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Revealing effects of starvation and oxidative stress on denitrifying bacteria: a basis for novel N2O mitigation and industrial applications

Alternative title: Innvirkning av sult og oksidativ stress på denitrifiserende bakterier: basis for innovativ lystgassreduksjon og industrielle applikasjoner

Awarded: NOK 12.0 mill.

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Project Period:

2021 - 2025

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The last reports from IPCC (the Intergovernmental Panel on Climate Change) describe steadily increasing emission of N2O, a potent greenhouse gas and in addition the main destructor of the stratospheric ozone. Denitrifying bacteria are responsible for the main part of the global N2O emissions from soils, primarily driven by excessive use of fertilizers. Denitrification is a microbial process where the organisms respire nitrate and other nitrogen oxides when oxygen becomes scarce. Many denitrifying bacteria are able to both produce and consume N2O, and the amounts that are emitted depend on their regulatory biology. Although much is known about this regulation, the current knowledge is mostly based on laboratory studies of organisms growing under optimal conditions. Surprisingly little is known about the regulation of denitrification in bacteria living under natural conditions, where they starve most of the time and are exposed to fluctuating oxygen concentrations that may harm the enzymes. This is the focus of STARVOX. The results will add new, basic knowledge to our understanding of the biogeochemical nitrogen cycle and will strengthen our ongoing development of novel methods for N2O mitigation where N2O reducing bacteria will be spread on farmland via biofertilization.

Denitrification is a key microbial metabolism in the global N-cycle. Microbes use this pathway to sustain respiration in anoxia, reducing NO3- to N2 via the free intermediates NO2-, NO and N2O. Depending on their regulation of the pathway, denitrifying organisms are either strong sinks or sources of the climate gas N2O, and this regulation is the core issue of STARVOX. Much of what is known about this regulation is biased because experiments were conducted under optimal conditions, and focused only on a single transition from oxic to anoxic respiration, while bacteria in soils and sediments are starved and faced with frequent fluctuations in oxygen availability. STARVOX will study the effect of these stressors on the regulation of denitrification and the persistence of denitrification enzymes under oxic conditions. We will study model strains and newly isolated strains as well as complex soil microbial communities. Our tools include time-resolved gas kinetics, bioassays, absolute quantification of enzyme abundance by MS; and metagenomics/ transcriptomics/proteomics to identify active organisms and quantify reductases in complex systems. We will implement a CRISPRi technology which makes it possible switch off all de novo synthesis of the denitrification reductases of interest. The research will provide new and more nature-relevant knowledge about regulation of denitrification, the potential utilization of H2 to relieve starvation, and the effects of oxygen and reactive oxygen species on denitrification reductases. The primary aim is to understand regulation of denitrification in natural environments, and strengthen ongoing attempts to bioengineer soil communities to reduce the emissions of N2O, but the project will also provide bench mark enzyme parameters (kcat and km in vivo) for future predictive modelling (systems biology), and strengthen the endeavors to develop efficient single cell protein production by anaerobic respiration.