Notes

  1. Substrate enrichment level. Typical SIP experiments involve using high substrate concentrations to achieve maximum labelling. Since 15N2 is also non-toxic, there is no limitation of supplying the incubation vials with atmospheric or even super-atmospheric concentrations of 15N2 gas (e.g. in anoxic incubations). However, this might not be necessary since even in very active systems only a small fraction of the dinitrogen gas eventually gets fixed. To save up on costs, some of the gas can be replaced with another inert gas such as helium or argon. We have incubated several types of soil under an atmosphere of 40:40:20 (15N2, He, O2) and noticed no difference in labelling compared to incubating the samples under 80:20 (15N2, O2; data not shown), although this should probably be best confirmed for every type of sample.
  2.  Substrate contamination issues. Bottles of 15N2 are nearly always sold at a purity of around 99% (and >97% isotopic enrichment). However, that single remaining per cent of foreign substance can turn out to be detrimental since it was found out that a significant fraction of it is in the form of 15N-labelled ammonia and nitrate \cite{Dabundo_2014}. Ideally, every batch of labelled gas should be tested for potential contamination either by direct measurement of ammonia, or indirectly by incubating a culture of non-diazotrophic microorganism in the presence of the gas as a sole nitrogen source and then testing if label has accumulated in the biomas.