A novel dual enrichment strategy provides soil- and digestate- competent N2O-respiring bacteria for mitigating climate forcing in agriculture

Manipulating soil metabolism by heavy inoculation with microbes is deemed realistic if waste from anaerobic digestion (digestate) is utilized as substrate and vector, but requires organisms that can grow both in digestate and soil (=generalist). We designed a strategy to enrich and isolate such generalist N2O-respiring bacteria (NRB) in soil and digestate, to provide inoculum for reducing N2O-emissions from agricultural soil. Sequential anaerobic enrichment cultures were provided with a small dose of O2 and unlimited N2O, alternating between sterilized digestate and soil as substrates. The cultures were monitored for gas kinetics and community composition (16SrDNA), and cluster-analysis identified generalist-OTUs which became dominant, digestate/soil-specialists which did not, and a majority that were diluted out. Several NRBs circumscribed by generalist-OTUs were isolated, genome sequenced to screen for catabolic capacity, and phenotyped, to assess their capacity as N2O-sinks in soil. The two isolates Cloacibacterium sp., carrying only N2O-reductase (Clade-II) and Pseudomonas sp., with full-fledged denitrification-pathway, were both very effective N2O-sinks in soil, with Pseudomonas sp., showing a long-lasting sink effect, suggesting better survival in soil. This avenue for utilizing waste to bioengineer the soil microbiota holds promise to effectively combat N2O-emissions but could also be utilized for enhancing other metabolic functions in soil. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY