Exploring heterotrophic bacterial diversity of Antarctic samples through cultivation

The microbial diversity on Antarctica is largely under-explored. As part of the AMBIO-project that aims to explore the factors affecting bacterial distribution patterns in Antarctica, nine samples from different regions were investigated. They include two samples from the Belgian Base site at Utsteinen: BB50 from a green microbial/algal mat on gravel from the nunatak and BB115 from a black mat on gravel and rock debris from a frozen lake on the south side of the nunatak Utsteinen; two samples from the Trans-Antarctic Mountains: TM2 from a cyanobacterial mat at the bottom of Forlidas Pond (Pensacola Mountains) and TM4 from Lundström Lake (Shackleton Mountains); a littoral sample PQ1 from Pourquoi-Pas Lake (Pourquoi-Pas Island); samples LA3 (Langhovde Peninsula), SK5 (Skarvsness Peninsula) and WO10 (West Ongul Island) from three lakes at Lützow-Holm Bay, Syowa and sample SO6 from the Schirmacher Oasis. Samples were investigated with a culture-dependent approach. Dilution series were plated on four media (MA, R2A, R2A/10 and PYGV) and incubated at three temperatures (4, 15 and 20ºC). Different types of colonies were picked up for all conditions. As a fast way of screening the isolates, the genotypic typing method rep-PCR fingerprinting was used. Cluster analysis of fingerprint patterns using Bionumerics software revealed a number of clusters (cut-off level 80%) of similar strains and a number of separate isolates. Representatives of each rep-cluster and the separate isolates were used in partial 16S rDNA sequencing to obtain a first approximate identification. For the different samples, between 338 (BB115) and 669 (WO10) isolates were obtained and purified. Rep-PCR fingerprinting analysis resulted in 33 (LA3) to 101 (BB50) clusters and 54 (BB115) to 151 (PQ1) separate isolates (excluding samples WO10 and SO6 for which the analysis is not finished yet). The preliminary results of the 16S rDNA sequencing show a large diversity, distributed over the major phylogenetic groups. The BB samples and TM4 are dominated by isolates of the class of Actinobacteria, most TM2 isolates belong to the Firmicutes class. The PQ1 and LA3 samples are dominated by the class of Bacteroidetes and the most SK5 isolates belong to the class of Alphaproteobacteria. Other phylogenetic groups were found in smaller amounts. Despite the large diversity and the fact that some samples originate from the same general area, only little overlap between the samples was observed. Some of the isolated clusters show low similarity values with neighbouring sequences in the EMBL-database and may represent new species or even new genera