Department of Biological Sciences, The University of Waikato, Hamilton 3240, NEW ZEALAND.
The goal of this study was to undertake a microbial investigation of the Historic Huts areas on Ross Island, to gain knowledge of the fungal biodiversity and biochemical framework, focusing on the wood degrading potential of these fungi at both psychrophilic (cold) and mesophilic (moderate) temperatures. Eight hundred and forty nine samples were collected from three Heroic Era Historic Huts of Antarctica, from a variety of substrates but predominantly structural wood. The huts, Discovery Hut at Hut Point, Terra Nova Hut at Cape Evans and Nimrod Hut at Cape Royds, are located on Ross Island and were all assembled in the early 20th century by the Heroic Era explorers to house the expeditions, stores and animals. These wooden huts were abandoned when the expeditions left. The introduction of wood and other organic material to a pristine environment along with the creation of a microclimate within the harsh Antarctica environment created interesting sites for studying fungal diversity, wood decay and fungal cellulase enzymes in an extreme environment. Each hut can be classified as offering different conditions and circumstances for fungal propagules. Of the three huts, Terra Nova Hut is the only hut where there are visible fungal blooms within the hut and it, with Discovery Hut, had the greatest number of samples that contained fungi compared to Nimrod Hut which had the least. Discovery Hut, at less than 500 metres from the United States McMurdo Station, is the most visited by scientist and base staff and has been the most demonstrably affected by human impact of the three huts due to its closeness to the research stations on Ross Island.
To ensure a full understanding of the fungal diversity of the Historic Hut sites, a variety of sampling techniques were used along with a variety of culture media. Two thousand and seventy six isolates consisting of 1177 filamentous fungi and 899 single celled microorganisms (yeast and bacteria) were isolated; all these cultures were frozen and now form the University of Waikato Antarctic Culture Collection. Five genera dominated the fungal isolates that were identified and these were Cladosporium, Geomyces, Cadophora, Penicillium and Thelebolus. The fungal diversity of these Historic Huts’ communities is low but the members present are metabolically active, consistent with other microbial communities in the Antarctic. The Historic Huts and surroundings contain a diverse array of provision in the way of wood and supplies, which provide nutrient sources for fungal growth. Endemic organisms present in the soil could have been enriched by using the introduced nutrient sources as primary and/or second metabolic substrates. In addition, fungi could have been introduced with the wooden huts and supplies when they were brought to Antarctica by the Heroic Era explorers, or introduced in the subsequent years with visitors and conservation work conducted at the sites. These introduced organisms, though, would have had to adapt to the change in climate and conditions posed by the Antarctic in order to survive and be subsequently isolated in this study.
A screen for carboxymethylcellulase (CMCase) activity was done on a selection of the fungal isolates as the first step to understand the cellulytic potential of the Antarctica fungal community inhabiting the huts. One hundred and six fungal isolates from a total of 404, that were screened were deemed to be CMCase positive, 27 fungal isolates were chosen for further study including quantifying the activity of extracellular endo-1,4-β-glucanase at psychrophilic and mesophilic incubation temperatures. All but one isolate could produce endo-1,4-β-glucanase activity at 4°C and many produced more endo-1,4-β-glucanase activity at 4°C than at 15°C.
Cadophora malorum 182, Cadophora malorum 242, Penicillium roquefortii 405, Penicillium roquefortii 408, Geomyces sp. 711, Geomyces sp. 824 and Cladosporium oxysporium 805 were selected for in-depth study of growth characteristics including growth temperature preferences, growth on a variety of cellulose substrates, water activity, and carbon sources, the latter done by using a commercially available microtitre plate containing 95 carbon sources. All seven of the fungal isolates were classified as psychrotolerant and produced, when cultured at either 4°C or at 15°C, cellulase, protease, amylase, xylanase, and pectinase and mannanase enzyme activities. The range of water activity that the Antarctic Penicillium roquefortii isolates could grow at was distinctive when compared with food Penicillium roquefortii isolates. The utilisation of different carbon sources showed that like many studies of Antarctica organism they have a diverse range of enzymatic activity, but interestingly the activity does not differ greatly with incubation temperature with most carbon sources being used or not used at both incubation temperatures tested. Although it took longer for the fungi to grow at the psychrophilic temperatures, the range of carbon sources they utilised was not reduced.
The protein composition of the extracellular supernatants was visualised using various electrophoretic and staining techniques. The cellulase activity of the protein bands was visualised by cellulose-containing zymograms, which illustrated that the cellulase complex in all fungi tested was multi-enzyme and differed between species, isolates and temperatures of culturing. The cellulase activity of Cadophora malorum 182 was enriched by purification techniques including ion exchange chromatography and native preparative electrophoresis. The protein complex was not purified to homogeneity, but enriched for a mixture of proteins and the mixture was described as having the following properties; a temperature range of β-1,4-glucan cellobiohydrolase activity from 20°C to 80°C with the optimum activity seen at 60°C, β-1,4-glucan cellobiohydrolase activity that is stable at 4, 25 and 40°C for at least 24 hrs, lost at 50 °C and 80°C within 24 hrs and 2 minutes respectively. Along with β-1,4-glucan cellobiohydrolase activity, the protein mixture contained Avicelase, CMCase, xylanase and mannanase activity.
The thesis research showed that there was limited fungal diversity in the Historic Huts and artefacts (a total of five dominant genera were identified) but the fungi are actively growing and producing viable spores in the cold of Antarctica and producing the necessary enzymes for degradation of wood. Although the metabolism and growth rate is slower at psychrophilic temperatures, the fungal isolates studied as part of this thesis research could still function enzymatically at cold temperatures and this includes the degradation of wood as evidenced by in vitro wood decay studies examined by scanning electron microscopy where two isolates of one species demonstrated the ability to degrade wood. The cellulase complex of the investigated fungal isolate was multi-enzymed and although the components were not purified to homogeneity, an enriched mixture of proteins had enzyme activity and stability in a broad temperature range, and activity to a variety of cellulosic substrates.
This thesis research adds to the knowledge of the fungal biodiversity in the Antarctic and increases the understanding of the biochemical framework, participating in relation to wood decay potential of these Antarctic fungal isolates.