A Dual Enrichment Strategy Provides Soil- and Digestate-Competent Nitrous Oxide-Respiring Bacteria for Mitigating...

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.