Josie van Dorst

Community fingerprinting in a sequencing world

Despite decreasing costs, generating large-scale, well-replicated and multivariate microbial ecology investigations with sequencing remains an expensive and time-consuming option. As a result, many microbial ecology investigations continue to suffer from a lack of appropriate replication. We evaluated two fingerprinting approaches - terminal restriction fragment length polymorphism (T-RFLP) and automated ribosomal intergenic spacer analysis (ARISA) against 454 pyrosequencing, by applying them to 225 polar soil samples from East Antarctica and the high Arctic. By incorporating local and global spatial scales into the dataset, our aim was to determine whether various approaches differed in their ability and hence utility, to identify ecological patterns. Through the reduction in the 454 sequencing data to the most dominant OTUs, we revealed that a surprisingly small proportion of abundant OTUs (< 0.25%) was driving the biological patterns observed. Overall, ARISA and T-RFLP had a similar capacity as sequencing to separate samples according to distance at a local scale, and to correlate environmental variables with microbial community structure. Pyrosequencing had a greater resolution at the global scale but all methods were capable of significantly differentiating the polar sites. We conclude fingerprinting remains a legitimate approach to generating large datasets as well as a cost-effective rapid method to identify samples for elucidating taxonomic information or diversity estimates with sequencing methods.

Recovering Greater Fungal Diversity from Pristine and Diesel Fuel Contaminated Sub-Antarctic Soil Through Cultivation Using Both a High and a Low Nutrient Media Approach

Novel cultivation strategies for bacteria are widespread and well described for recovering greater diversity from the “hitherto” unculturable majority. While similar approaches have not yet been demonstrated for fungi it has been suggested that of the 1.5 million estimated species less than 5% have been recovered into pure culture. Fungi are known to be involved in many degradative processes, including the breakdown of petroleum hydrocarbons, and it has been speculated that in Polar Regions they contribute significantly to bioremediation of contaminated soils. Given the biotechnological potential of fungi there is a need to increase efforts for greater species recovery, particularly from extreme environments such as sub-Antarctic Macquarie Island. In this study, like the yet-to-be cultured bacteria, high concentrations of nutrients selected for predominantly different fungal species to that recovered using a low nutrient media. By combining both media approaches to the cultivation of fungi from contaminated and non-contaminated soils, 91 fungal species were recovered, including 63 unidentified species. A preliminary biodegradation activity assay on a selection of isolates found that a high proportion of novel and described fungal species from a range of soil samples were capable of hydrocarbon degradation and should be characterized further.

Bacterial Targets as Potential Indicators of Diesel Fuel Toxicity in Subantarctic Soils

ABSTRACT Appropriate remediation targets or universal guidelines for polar regions do not currently exist, and a comprehensive understanding of the effects of diesel fuel on the natural microbial populations in polar and subpolar soils is lacking. Our aim was to investigate the response of the bacterial community to diesel fuel and to evaluate if these responses have the potential to be used as indicators of soil toxicity thresholds. We set up short- and long-exposure tests across a soil organic carbon gradient. Utilizing broad and targeted community indices, as well as functional genes involved in the nitrogen cycle, we investigated the bacterial community structure and its potential functioning in response to special Antarctic blend (SAB) diesel fuel. We found the primary effect of diesel fuel toxicity was a reduction in species richness, evenness, and phylogenetic diversity, with the resulting community heavily dominated by a few species, principally Pseudomonas. The decline in richness and phylogenetic diversity was linked to disruption of the nitrogen cycle, with species and functional genes involved in nitrification significantly reduced. Of the 11 targets we evaluated, we found the bacterial amoA gene indicative of potential ammonium oxidation, the most suitable indicator of toxicity. Dose-response modeling for this target generated an average effective concentration responsible for 20% change (EC20) of 155 mg kg−1, which is consistent with previous Macquarie Island ecotoxicology assays. Unlike traditional single-species tolerance testing, bacterial targets allowed us to simultaneously evaluate more than 1,700 species from 39 phyla, inclusive of rare, sensitive, and functionally relevant portions of the community.

Atmospheric trace gases support primary production in Antarctic desert surface soil

Cultivation-independent surveys have shown that the desert soils of Antarctica harbour surprisingly rich microbial communities. Given that phototroph abundance varies across these Antarctic soils, an enduring question is what supports life in those communities with low photosynthetic capacity. Here we provide evidence that atmospheric trace gases are the primary energy sources of two Antarctic surface soil communities. We reconstructed 23 draft genomes from metagenomic reads, including genomes from the candidate bacterial phyla WPS-2 and AD3. The dominant community members encoded and expressed high-affinity hydrogenases, carbon monoxide dehydrogenases, and a RuBisCO lineage known to support chemosynthetic carbon fixation. Soil microcosms aerobically scavenged atmospheric H2 and CO at rates sufficient to sustain their theoretical maintenance energy and mediated substantial levels of chemosynthetic but not photosynthetic CO2 fixation. We propose that atmospheric H2, CO2 and CO provide dependable sources of energy and carbon to support these communities, which suggests that atmospheric energy sources can provide an alternative basis for ecosystem function to solar or geological energy sources. Although more extensive sampling is required to verify whether this process is widespread in terrestrial Antarctica and other oligotrophic habitats, our results provide new understanding of the minimal nutritional requirements for life and open the possibility that atmospheric gases support life on other planets.

Geological connectivity drives microbial community structure and connectivity in polar, terrestrial ecosystems

Landscape heterogeneity impacts community assembly in animals and plants, but it is not clear if this ecological concept extends to microbes. To examine this question, we chose to investigate polar soil environments from the Antarctic and Arctic, where microbes often form the major component of biomass. We examined soil environments that ranged in connectivity from relatively well-connected slopes to patchy, fragmented landforms that comprised isolated frost boils. We found landscape connectedness to have a significant correlation with microbial community structure and connectivity, as measured by co-occurrence networks. Soils from within fragmented landforms appeared to exhibit less local environmental heterogeneity, harboured more similar communities, but fewer biological associations than connected landforms. This effect was observed at both poles, despite the geographical distances and ecological differences between them. We suggest that microbial communities inhabiting well-connected landscape elements respond consistently to regional-scale gradients in biotic and edaphic factors. Conversely, the repeated freeze thaw cycles that characterize fragmented landscapes create barriers within the landscape and act to homogenize the soil environment within individual frost boils and consequently the microbial communities. We propose that lower microbial connectivity in the fragmented landforms is a function of smaller patch size and continual disturbances following soil mixing.

Microbial Diversity of Browning Peninsula, Eastern Antarctica Revealed Using Molecular and Cultivation Methods

Browning Peninsula is an ice-free polar desert situated in the Windmill Islands, Eastern Antarctica. The entire site is described as a barren landscape, comprised of frost boils with soils dominated by microbial life. In this study, we explored the microbial diversity and edaphic drivers of community structure across this site using traditional cultivation methods, a novel approach the soil substrate membrane system (SSMS), and culture-independent 454-tag pyrosequencing. The measured soil environmental and microphysical factors of chlorine, phosphate, aspect and elevation were found to be significant drivers of the bacterial community, while none of the soil parameters analyzed were significantly correlated to the fungal community. Overall, Browning Peninsula soil harbored a distinctive microbial community in comparison to other Antarctic soils comprised of a unique bacterial diversity and extremely limited fungal diversity. Tag pyrosequencing data revealed the bacterial community to be dominated by Actinobacteria (36%), followed by Chloroflexi (18%), Cyanobacteria (14%), and Proteobacteria (10%). For fungi, Ascomycota (97%) dominated the soil microbiome, followed by Basidiomycota. As expected the diversity recovered from culture-based techniques was lower than that detected using tag sequencing. However, in the SSMS enrichments, that mimic the natural conditions for cultivating oligophilic “k-selected” bacteria, a larger proportion of rare bacterial taxa (15%), such as Blastococcus, Devosia, Herbaspirillum, Propionibacterium and Methylocella and fungal (11%) taxa, such as Nigrospora, Exophiala, Hortaea, and Penidiella were recovered at the genus level. At phylum level, a comparison of OTUs showed that the SSMS shared 21% of Acidobacteria, 11% of Actinobacteria and 10% of Proteobacteria OTUs with soil. For fungi, the shared OTUs was 4% (Basidiomycota) and <0.5% (Ascomycota). This was the first known attempt to culture microfungi using the SSMS which resulted in an increase in diversity from 14 to 57 microfungi OTUs compared to standard cultivation. Furthermore, the SSMS offers the opportunity to retrieve a greater diversity of bacterial and fungal taxa for future exploitation.

Flow cytometry in environmental microbiology: a rapid approach for the isolation of single cells for advanced molecular biology analysis.

The isolation and subsequent characterization of microbial cells from within environmental samples is a difficult process. Flow cytometry and cell sorting, when combined with the application of fluorescent probes, have the capability for the detection and separation of diverse microbial populations from within complex mixtures. The isolation of single cells allows for downstream investigations towards system-level characterization of unknown Bacterial Phyla to occur. We describe here the combination of fluorescent in situ hybridization and cell sorting for the detection and isolation of Candidate Division TM7 bacteria from an enriched soil sample. The result is the isolation of rare cells suitable for advanced molecular analysis including whole genome amplification and high-throughput pyrosequencing.

New Insights into the Microbial Diversity of Polar Desert Soils: A Biotechnological Perspective

Microorganisms represent the most abundant cold-adapted life-forms on earth. Far from just surviving, microorganisms appear to be thriving in cold climates, with microbial richness present in polar soils often in line with temperate soils. Recent advances in molecular techniques have allowed the true extent of global microbial diversity to be revealed. Antarctica in particular, has been found to harbour diverse and unique microbial populations comprise of high proportions of Chloroflexi, Actinobacteria, and unknown, previously uncultured taxa. Microorganisms have been the targets for bioprospecting for many years but efforts have thus far largely focused on easily obtainable temperate organisms, readily cultured within the laboratory. The extreme conditions that push the limits of life within cold environments leads to the evolution of unique physiologies and functional capabilities. Actinobacteria are well known to be prolific producers of useful natural products. Their high relative abundance along with the plethora of rare and previously unknown organisms highlights the potential for new biotechnological discoveries within cold adapted microorganisms. With limited to no higher organisms, Polar soils also provide ideal model ecosystems to examine the mechanisms driving microbial patterns of distribution. Thus far microbial communities have been found to be largely endemic and exhibit spatial patterns over meter, kilometre, regional and continental scales. While the mechanisms driving the patterns are not completely understood, a number of key biotic and abiotic factors, in particular pH, C/N ratio, NH4 and N concentrations, phosphorus and plant cover, have been identified as influencing polar microbial communities and their survival in these extreme environments. Identifying and understanding key environmental drivers of microbial populations through biogeochemical analysis, structural equation models, microbial co-occurrence models, or space for time substitution studies, are providing the first step towards identifying the distribution of populations with desirable genetic or functional capacity and likewise polar regions that may contain unique communities for protection. At the same time this research is improving our capacity to predict microbial responses to disturbance due to both a changing climate and anthropogenic contamination.