Optimising and applying RNA based approaches to identify active nitrifiers in coastal sediments

Fabien Cholet
Nitrogen is an essential element for all forms of life on earth. Since the beginning of the industrial revolution, nitrogen has become a major pollutant of marine and coastal ecosystems due to the huge rise in the use of inorganic fertiliser. Like many other nutrients, the transformations of the nitrogen cycle are mainly controlled by the activity of microorganisms. Understanding the factors influencing the activity of microbes involved in the biochemical transformation of nitrogen in the
more » ... nment is therefore crucial. The aim of this thesis is to establish a robust workflow for the study of microbial activity in coastal sediment using transcriptomics. In particular, this work focuses on nitrification, the aerobic chemo-litho-autotrophic oxidation of ammonia to nitrite carried out by ammonia oxidizing bacteria and archaea (AOB and AOA respectively). The First part of the thesis (Chapter I) will consist of a review of the literature on the nitrogen cycle, with a particular focus on nitrification. A review of the techniques used to measure microbial activity in natural environment will also be presented and the knowledge gap that exist in transcriptomic workflow in environmental microbiology identified alongside current understanding of active nitrifiers in coastal sediments. The second part of the thesis (Chapters II and III) will present the first experimental work package which consists of the optimisation of reverse-transcription (RT)- based protocol for the study of microbial activity via transcriptomics. First, a new technique to evaluate RNA integrity, extracted from environmental samples, based mRNA will be developed and tested in a controlled-RNA degradation experiment. We show that this technique can provide a useful complement to the commercial approaches that evaluate RNA integrity mainly through the 16S/23S rRNA ratio. Then, the effect of the RT protocol itself on RT-Q-PCR and RT-PCR- sequencing results will be evaluated by testing a combination of four different RT enzymes and two priming strategie [...]
doi:10.5525/gla.thesis.82100 fatcat:xqtutvvw2nbo3jqoqpllotzm2a