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Understanding functional drivers in two terrestrial key processes- nitrogen fixation and cellulose degradation- by a single cell approach

Final Report Summary - DW_FDTP_UVA (Understanding functional drivers in two terrestrial key processes- nitrogen fixation and cellulose degradation- by a single cell approach)

The terrestrial carbon (C) and nitrogen (N) cycles are essential for the Earth’s biosphere and intimately linked by microbial activity. The overarching goal of this project was to elucidate the active participants of two microbial key processes - plant polymeric C degradation and nitrogen fixation - in soil and their contributions to these processes. To identify the active participants of the two processes we sought to combine stable isotope probing experiments (with 13C-cellulose and 15N2 gas, respectively), functional gene sequencing, cell identification with FISH, and Raman microspectroscopy or NanoSIMS for measuring the activity of single cells. However, single-cell techniques such as NanoSIMS and Raman microspectroscopy found limited application in soils presumably due to the dispersal of microbial cells in a large background of particles. It was therefore one goal of this project to develop a procedure that allows the application of these methods to soil microorganisms by separating the cells from soil particles and concentrate them for efficient analysis. We have developed a soil sample preparation procedure that generates samples with reduced load of particles that are suited for NanoSIMS analysis as well as Raman microspectroscopy.
One of the two microbial processes this project focused on was N2 fixation (diazotrophy) in soils. In a first step we have conducted surveys to identify soil ecosystems with N2 fixation activity, such as forest and grassland soils. We have recently extended the investigation of N2 fixation activity to another important terrestrial environment - the soil-plant interphase. In our grassland site, we have included the plant-associated (but not symbiotic) diazotrophs, as their diversity and contribution to N2 fixation is still largely unknown. In order to identify the microorganisms with the genetic capability to fix N2 and the active diazotroph community in soils, we use a combined molecular, stable isotope tracer, FISH, and NanoSIMS approach. In this project, we established a pipeline to investigate the diversity of diazotrophs using the latest generation of sequencing technology (Illumina MiSeq sequencing), which involved primer testing and the bioinformatics analysis of the functional genes for N2 fixation - the dinitrogenase reductase gene (nifH). In order to identify active diazotrophs, one can also make use of the rare isotope of N (15N) by incubating soil samples with 15N2. If a cell can fix and incorporate 15N2, the 15N tracer should be found in microbial biomass. This can be detected in different ways, and another goal of this project was to test whether Raman microspectroscopy can be used to detect cells that have incorporated 15N. By measuring bacteria of different phyla grown under different 15N levels via Raman microspectroscopy, we learned that while labelling of bacterial cells with 15N leads to shifts in Raman spectra, the sensitivity of the method might be too low for many environmental applications.
As a backup plan we established a 15N-RNA stable isotope probing protocol using 15N2 gas. This represents another method that allows the identification of microorganisms that took up the 15N2 tracer and incorporated the 15N tracer in their 16S rRNA, which can then be analysed. Based on this information FISH probes can be chosen, and the targeted population can be further investigated for their 15N2 fixation activity by NanoSIMS.

The degradation of cellulose was the other microbial process this project focused on, and it is known that fungi as well as some bacteria have the capacity for this activity. It was the goal in this project to identify the active cellulose degraders and to understand their contribution to this process. To achieve this, we applied a combined enzymatic, molecular and activity-based approach to identify cellulose-degrading guilds over time in differing nutrient regimes in an Austrian beech forest soil by using 13C-labeled cellulose. We hypothesized that by varying certain edaphic properties (such as N and C content) that can limit cellulose degradation, we will uncover different active cellulose-degrading guilds of bacteria and fungi and thereby elucidate their ecological niches. Our combined data show that the N-amended microcosms exhibited the highest rates of cellulose degradation activity with a change in the cellulolytic community (fungi as well as bacteria) in comparison to the other conditions. These data support our hypothesis that fungi and bacteria exploit different physiological niches in this process due to nutrient availability. Additionally, this effect can also be seen within the fungi and bacteria, as different fungal and bacterial groups are active in cellulose degradation at different nutrient regimes. We are currently investigating the interactions of bacterial and fungal groups across treatments and time by applying network analysis. This may allow us to better identify those organisms that potentially interact synergistically during the cellulolytic process or those that are competitive and might have negative effects on others. Additionally, with the established procedure for single-cell investigations on soil microorganism and based on the OTUs identified in our 13C-DNA SIP experiment, we can now target these specific groups using FISH and investigate their degree of activity at different conditions via NanoSIMS.

The main achievements of this CIG project are a pipeline for the analysis of Illumina amplicon sequences of the marker gene for diazotrophs (nifH), from work in the laboratory to bioinformatic analysis, which should be of assistance for other researchers that investigate diazotrophs in diverse environments. Furthermore, a 15N-RNA SIP protocol for the identification of active diazotrophs was established. Regarding the application of single-cell methods to soil microorganisms that was greatly lacking in the literature, we established a sample preparation procedure of soil samples that generates a cell fraction that is amenable for NanoSIMS and Raman microspectroscopy. The combination of a staining method (CARD-FISH) with NanoSIMS on soil microorganisms was also demonstrated, which allows the targeted analysis of specific microbial groups and thus can elucidate their function in the soil environment. We hope that these results will encourage other soil scientists to apply these techniques in their research projects. And last, we identified specific ecological niches for cellulolytic bacteria and fungi in a combined biogeochemical, enzymatic, and activity-based approach, the ladder encompassing 13C-DNA SIP followed by amplicon sequencing.