Metabolic investigation of nitrogen fixation in the free-living plant associative bacteria, Klebsiella oxytoca

Abstract

Biologically available nitrogen is among the most limiting factors to crop growth. Currently this is overcome by the widespread use of inorganic fertilisers supplemented with ammonium, which comes at a great environmental cost. Biological nitrogen fixation, the conversion of atmospheric dinitrogen to ammonium by specialist microorganisms, can reduce reliance on this fertiliser and the impact on the climate. Here, the nitrogen fixing bacteria Klebsiella oxytoca is used as a model organism to investigate nitrogen fixation and its effect on global cell metabolism.

In the third chapter of this thesis, a series of sampling and liquid chromatography mass spectrometry methods were tested to investigate how to accurately assess the metabolic state of K. oxytoca under nitrogen fixing conditions. In Chapter 4 K. oxytoca were cultured under fixing and non-fixing conditions and Nuclear Magnetic Resonance (NMR) analysis was carried out on the supernatant. The results showed butan-2-3-diol, a metabolite known to improve draught resistance in plants, was released by cells fixing nitrogen.

In Chapter 5, analysis of K. oxytoca batch cultures using the methods developed in Chapter 3 revealed distinct intracellular metabolic profiles for nitrogen fixing and non-fixing cells. Recent work has shown that some species of nitrogen fixing bacteria can form biofilms and fix nitrogen in air. To the best of the authors knowledge, the work in Chapter 6 is the first reporting of K. oxytoca biofilms fixing nitrogen in air. Targeted LC-MS analysis found the metabolic profiles of these biofilms varied over time and between nitrogen fixing and non-fixing biofilms.

Collectively, the results presented in this thesis provide insights into the profound impact nitrogen fixation has on global cell metabolism in K. oxytoca. The results can be utilised to improve biotechnological solutions to the nitrogen crisis, helping to reduce the environmental impact of agricultural practices as we expand food production.