@phdthesis{Heise2017,
  author    = {Heise, Janine},
  title     = {Phylogenetic and physiological characterization of deep-biosphere microorganisms in El'gygytgyn Crater Lake sediments},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-403436},
  school      = {Universit{\"a}t Potsdam},
  pages     = {117},
  year      = {2017},
  abstract  = {The existence of diverse and active microbial ecosystems in the deep subsurface - a biosphere that was originally considered devoid of life - was discovered in multiple microbiological studies. However, most of the studies are restricted to marine ecosystems, while our knowledge about the microbial communities in the deep subsurface of lake systems and their potentials to adapt to changing environmental conditions is still fragmentary. This doctoral thesis aims to build up a unique data basis for providing the first detailed high-throughput characterization of the deep biosphere of lacustrine sediments and to emphasize how important it is to differentiate between the living and the dead microbial community in deep biosphere studies. In this thesis, up to 3.6 Ma old sediments (up to 317 m deep) of the El'gygytgyn Crater Lake were examined, which represents the oldest terrestrial climate record of the Arctic. Combining next generation sequencing with detailed geochemical characteristics and other environmental parameters, the microbial community composition was analyzed in regard to changing climatic conditions within the last 3.6 Ma to 1.0 Ma (Pliocene and Pleistocene). DNA from all investigated sediments was successfully extracted and a surprisingly diverse (6,910 OTUs) and abundant microbial community in the El'gygytgyn deep sediments were revealed. The bacterial abundance (10³-10⁶ 16S rRNA copies g⁻¹ sediment) was up to two orders of magnitudes higher than the archaeal abundance (10¹-10⁵) and fluctuates with the Pleistocene glacial/interglacial cyclicality. Interestingly, a strong increase in the microbial diversity with depth was observed (approximately 2.5 times higher diversity in Pliocene sediments compared to Pleistocene sediments). The increase in diversity with depth in the Lake El'gygytgyn is most probably caused by higher sedimentary temperatures towards the deep sediment layers as well as an enhanced temperature-induced intra-lake bioproductivity and higher input of allochthonous organic-rich material during Pliocene climatic conditions. Moreover, the microbial richness parameters follow the general trends of the paleoclimatic parameters, such as the paleo-temperature and paleo-precipitation. The most abundant bacterial representatives in the El'gygytgyn deep biosphere are affiliated with the phyla Proteobacteria, Actinobacteria, Bacteroidetes, and Acidobacteria, which are also commonly distributed in the surrounding permafrost habitats. The predominated taxon was the halotolerant genus Halomonas (in average 60\% of the total reads per sample). Additionally, this doctoral thesis focuses on the live/dead differentiation of microbes in cultures and environmental samples. While established methods (e.g., fluorescence in situ hybridization, RNA analyses) are not applicable to the challenging El'gygytgyn sediments, two newer methods were adapted to distinguish between DNA from live cells and free (extracellular, dead) DNA: the propidium monoazide (PMA) treatment and the cell separation adapted for low amounts of DNA. The applicability of the DNA-intercalating dye PMA was successfully evaluated to mask free DNA of different cultures of methanogenic archaea, which play a major role in the global carbon cycle. Moreover, an optimal procedure to simultaneously treat bacteria and archaea was developed using 130 µM PMA and 5 min of photo-activation with blue LED light, which is also applicable on sandy environmental samples with a particle load of ≤ 200 mg mL⁻¹. It was demonstrated that the soil texture has a strong influence on the PMA treatment in particle-rich samples and that in particular silt and clay-rich samples (e.g., El'gygytgyn sediments) lead to an insufficient shielding of free DNA by PMA. Therefore, a cell separation protocol was used to distinguish between DNA from live cells (intracellular DNA) and extracellular DNA in the El'gygytgyn sediments. While comparing these two DNA pools with a total DNA pool extracted with a commercial kit, significant differences in the microbial composition of all three pools (mean distance of relative abundance: 24.1\%, mean distance of OTUs: 84.0\%) was discovered. In particular, the total DNA pool covers significantly fewer taxa than the cell-separated DNA pools and only inadequately represents the living community. Moreover, individual redundancy analyses revealed that the microbial community of the intra- and extracellular DNA pool are driven by different environmental factors. The living community is mainly influenced by life-dependent parameters (e.g., sedimentary matrix, water availability), while the extracellular DNA is dependent on the biogenic silica content. The different community-shaping parameters and the fact, that a redundancy analysis of the total DNA pool explains significantly less variance of the microbial community, indicate that the total DNA represents a mixture of signals of the live and dead microbial community. This work provides the first fundamental data basis of the diversity and distribution of microbial deep biosphere communities of a lake system over several million years. Moreover, it demonstrates the substantial importance of extracellular DNA in old sediments. These findings may strongly influence future environmental community analyses, where applications of live/dead differentiation avoid incorrect interpretations due to a failed extraction of the living microbial community or an overestimation of the past community diversity in the course of total DNA extraction approaches.},
  language  = {en}
}