A dual enrichment strategy provides soil- and digestate-competent nitrous oxide-respiring bacteria for mitigating climate forcing in agriculture.

Manipulating soil metabolism through heavy inoculation with microbes is feasible if organic wastes can be utilized as the substrate for growth and vector as a fertilizer. This, however, requires organisms active in both digestate and soil (generalists). Here, we present a dual enrichment strategy to enrich and isolate such generalists among N2O-respiring bacteria (NRB) in soil and digestates, to be used as an inoculum for strengthening the N2O-reduction capacity of soils. The enrichment strategy utilizes sequential batch enrichment cultures alternating between sterilized digestate and soil as substrates, with each batch initiated with limited O2 and unlimited N2O. The cultures were monitored for gas kinetics and community composition. As predicted by a Lotka-Volterra competition model, cluster analysis identified generalist operational taxonomic units (OTUs) which became dominant, digestate/soil-specialists which did not, and a majority that were gradually diluted out. We isolated several NRBs circumscribed by generalist OTUs. Their denitrification genes and phenotypes predicted a variable capacity to act as N2O-sinks, while all genomes predicted broad catabolic capacity. The latter contrasts with previous attempts to enrich NRB by anaerobic incubation of unsterilized digestate only, which selected for organisms with a catabolic capacity limited to fermentation products. The two isolates with the most promising characteristics as N2O sinks were a Pseudomonas sp. with a full-fledged denitrification-pathway and a Cloacibacterium sp. carrying only N2O reductase (clade II), and soil experiments confirmed their capacity to reduce N2O-emissions from soil. The successful enrichment of NRB with broad catabolic spectra suggests that the concept of dual enrichment should also be applicable for enrichment of generalists with traits other than N2O reduction. IMPORTANCE N2O emissions from farmed soils are a major source of climate forcing. Here, denitrifying bacteria act as both source and sink for N2O, determined by regulatory traits or the absence of genes coding for the enzymes producing or reducing N2O. One approach to reducing emissions is to amend large numbers of N2O-reducing bacteria (NRB) to soil. This was shown to be feasible by growing NRB to high densities in organic wastes and then applying them as fertilizers. The effect on N2O emissions, however, was transient because the isolated NRBs were unsuited to soil. Here, we have developed an enrichment strategy selecting for organisms with generalist lifestyles, tolerant of rapid environmental changes. This was used to isolate robust NRBs that grow both in digestate and when amended to soils. This strategy opens an avenue for obtaining not just robust NRBs to reduce N2O emissions, but any organism destined for application to complex environments

Regulation of the emissions of the greenhouse gas nitrous oxide by the soybean endosymbiont Bradyrhizobium diazoefficiens

The greenhouse gas nitrous oxide (N2O) has strong potential to drive climate change. Soils are a major source of N2O, with microbial nitrification and denitrification being the primary processes involved in such emissions. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model microorganism to study denitrification, a process that depends on a set of reductases, encoded by the napEDABC, nirK, norCBQD, and nosRZDYFLX genes, which sequentially reduce nitrate (NO3) to nitrite (NO2), nitric oxide (NO), N2O, and dinitrogen (N2). In this bacterium, the regulatory network and environmental cues governing the expression of denitrification genes rely on the FixK2 and NnrR transcriptional regulators. To understand the role of FixK2 and NnrR proteins in N2O turnover, we monitored real-time kinetics of NO3, NO2, NO, N2O, N2, and oxygen (O2) in a fixK2 and nnrR mutant using a robotized incubation system. We confirmed that FixK2 and NnrR are regulatory determinants essential for NO3 respiration and N2O reduction. Furthermore, we demonstrated that N2O reduction by B. diazoefficiens is independent of canonical inducers of denitrification, such as the nitrogen oxide NO3, and it is negatively affected by acidic and alkaline conditions. These findings advance the understanding of how specific environmental conditions and two single regulators modulate N2O turnover in B. diazoefficiens.This research was funded by MCIN/AEI/10.13039/501100011033, “ERDF A way of making Europe”, grant AGL2017-85676-R to María J Delgado, grants AGL2015-63651-P and PID2020- 114330GB-100 to Socorro Mesa, and also Junta de Andalucía, grant P18-RT-1401 to María J Delgado and Socorro Mesa. EB was supported by a personal visiting researcher grant–IS-MOBIL (Oslo University, Norway) and the CSIC JAE-DOC Program co-financed by ESF