Microbial biodiversity in Tasmanian caves

Caves represent one of few remaining isolated planetary habitats, in terms of human impact and characterisation of microbial biodiversity. Caves are unique environments characterised by little or no light, low levels of organic nutrients, high mineral concentrations and a stable microclimate providing ecological niches for highly specialised organisms. Caves are not uniform environments in terms of geological and geochemical characteristics, as they can vary from one to the other, eg. rock type, method of formation, length, depth, number of openings to the surface, presence or absence of active streamways, degree of impact by human visitation etc. Furthermore, on a smaller scale, various microhabitats, with vast differences in community structure can exist within caves. Culture studies point to the dominance of actinomycetes in caves and reveals great taxonomic diversity within actinomycetes isolated. However it is widely accepted that only - 1 % of microbes are cultured in the laboratory. Culture-independent methods are being increasingly used to describe the composition of microbial communities and reveal significantly broader diversity than culture-based studies. Nevertheless, to date our knowledge of bacterial communities in caves is largely due to culture studies. Based on the literature available, this study was initially aimed at examining culturable vs. non-culturable diversity of actinomycetes in Entrance and Loons Caves and to gain an increased understanding of the composition of cave microbial communities employing classical isolation and advanced molecular detection methods. As the study progressed the focus evolved as it became apparent that actinomycetes dominated only very specific habitats, the dry sediment in Entrance Cave, and represented only a minor fraction of the microbial biodiversity of most other microhabitats studied. Entrance Cave dry sediments and inactive (dry) speleothems produced a higher number of actinomycete isolates compared to saturated sediments and wet formations from Entrance and Loons Caves. This was reinforced by the actinomycetes being the second most abundant group (26.8%) detected in clone analysis of the dry Entrance sediment and low abundances (4-16%) detected in saturated sediments from both Entrance and Loons Caves. Sediment phylotypes and isolates identified in this study closely resemble species associated with oligotrophic, chemolithotrophic and heterotrophic lifestyles indicating that these communities survive by utilising a combination of metabolic pathways. Bacteria involved in the nitrogen and sulfur cycles were important members of all sediment communities along with hydrogen-oxidising bacteria. Pair-wise comparisons of sediment communities demonstrated that they were more similar to each other within individual cave systems, Entrance and Loons, rather than between microhabitat types (dry vs. wet sediment) though saturated sediment from Entrance Cave did show a higher degree of similarity in community composition to Loons Cave samples than the dry sediment from Entrance Cave. Saturated sediments were dominated by oligotrophs able to fix atmospheric gases, methanotrophs and had a high proportion of rare phylotypes most likely representing new lineages related to microbes detected in anaerobic, anoxic environments, but low abundances of heterotrophic microbes. Geornicrobiological activities are no longer underestimated since studies have shown that bacterial metabolism may lead to mineral precipitation or dissolution. Questions remain as to the identity of these microbes and whether they are actively involved in speleothem formation, or simply buried during mineral precipitation. Results demonstrated a marked difference between sediment communities and those associated with calcite speleothem and calcite mat samples. Results of ESEM and XRD analysis demonstrated that calcite speleothem samples ME3 and MXl are true calcite moonrnilk (mondmilch). Phylogenetic analyses and isolation results demonstrated the unique composition of the microbial communities associated with moonrnilk deposits, predominantly composed of nitrogen-fixing ~-Proteobacteria and psychrotrophic heterotrophic CFBs and to a lesser extent, heterotrophic actinomycetes. Despite XRD and ESEM analysis showing similar calcite composition and crystal morphology, phylogenetic results indicated that sample ME2 represented a very different rnicrohabitat to moonmilk samples, dominated by oligotrophic a. -Proteobacteria and heterotrophic actinomycetes composing 84.2% of the total diversity. Phylogenetic analyses and biodiversity indices reveal the striking similarities between moonmilk samples from both Entrance and Exit Caves and the uniqueness of the calcite mat in Entrance Cave. The one similarity in composition between all three calcite communities was the presence of members of the Pseudonocardineae in particular of the genus Saccharothrix, in all calcite samples. 165 rRNA gene sequencing of cave isolates detected high levels of diversity and novelty, particularly of moonrnilk isolates. A total of two putatively novel genera (within the CFBs and Actinobacteria) and 18 putatively novel species (of genera: Paracoccus, Actinoplanes I Couchioplanes, Micromonospora, Amycolatopsis, Saccharothrix, Bacillus, Paenibacillus, Methylobacterium, Porphyrobacter, Sphingomonas, Alcaligenes, Stenotrophomonas, Xanthomonas) were identified. This study represents the first reported culture-independent analysis of moonrnilk microbial communities globally and of cave sediment communities in the Southern Hemisphere. Information gained from this study and the discovery of actively growing microbial communities appearing to precipitate CaC03 provides focus for important future studies and represents a unique opportunity to examine the nature and extent of complex microbe-mineral interactions in the formation of speleothems and implications for cave management. The biodiversity described acts as a baseline for assessing environmental impacts and to identify factors influencing microbial biodiversity.