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

Food production contributes to global warming, and farmlands’ emission of nitrous oxide (N2O) accounts for a large share because it is a strong climate gas. It has proven difficult to mitigate N2O emission beyond the marginal effects of optimizing fertilization and agronomic management. However, the NRBOW-team has developed an innovative approach that has now been proven to reduce emissions substantially. The concept is to use organic wastes destined for soils, as substrate and vectors for selected bacteria (NNRB) that can reduce N2O to harmless nitrogen gas (N2). We developed this approach, using waste material from the biogas production in a waste water treatment plant. For this digestate to be used, we needed suitable NNRB which are able to grow aerobically to high numbers in the digestate, and which remain metabolically active in soil, thus scavenging N2O that would otherwise be emitted to the atmosphere. Thanks to a robust enrichment and isolation strategy, we isolated an NNRB which was very effective: Fertilization with digestate in which this organism had grown aerobically to high cell densities resulted in 50-95% lower emissions than in the control treatment. 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. Ongoing work to isolate such NNRB has revealed that most of the isolates carry the genes for N2O-production from nitrite (Nir). This is unfortunate, because it may weaken their capacity to reduce the emissions. We are working to eliminate Nir in selected strains, by random mutations and by CRISPR gene editing, hypothesizing that this will strengthen their capacity to reduce N2O emissions. For organic fertilizers with NNRB to become commercial products, they must be stabilized by air drying (and pelleting), and this represents a special challenge: we need to secure that a substantial (or at least predictable) fraction of the NNRB survive the airdrying. Routines for controlled drying have been developed using commercially available straw pellets as substrate and vector for the NNRB-strain Cloacibacter. Preliminary results indicate that all waste materials must be “sanitized” by heat treated to eliminate microbes that would otherwise outcompete our NNRB strains. 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.