A functional approach to understand active non-symbiotic diazotrophs in soil

The terrestrial nitrogen (N) cycle is essential for the Earth`s biosphere and intimately linked by microbial activity. Understanding the microorganisms that provide biologically available N through N2 fixation (diazotrophy) is imperative as N is a limiting factor for primary production and thereby CO 2 sequestration. Non-symbiotic (free- living) microorganisms can contribute greatly to N2 fixation; however, we still have a very limited understanding of these microorganisms, because only a small fraction has been cultivated in the laboratory. The advent of molecular biological techniques has provided first insights into the vast genetic diversity and distribution of diazotrophs across numerous environments. Nevertheless, we still have not fully explored the depths of the diazotroph microbial seed bank in soils and the active drivers in this important process and the factors influencing their activity. Due to recent advances in the field, it is timely to now apply a functional approach to better understand the active drivers of N2 fixation in terrestrial ecosystems. This project will use a unique and cultivation- independent approach combining biogeochemical assays, stable isotope probing, next generation sequencing, and single cell techniques to better understand diazotrophy in soils. First, we will investigate a recent hypothesis suggesting that diazotroph diversity positively influences N2 fixation activity through massive parallel sequencing of the functional gene for N2 fixation (dinitrogenase reductase gene, nifH) across different ecosystems and seasons, and ultimately correlating these diversity data with soil properties and N2 fixation activity. Second, since N2 fixation is a highly energy demanding process often depending on external energy sources, we will investigate the influence of different carbon (C) sources on N2 fixation activity. We hypothesize that based on the addition of different C sources distinct groups of diazotrophs will respond and actively fix N2 . We will mimic the natural addition of C source through plant root exudates and litter in 15N2 incubation experiments and will identify the diazotrophic groups that uniquely respond to different carbon sources by combining 15N2 -DNA SIP, functional transcript sequencing with a FISH-NanoSIMS approach. And finally, we aim to develop a method that allows the detection of 15N-labeled cells by Raman microspectroscopy in a sequence information-independent approach that will facilitate new ways of studying non-cultured diazotrophs in terrestrial ecosystems. In summary, this integrated combination of cutting-edge methods has great potential to (i) advance our understanding of terrestrial diazotrophy and the factors controlling this important process, (ii) allow comparing the in situ activities of different microbial groups and the analysis of within population heterogeneities in an unprecedented way, and will (iii) encourage other scientists to apply this function-driven approach to terrestrial environments and tackle the many other un- answered questions of microbial function in these ecosystems.

The terrestrial nitrogen (N) cycle is essential for the Earths biosphere and intimately linked by microbial activity. Understanding the microorganisms that provide biologically available N through N2 fixation (diazotrophy) is imperative as N is a limiting factor for primary production and thereby CO2 sequestration. Non -symbiotic (free -living) microorganisms can contribute greatly to N2 fixation; however, we still have a very limited understanding of these microorganisms, because only a small fraction has been cultivated in the laboratory. The advent of molecular biological techniques has provided first insights into the vast genetic diversity and distribution of diazotrophs across numerous environments, and especially soils are expected to harbor a tremendous diversity of diazotrophs. Nevertheless, we still have not fully explored the depths of the diazotroph microbial seed bank in soils, the active drivers in this important process and the factors influencing their activity. In this project we have used a functional approach combining biogeochemical assays, stable isotope probing, next generation sequencing, and single cell techniques to investigate diazotrophy in soils. Through this project, we have (i) furthered our understanding of the identity and distribution of diazotrophs in terrestrial habitats, (ii) revealed the driving factors for the observed diazotroph community composition, (iii) identified the active diazotrophs in these habitats, and (iv) developed or optimized methods for the investigation of diazotrophs ranging from a sequences analysis pipeline to single -cell techniques. With this, we have deepened our knowledge on the distribution and activity of diazotrophs in several terrestrial systems and provide new tools that can be applied by other scientists to further investigate this important group of microorganisms.