Jonassen, K. R. | bioRxiv Science

bioRxiv2021

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

microbiology↗

bioRxiv2020

Bacteria in biogas digestates for reduced climate forcing

Inoculating agricultural soils with N2O-respiring bacteria (NRB) can reduce N2O-emissions, but would be impractical as a standalone operation. Here we demonstrate that digestates obtained after biogas production are suitable substrates and vectors for NRB. We show that indigenous NRB in digestates grew to high abundance during anaerobic enrichment under N2O. Gas-kinetics and meta-omic analyses showed that these NRB's, recovered as metagenome-assembled genomes (MAGs), grew by harvesting fermentation intermediates of the methanogenic consortium. Three NRB's were isolated, one of which matched the recovered MAG of a Dechloromonas, deemed by proteomics to be the dominant producer of N2O-reductase in the enrichment. While the isolates harbored genes required for a full denitrification pathway and could thus both produce and sequester N2O, their regulatory traits predicted that they act as N2O sinks in soil, which was confirmed experimentally. The isolates were grown by aerobic respiration in digestates, and fertilization with these NRB-enriched digestates reduced N2O emissions from soil. Our use of digestates for low-cost and large-scale inoculation with NRB in soil can be taken as a blueprint for future applications of this powerful instrument to engineer the soil microbiome, be it for enhancing plant growth, bioremediation, or any other desirable function.

microbiology↗