Enriching digestates with N2O reducing bacteria for reeduced climate forcing

N2O released from fertilized agricultural soils contributes to ˜1/3 of the total climate forcing by food production. Increasing the proportion of active N2O reducing bacteria in soil could quench these emissions but would be expensive as a standalone option. In this study we conceptualize integrating such a mitigation strategy with the established material pipeline of organic wastes to soil through anaerobic digestion using digestates as substrates for growth and as a vector for transfer of N2O reducing bacteria to soil. Metagenomic genome reconstructions showed that full fledged denitrifyers closely related to Dechloromonas sp. dominated the nitrous oxide reducing community in the enriched digestates. Three digestate-derived N2O reducing bacteria originating from the enrichments were obtained through isolation and their genomes sequenced and deposited under this Study acc. PREJEB41283. Among these an axenic culture of a fully-fledged denitrifying Azonexus sp. which matched the MAG of the dominant N2O reducer in the enrichment. This Azonexus sp. produced a disproportionately high complement of N2O reductase relative to other enriched organisms and maintained activity in the soil. Characterization of the isolated organisms' denitrifying phenotypes, and subsequent fertilization of soil with digestate enriched with the isolated strains identified interesting regulatory properties favorable for an inoculant in such applications. These include more efficient electron transfer to N2O reductase as opposed to nitrate reductase (NAP), as well as bet-hedging strategies in response to the oxic/anoxic transition whereby N2O reduction is favored over NO3 reduction by the majority of cells in a clonal population.