Characterisation of diverse microbial communities and application of novel detection techniques

Microbes are essential for all life on Earth. They are found in all viable habitats from deep sea sediments and bedrock to high up in the atmosphere with a variety that exceeds by far the eukaryotic diversity. Ecosystem services provided by microorganisms, such as degradation of organic material and mediation of biogeochemical cycles are fundamentally important for the whole biosphere and its inhabitants. Microbes also form symbiotic relationships with multicellular organisms, and play important roles in nutrition and disease. Recent developments in molecular techniques, especially the next generation sequencing technologies and microarray applications, have opened new possibilities in studying diverse microbial communities. In this thesis, the aim was to determine the diversity and community structure of environmental samples collected from the northern Baltic Sea water column and anaerobic digestion reactor, and to assess how the prevailing abiotic factors affect the microbial community structure. We applied 16S rRNA and ITS gene amplicon sequencing method with 454 sequencing technology to form a detailed taxonomic description of studied communities. The produced sequence data was further utilised in designing probes for a new padlock probe based ligation detection reaction (LDR) microarray that could be employed for specific and sensitive taxonomic identification of microbial groups in diverse communities. The functionality, specificity and sensitivity of the microarray were assessed using artificial and real environmental samples. Additionally, selected amplicon sequencing data analysis methods were compared in order to discover which algorithms work most reliably. In this subproject, we aimed to clarify how significantly the selected analysis methods, specifically denoising and clustering algorithms, affect the results and how comparable the results derived from different analysis pipelines are. Amplicon sequencing revealed diverse microbial communities in the northern Baltic Sea water column and anaerobic digestion reactor. The pelagic bacterial communities in the northern Baltic Sea were strongly stratified, with aerobic Bacteria such as Pseudomonas and Flavobacterium dominating in the surface layer and Oleispira and sulfate-reducing bacteria in the anoxic deep waters. Based on the sequence data the diversity was assessed one order of magnitude less diverse compared to Atlantic and Pacific ocean bacterial communities. The anaerobic digestion reactor communities were dominated by Bacteria belonging to phyla Bacteroidetes, Firmicutes and Thermotogae and methanogenic Archaea, all essential and typical degraders in anaerobic digestion. The process also supported a diverse fungal community of phyla Ascomycota and Basidiomycota, including several taxa capable of degrading organic material in anaerobic conditions. The LDR microarray technology proved sensitive, specific and semiquantitative method for identifying microbes in diverse communities. The proof of principle tests and experiments with real environmental samples showed that if the probes are designed carefully, the detection is comparable to qPCR and amplicon sequencing. The detection limit was 0.01 fmol/µl/template. Data analysis method comparisons revealed prominent differences in observed operational taxonomic units and relative abundance of identified taxa. The majority of tested methods assessed the species richness too high. Using a functioning denoising method evened out the differences in the number of observed OTUs caused by various clustering algorithms. The ability to filter out the spurious taxa produced by amplification and sequencing, but still retain all the real diversity varied between methods. This study shows both the potential and the challenges in the use of amplicon sequencing and microarray technologies in studying diverse microbial communities. The results indicate that the padlock based LDR microarray can be designed for very accurate and sensitive identification of microbial groups of interest. The data suggest that amplicon sequencing is a powerful tool in identifying microbes and assessing the diversity but distinguishing between spurious and true community members remain a challenge. There is still work to be done in the development and application of data analysis tools.

Grantor dc:publisher

Helsingin yliopisto

Year dc:date.issued

2013

Author dc:creator

• Koskinen, Kaisa

• ympäristöekologia

Statement dc:rights

• Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.
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Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/10138/41568

OAI record oai:identifier

https://helda.helsinki.fi/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Ahelda.helsinki.fi%3A10138%2F41568