Bjorn Tytgat

Bacterial Diversity Assessment in Antarctic Terrestrial and Aquatic Microbial Mats: A Comparison between Bidirectional Pyrosequencing and Cultivation

The application of high-throughput sequencing of the 16S rRNA gene has increased the size of microbial diversity datasets by several orders of magnitude, providing improved access to the rare biosphere compared with cultivation-based approaches and more established cultivation-independent techniques. By contrast, cultivation-based approaches allow the retrieval of both common and uncommon bacteria that can grow in the conditions used and provide access to strains for biotechnological applications. We performed bidirectional pyrosequencing of the bacterial 16S rRNA gene diversity in two terrestrial and seven aquatic Antarctic microbial mat samples previously studied by heterotrophic cultivation. While, not unexpectedly, 77.5% of genera recovered by pyrosequencing were not among the isolates, 25.6% of the genera picked up by cultivation were not detected by pyrosequencing. To allow comparison between both techniques, we focused on the five phyla (Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Deinococcus-Thermus) recovered by heterotrophic cultivation. Four of these phyla were among the most abundantly recovered by pyrosequencing. Strikingly, there was relatively little overlap between cultivation and the forward and reverse pyrosequencing-based datasets at the genus (17.1–22.2%) and OTU (3.5–3.6%) level (defined on a 97% similarity cut-off level). Comparison of the V1–V2 and V3–V2 datasets of the 16S rRNA gene revealed remarkable differences in number of OTUs and genera recovered. The forward dataset missed 33% of the genera from the reverse dataset despite comprising 50% more OTUs, while the reverse dataset did not contain 40% of the genera of the forward dataset. Similar observations were evident when comparing the forward and reverse cultivation datasets. Our results indicate that the region under consideration can have a large impact on perceived diversity, and should be considered when comparing different datasets. Finally, a high number of OTUs could not be classified using the RDP reference database, suggesting the presence of a large amount of novel diversity.

Bacterial community composition in relation to bedrock type and macrobiota in soils from the Sør Rondane Mountains, East Antarctica.

Antarctic soils are known to be oligotrophic and of having low buffering capacities. It is expected that this is particularly the case for inland high-altitude regions. We hypothesized that the bedrock type and the presence of macrobiota in these soils enforce a high selective pressure on their bacterial communities. To test this, we analyzed the bacterial community structure in 52 soil samples from the western Sør Rondane Mountains (Dronning Maud Land, East Antarctica), using the Illumina MiSeq platform in combination with ARISA fingerprinting. The samples were taken along broad environmental gradients in an area covering nearly 1000 km(2) Ordination and variation partitioning analyses revealed that the total organic carbon content was the most significant variable in structuring the bacterial communities, followed by pH, electric conductivity, bedrock type and the moisture content, while spatial distance was of relatively minor importance. Acidobacteria (Chloracidobacteria) and Actinobacteria (Actinomycetales) dominated gneiss derived mineral soil samples, while Proteobacteria (Sphingomonadaceae), Cyanobacteria, Armatimonadetes and candidate division FBP-dominated soil samples with a high total organic carbon content that were mainly situated on granite derived bedrock.

Analysis of cbbL, nifH, and pufLM in soils from the Sør Rondane Mountains, Antarctica, reveals a large diversity of autotrophic and phototrophic bacteria

Cyanobacteria are generally thought to be responsible for primary production and nitrogen fixation in the microbial communities that dominate Antarctic ecosystems. Recent studies of bacterial communities in terrestrial Antarctica, however, have shown that Cyanobacteria are sometimes only scarcely present, suggesting that other bacteria presumably take over their role as primary producers and diazotrophs. The diversity of key genes in these processes was studied in surface samples from the Sør Rondane Mountains, Dronning Maud Land, using clone libraries of the large subunit of ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) genes (cbbL, cbbM) and dinitrogenase-reductase (nifH) genes. We recovered a large diversity of non-cyanobacterial cbbL type IC in addition to cyanobacterial type IB, suggesting that non-cyanobacterial autotrophs may contribute to primary production. The nifH diversity recovered was predominantly related to Cyanobacteria, particularly members of the Nostocales. We also investigated the occurrence of proteorhodopsin and anoxygenic phototrophy as mechanisms for non-Cyanobacteria to exploit solar energy. While proteorhodopsin genes were not detected, a large diversity of genes coding for the light and medium subunits of the type 2 phototrophic reaction center (pufLM) was observed, suggesting for the first time, that the aerobic photoheterotrophic lifestyle may be important in oligotrophic high-altitude ice-free terrestrial Antarctic habitats.

A Doubling of Microphytobenthos Biomass Coincides with a Tenfold Increase in Denitrifier and Total Bacterial Abundances in Intertidal Sediments of a Temperate Estuary

Surface sediments are important systems for the removal of anthropogenically derived inorganic nitrogen in estuaries. They are often characterized by the presence of a microphytobenthos (MPB) biofilm, which can impact bacterial communities in underlying sediments for example by secretion of extracellular polymeric substances (EPS) and competition for nutrients (including nitrogen). Pyrosequencing and qPCR was performed on two intertidal surface sediments of the Westerschelde estuary characterized by a two-fold difference in MPB biomass but no difference in MPB composition. Doubling of MPB biomass was accompanied by a disproportionately (ten-fold) increase in total bacterial abundances while, unexpectedly, no difference in general community structure was observed, despite significantly lower bacterial richness and distinct community membership, mostly for non-abundant taxa. Denitrifier abundances corresponded likewise while community structure, both for nirS and nirK denitrifiers, remained unchanged, suggesting that competition with diatoms for nitrate is negligible at concentrations in the investigated sediments (appr. 1 mg/l NO3 -). This study indicates that MPB biomass increase has a general, significantly positive effect on total bacterial and denitrifier abundances, with stimulation or inhibition of specific bacterial groups that however do not result in a re-structured community.

Bacterial and eukaryotic biodiversity patterns in terrestrial and aquatic habitats in the Sør Rondane Mountains, Dronning Maud Land, East Antarctica

The bacterial and microeukaryotic biodiversity were studied using pyrosequencing analysis on a 454 GS FLX+ platform of partial SSU rRNA genes in terrestrial and aquatic habitats of the Sør Rondane Mountains, including soils, on mosses, endolithic communities, cryoconite holes and supraglacial and subglacial meltwater lenses. This inventory was complemented with Denaturing Gradient Gel Electrophoresis targeting Chlorophyta and Cyanobacteria. OTUs belonging to the Rotifera, Chlorophyta, Tardigrada, Ciliophora, Cercozoa, Fungi, Bryophyta, Bacillariophyta, Collembola and Nematoda were present with a relative abundance of at least 0.1% in the eukaryotic communities. Cyanobacteria, Proteobacteria, Bacteroidetes, Acidobacteria, FBP and Actinobacteria were the most abundant bacterial phyla. Multivariate analyses of the pyrosequencing data revealed a general lack of differentiation of both eukaryotes and prokaryotes according to habitat type. However, the bacterial community structure in the aquatic habitats was dominated by the filamentous cyanobacteria Leptolyngbya and appeared to be significantly different compared with those in dry soils, on mosses, and in endolithic habitats. A striking feature in all datasets was the detection of a relatively large amount of sequences new to science, which underscores the need for additional biodiversity assessments in Antarctic inland locations.

Diversity of Phototrophic Genes Suggests Multiple Bacteria May Be Able to Exploit Sunlight in Exposed Soils from the Sør Rondane Mountains, East Antarctica

Microbial life in exposed terrestrial surface layers in continental Antarctica is faced with extreme environmental conditions, including scarcity of organic matter. Bacteria in these exposed settings can therefore be expected to use alternative energy sources such as solar energy, abundant during the austral summer. Using Illumina MiSeq sequencing, we assessed the diversity and abundance of four conserved protein encoding genes involved in different key steps of light-harvesting pathways dependent on (bacterio)chlorophyll (pufM, bchL/chlL, and bchX genes) and rhodopsins (actinorhodopsin genes), in exposed soils from the Sør Rondane Mountains, East Antarctica. Analysis of pufM genes, encoding a subunit of the type 2 photochemical reaction center found in anoxygenic phototrophic bacteria, revealed a broad diversity, dominated by Roseobacter- and Loktanella-like sequences. The bchL and chlL, involved in (bacterio)chlorophyll synthesis, on the other hand, showed a high relative abundance of either cyanobacterial or green algal trebouxiophyceael chlL reads, depending on the sample, while most bchX sequences belonged mostly to previously unidentified phylotypes. Rhodopsin-containing phototrophic bacteria could not be detected in the samples. Our results, while suggesting that Cyanobacteria and green algae are the main phototrophic groups, show that light-harvesting bacteria are nevertheless very diverse in microbial communities in Antarctic soils.

Abditibacterium utsteinense sp. nov., the first cultivated member of candidate phylum FBP, isolated from ice-free Antarctic soil samples

Most bacterial lineages are known only by molecular sequence data from environmental surveys and represent the uncultivated majority. One of these lineages, candidate phylum FBP, is widespread in extreme environments on Earth, ranging from polar and desert ecosystems to wastewater and contaminated mine sites. Here we report on the characterization of strain LMG 29911T, the first cultivated representative of the FBP lineage. The strain was isolated from a terrestrial surface sample from Utsteinen, Sør Rondane Mountains, East Antarctica and is a Gram-negative, aerobic, oligotrophic chemoheterotrophic bacterium. It displays growth in a very narrow pH range, use of only a limited number of carbon sources, but also a metabolism optimized for survival in low-nutrient habitats. Remarkably, phenotypic and genome analysis indicated an extreme resistance against antibiotics and toxic compounds. We propose the names Abditibacterium utsteinense for this bacterium and Abditibacteriota for the former candidate phylum FBP. Furthermore, inter- and intra-phylum relationships indicate Armatimonadetes, a neighboring lineage to the Abditibacteriota, to be a superphylum.