terricola, Botrytis cinerea,2Chaetophoma sp., Chrysosporium Medium 6 (yeast extract 0.2%, malt extract 1.5%, agar pannorum, Cladosporium sphaerospermum,andFusarium 1.8%, chloramphenicol 0.2 g l-1, streptomycin sulphate lateritium) were grown on and were able to cause clearing 0.1 g l-1, cycloheximide 0.4 g l-1 ) for isolation of of cellulose agar plates at 208C. In addition they reported cycloheximide resistant fungi (Harrington 1981), cellulase activity at 18CforB. cinerea, C. pannorum, Medium 7 (yeast extract 0.2%, malt extract 1.5%, agar Chaetophoma,andC. sphaerospermum. Fenice et al. (1997) 1.8%, chloramphenicol 0.2 g l-1, benlate 0.06 g l-1, reported rather low cellulase activity in 12 of 33 fungal streptomycin sulphate 0.1 g l-1, lactic acid 2 ml) for strains tested and Bradner et al. (1999) reported testing for selection of Basidiomycetes, and cellulase activity along with other hemicellulolytic activity Vogel Bonner minimal medium (-25% glucose, 2.0% agar, but did not present results for cellulolytic activity. 20 ml VB concentrate/litre (50% K HPO (anhydrous), This investigation focused on fungi isolated from cellulose 2 4 17.5% NaNH PO . 4H O, 10% citric acid . H O, 1% containing samples (wood, straw and food stuffs) from 4 4 2 2 MgSO . 7H O in 670 mls distilled water) for the selection Discovery Hut. It was proposed that isolated fungi would 4 2 of slow growing fungi (Vogel & Bonner 1956). be able to grow at temperatures typical of the summer months and that they would be able to degrade cellulose. For fungal isolations wood samples were surface sterilized by The isolated fungi were screened for the ability to degrade soaking for one minute in a 5% hypochlorite solution, carboxymethyl cellulose, production of the related enzyme followed by two rinses in sterile, distilled water, sliced and endo-1, 4-b-glucanase (EC 3.2.1.4), and their ability to use cultured on a variety of enriched and semi-selective media a variety of synthetic celluloses as a nutrient source to prepared as agar plates. Organic material samples were cut determine their ability to degrade cellulose. with a sterile scalpel and placed onto culture media; swab samples were wiped over the surface of the media; wood scraping samples were aseptically placed onto the media. Materials and methods The agar plates were then incubated at 48C, 158Cor258C for up to six weeks. Organisms growing on the agar plates Sample collection were transferred by subculturing from hyphal tips, colonies In January 1999, December 1999 and December 2000 samples or spores to fresh YM agar plates.