Nitrous oxide respiring bacteria in biogas digestates for reduced agricultural emissions

Nitrous oxide (N₂O), a potent greenhouse gas, is largely emitted from agricultural soils due to microbial processes in the nitrogen cycle. While minimizing fertilizer nitrogen use and reducing meat consumption can help, these measures alone are insufficient to significantly reduce global N₂O emissions. More innovative approaches are needed, focusing on manipulating soil microbiomes, specifically the organisms involved in N₂O production and consumption. N₂O turnover in soil is complex, involving various metabolic pathways influenced by fluctuating environmental conditions. Heterotrophic denitrification is a primary N₂O source, while autotrophic ammonia oxidation can also contribute. Heterotrophic denitrifiers can be both N₂O producers and sinks, as N₂O is an intermediate in their nitrate-to-dinitrogen reduction. This process involves four enzymes (denitrification reductases), encoded by specific genes. Oxygen strongly represses denitrification. Many organisms have incomplete denitrification pathways, lacking one or more reductase genes, potentially leading to N₂O production or reduction. Organisms possessing only *nosZ* (encoding nitrous oxide reductase) are of particular interest as N₂O sinks. Even organisms with complete pathways can be N₂O sinks, depending on enzyme regulation. However, practical methods for utilizing N₂O-reducing organisms to reduce emissions have been lacking. A soil with strong N₂O-reducing capacity emits less N₂O, as shown in previous research. Large-scale production and distribution of N₂O-respiring bacteria is impractical, but integrating them into existing fertilization systems, like using nitrogen- and phosphate-rich digestate from biogas production, could be feasible. Anaerobic digestion (AD) is a widely used technology, with potential for significant expansion in the agricultural sector. This study investigates the use of digestates enriched with N₂O-respiring bacteria as a cost-effective, large-scale soil amendment to reduce N₂O emissions.

The introduction thoroughly reviews the existing literature on nitrous oxide emissions from agricultural soils, the microbial processes involved in N2O production and consumption (denitrification and ammonia oxidation), and the role of various denitrification enzymes and genes. It highlights the limitations of current mitigation strategies and the potential of utilizing N2O-reducing organisms. The authors cite relevant research demonstrating the importance of soil microbial communities in N2O emissions, the complexities of N2O turnover, the role of oxygen in regulating denitrification, and the potential benefits of inoculating soils with N2O-reducing bacteria. The review also covers existing research on biogas production and the potential for using digestate as a fertilizer, setting the stage for the proposed novel approach.

The study employed a multi-faceted approach combining enrichment culturing, metagenomics, metaproteomics, isolation and characterization of N₂O-respiring bacteria, and soil incubation experiments. Digestates from mesophilic (37°C) and thermophilic (52°C) anaerobic digesters treating municipal wastewater sludge were used. A robotized incubation system monitored gas kinetics (O₂, N₂, N₂O, NO, CO₂, CH₄) in parallel stirred batch cultures. Anaerobic enrichment cultures were established by incubating digestates under N₂O, with regular N₂O replenishment. Liquid samples were taken for metagenomic and metaproteomic analyses, VFA quantification, and 16S rRNA gene abundance determination. Metagenomic sequencing (Illumina HiSeq4000) was performed, and metagenome-assembled genomes (MAGs) were binned and phylogenetically placed. Proteomic analysis involved nanoLC-MS/MS. Isolation of N₂O-respiring bacteria was achieved by spreading diluted enrichment samples on agar plates and incubating them anaerobically with N₂O. Isolated strains were characterized through genome sequencing, Biolog Phenotype MicroArray analysis (to assess carbon source utilization), and denitrification phenotyping experiments monitoring gas kinetics during aerobic-to-anaerobic transitions. Finally, soil incubation experiments evaluated the effect of NRB-enriched digestates on N₂O emissions, comparing soils amended with different digestates (live, sterilized, NRB-enriched) under controlled conditions. Statistical analysis was used to evaluate the significance of results.

The study successfully enriched indigenous N₂O-respiring bacteria (NRB) in digestates under anaerobic conditions with N₂O. Gas kinetics modeling indicated exponential growth of NRB, reaching high cell densities. Metagenomic and metaproteomic analyses revealed that these NRBs, mainly *Dechloromonas* sp. (MAG260), thrived by utilizing fermentation intermediates from the methanogenic consortium. The dominance of *Dechloromonas* sp. in the enrichment was confirmed by proteomics, showing it to be the major producer of N₂O reductase. Three NRB isolates (*Pseudomonas* sp., *Azospira* sp., and *Azonexus* sp.) were obtained; *Azonexus* sp. showed 98.2% ANI with MAG260. While these isolates possessed genes for a full denitrification pathway, their regulatory characteristics indicated a strong preference for N₂O reduction as a sink, confirmed through experimental validation. Soil incubation experiments using NRB-enriched digestates showed significant reductions in N₂O emissions compared to controls, particularly in near-neutral pH soils. The *Pseudomonas* sp. isolate showed the most consistent N2O reduction across different pH conditions. The study demonstrated the sustained effect of NRB-enriched digestate on reducing N2O emissions even after 70 hours of aerobic storage. Methane production was inhibited by N2O during enrichment but resumed when N2O was depleted, indicating a potential interplay between NRB and methanogens.

The findings demonstrate the feasibility of using biogas digestates as a cost-effective vector for delivering N₂O-reducing bacteria to soil, providing a scalable approach to mitigate N₂O emissions from agriculture. The successful enrichment and isolation of NRB, particularly *Dechloromonas* sp., highlight the potential for manipulating soil microbial communities to enhance N₂O reduction. The observation that the methanogenic consortium remains active during the enrichment process suggests a complex interaction between NRB and methanogens, with NRB competing for fermentation intermediates. The variable effects of different NRB isolates on N₂O emissions, particularly in relation to soil pH, underscore the importance of selecting suitable strains for optimal performance. This study's approach presents a valuable strategy for engineering soil microbiomes to enhance desired functions beyond N₂O mitigation, such as bioremediation or plant growth promotion.

This study successfully demonstrated the feasibility of using biogas digestates as a low-cost, scalable method for delivering N₂O-reducing bacteria to agricultural soils, thus mitigating N₂O emissions. The identification and characterization of key NRB, such as *Dechloromonas* sp., provide a foundation for selecting strains with optimal N₂O reduction capabilities. Future research should focus on optimizing NRB selection, incorporating field trials to validate the long-term impact of the approach, and exploring the potential for integrating additional beneficial functions into the digestate microbiome.

The study's findings were primarily based on laboratory experiments using controlled conditions. The long-term effects of this approach on soil microbial communities and N₂O emissions under field conditions require further investigation. The influence of various soil properties beyond pH on NRB activity and their persistence in the soil environment warrant additional study. The observed variable effects of different NRB isolates on N2O emissions highlight the need for further research on strain selection and optimization for sustainable long-term effects.