N2O-respiring bacteria in organic fertilizers, for reducing N2O emissions

Food production contributes to global warming, and the farmland’s emission of the climate gas N2O accounts for a large share. It has proven difficult to mitigate these N2O emissions beyond the marginal effects of optimizing fertilization. However, the NRBOW-team has developed an innovative approach that shows promise in achieving significant reductions in emissions. The concept is to use organic wastes, that are destined for soils anyway, as vectors for special bacteria (NNRB) that can reduce N2O to harmless nitrogen gas (N2). Suitable NNRB must be able to grow to high numbers in the organic wastes, and must also be metabolically active in soil, thus scavenging N2O that would otherwise be emitted to the atmosphere. A major challenge for the NRBOW-project is to isolate suitable NNRB for the variety of organic wastes that are applied to farmed soils, including organic fertilizer commodities that are expected to take an increasing share of the fertilizer market. These products, which are necessarily stabilized by airdrying (and pelleting), represent a special challenge because the NNRB must be able to survive the airdrying. The technology for selecting suitable NNRB is powerful in enriching and isolating N2O-reducing bacteria that grow fast in the organic wastes, and survive in soil, but most of the isolates carry the genes for N2O-production as well. These genes will be eliminated, by random mutations and by gene editing, using CRISPR, expecting the use of such gene edited bacteria to be legal in the near future. For the NNRB technology to become profitable for the farmers and the fertilizer industries, economic incentives must be implemented by the authorities, and this will require verification of the effects on N2O emissions under realistic farming conditions. This is an important task for the NRBOW project.

The project will develop feasible technologies that effectively reduce the emission of the greenhouse gas N2O from farmland, thus reducing the climate footprints of food production. The starting point is our recent groundbreaking research showing significant impact on N2O emissions by using waste from biogas digesters (digestates) as a substrate and vector for specially selected Non-denitrifying N2O-Respiring Bacteria (NNRB). These organisms are effective N2O-sinks because they respire the gas (to harmless N2) but are unable to produce it. We will expand the technology to a range of agronomically relevant waste materials as vectors for NNRB and adapt it for production of dry organic fertilizers. We will isolate new NNRB strains using our Sequential Dual Substrate Enrichment Culturing (SDEC), alternating between soil and organic waste as substrate for isolation of NNRB, guided by the genomics of the enrichment cultures. To selectively enrich drought-tolerant NNRB we will modify the SDEC technology by including heat treatment (selecting for endospore formers) and drought-treatment (selecting for drought tolerance) after each culturing. Our experience so far is that the majority of bacteria enriched by SDEC are not NNRB as they have the ability not only to consume N2O but to also produce it through denitrification. To make them useful NNRB, we will knock out their gene(s) coding for denitrification by gene editing (CRISPR) and random mutations. We will adopt a tiered approach to identify the most effective NNRB, starting with short term laboratory incubations, followed by chamber-incubations for the most promising, and finally testing under realistic agronomic conditions in field plot experiments. Our goal is to develop robust NNR-technology for the most relevant organic wastes, to act as strong sinks for N2O which persist in soil over at least a growth season. Finally, we will develop a sustainable packaging for the products to secure a desired shelf-life.