Dinitrogen (N2) fixation, the biological reduction of atmospheric N2 to ammonium, is the main route of new nitrogen (N) to ecosystems. It influences plant growth and carbon exchange at local, regional and global scales. Our understanding of natural N2 fixation however remains limited, especially in terrestrial environments where it is systematically underestimated. Because fully understanding, or "closing" the N budget of terrestrial ecosystems depends on a better measurement of non-symbiotic N2 fixation, there is a pressing need for new analytical techniques allowing for an affordable, highly sensitive, continuous and non disturbing assessment of non-symbiotic N2 fixation. This is the objective of the proposed work. Briefly, the PIs propose to develop a new method to monitor the reduction of acetylene to ethylene (the most commonly used surrogate for N2 fixation measurements) by wavelength-scanned cavity ring down spectroscopy (WS-CRDS). The portability and high sensitivity of the device will allow for high frequency surveying of large surfaces and provide an innovative tool to study the poorly-resolved spatial variability and short-term responses to environmental gradients of terrestrial N2 fixation. This work also answers a pressing demand for improved tools to assess on-site variability of N2 fixation.
The main objective of our grant was to design and field test a new method for high resolution, high sensitivity, continuous and non disturbing measurements of N2 fixation. Our goal was achieved in two ways. We successfully developed and published the Method (Cassar et al. 2012). We tested with the approach using field samples of mosses, lichens, and chemoheterotrophic and photoautotrophic bacteria (Jean et al. 2012). The new method, Acetylene Reduction Assays by Cavity ring-down laser Absorption Spectroscopy (ARACAS) provides continuous and precise measurements of N2 fixation, allows for simple field deployment (portability, ruggedness and stability), and can be applied by other research groups interested in N2 fixation processes and environments. ARACAS provides high resolution, high sensitivity, continuous and non- disturbing measurements of N2 fixation. We tested various designs relative to the conventional gas chromatography hydrogen flame ionization detection (GC-FID) method with high-purity standards, and diazotrophic cultures, lichens, and mosses. We shared our method at conferences and with the publication of a method paper in Oecologia. In our method paper, we illustrate its use for measuring N2 fixation in the diazotrophic bacterium Azotobacter vinelandii and the lichen Peltigera praetextata (Cassar et al. 2012). Our experiments demonstrate that the method can be used over a large dynamic range in ethylene concentration (ppb to ppm) with high sensitivity over long periods of incubation. We have also used the method to study latitudinal gradients in N2 fixation in mosses. A preliminary study was published last year discussing environmental forcings on N2 fixation in temperate forest mosses (Jean et al. 2012). Using this new method with higher sensitivity and sampling frequency than conventional approaches, we characterized the short-term kinetics of N2 fixation by moss carpets from contrasting ecosystems, and reported the identification of a formerly unknown true moss-diazotrophic cyanobacteria association in a widespread moss species. We are currently working up data from a more detailed latitudinal transect. Three undergraduate students at Duke University have benefitted from this work, conducting lab work for their senior theses using ARACAS to study N2 fixation kinetics in a variety of diazotrophs.
