The development of a new 15N-stable isotope probing method for the quantification of biological nitrogen fixation in soils

  • Mashita Nina Chiewattanakul

Student thesis: Master's ThesisMaster of Science (MSc)


Biological nitrogen fixation (BNF) performed by diazotrophs is vital to our understanding of ecosystem functions, as nitrogen (N) is commonly a limiting factor in primary productivity. Yet, significant limitations remain in our knowledge of the controls and rates of this process. To address this problem, we developed a novel 15N-stable isotope probing method via gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) for the quantification of BNF in soils. Through initial method development on both symbiotic and free-living diazotrophs in clover root nodules and peat, respectively, the key properties governing N2 fixation were identified and more comprehensively understood. Refinements were then made to the protocol and retested on peat: longer incubations and the addition of an energy source proved successful as the level of 15N enrichment, i.e. N2 fixed, was greatly enhanced. Further method refinements were carried out on British grassland soils, where root exudates were found to be the most effective source of energy in fuelling N2 fixation. The process is also limited by the availability of N within the environment – whether through inputs of N fertiliser or atmospheric N deposition. Lastly, the validated method was applied to a more diverse environment by assaying a range of soils from southwest China, in order to assess the responses of free-living diazotrophs in a recovering ecosystem. Results correlated well with vegetation recovery phases where N2 fixation increased in order of: sloping farmland > recently abandoned farmlands > secondary forest. Anomalies could be explained through other conditions such as soil moisture levels and inactive nifH genes. Taken together, the successful development of a compound-specific GC-C-IRMS method to quantify BNF allows an improved interpretation of the process in a highly sensitive manner – thereby enabling further implications for sustainable agriculture and food security.
Date of Award24 Mar 2020
Original languageEnglish
Awarding Institution
  • The University of Bristol
SupervisorRichard P Evershed (Supervisor)

Cite this