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Investigating factors that govern biological N2 fixation in soil by CHIP-SIP

 

Supervisor: Dagmar Wöbken

PhD student: Marlies Dietrich

Group: Division of Microbial Ecology, Centre for Microbiology and Environmental Systems Science

 

 

Biologically available nitrogen (N) is often a limiting factor for primary production in soils. Diazotrophs in terrestrial ecosystems account for ca. 65% of the Earth’s N2 fixation activity. Recent molecular surveys suggest that non-symbiotic (free-living) diazotrophs, in addition to symbiotic diazotrophs, have great importance in terrestrial N2 fixation. Terrestrial ecosystems harbor a large but unknown diversity of diazotrophs, a diversity of microorganisms that we still have to elucidate. The activity of diazotrophs has shown to be affected/limited by various environmental factors, such as O2 concentrations (since nitrogenase is a highly O2 sensitive enzyme), the availability of phosphorous (P) and molybdenum (Mo) and carbon (C) sources, the latter presumably due to the high-energy demand of N2 fixation. Most likely, the C quality and quantity will differentially influence the rate of fixation and the active diazotroph community composition. In a native setting, potential C sources in soil are plant roots exudates (readily oxidizable C) and plant polymeric C from litter (such as xylan, cellulose).

Information about the influence of oxygen, C sources and the availability of P and Mo on the active diazotroph community is still scarce. This project aims to shed light on this aspect by applying the CHIP-SIP approach (Mayali et al., 2012, ISME J) to soil diazotroph communities. This approach consists of incubation of the microbial community with isotopically labelled substrates (such as 15N2), hybridization of the extracted community rRNA to a microarray and measurement of isotope incorporation (and thereby 15N2 incorporation) by secondary ion mass spectrometry imaging (NanoSIMS). In summary, this microarray and NanoSIMS-based method allows linking microbial phylogeny and function, and thus testing the 15N2-incoporation activity of a diverse diazotroph community.

The work in this project will be performed in close collaboration with the project of Andreas Richter. For both projects, a microarray will be designed to target the microbial community in bulk soil of a grassland site and for the rhizosphere community of a specific grass species inhabiting this ecosystem. In soil microcosm experiments, we will test the effect of differing oxygen concentrations, P and Mo levels, as well as different C sources on the active diazotrophic population. For instance, different 13C-labeled C sources (such as fructose, cellobiose and artificial root exudate mix, as well as cellulose and xylan as complex C sources) will be applied together with 15N2 in stable isotope incubation experiments of bulk soil and rhizosphere soil. By this approach, the actively 15N2 fixing taxa will be identified together with their utilized C sources. The 15N2 fixing activity of the identified taxa can be analysed in more detail by subsequent FISH-NanoSIMS analysis on the single-cell level.

 

Co-Supervision by Andreas Richter and Michael Wagner, secondment by Dr. Jennifer Pett-Ridge (Lawrence Livermore National Laboratory, USA) on the CHIP-SIP procedure.

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