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

De siste rapportene fra FN:s Klimapanel IPCC (Intergovernmental Panel on Climate Change) beskriver en stadig økning i lystgassutslipp, en av de verste klimagassene som i tillegg ødelegger ozonlaget i stratosfæren. Denitrifiserende bakterier står for mesteparten av de globale lystgassutslippene, drevet av overforbruk av kunstgjødsel. Denitrifikasjon er en mikrobiell prosess hvor organismene respirerer nitrat og andre nitrogenoksyder når det blir mangel på oksygen. Mange denitrifiserende bakterier kan både danne og konsumere lystgass, og mengden som slippes ut avhenger av deres regulatoriske biologi. Dette vet man mye om fra laboratorieforsøk under optimale forhold, mens man vet veldig lite om denne reguleringen hos bakterier i naturlige miljøer hvor de sulter for det meste av tiden, og må takle fluktuerende oksygenkonsentrasjoner som risikerer å skade enzymene. Dette skal vi undersøke i STARVOX. Resultatene kommer å øke grunnleggende forståelse av den biogeokjemiske nitrogensyklusen og være til stor nytte i vår pågående utvikling av innovative metoder for å minke lystgassemisjoner, som bygger på at lystgass-reduserende bakterier spres i landbruksjord via biofertilisering.

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.