Nitrous oxide respiring bacteria in biogas digestates for reduced agricultural emissions

Nitrous oxide (N₂O) is a potent greenhouse gas, with agricultural soils contributing about one-third of anthropogenic emissions. Heterotrophic denitrification is a major source of soil N₂O, but denitrifiers also act as sinks via nitrous oxide reductase (NosZ). Many organisms have truncated denitrification pathways, and organisms harboring nosZ alone (non-denitrifying N₂O-reducers) have been proposed as N₂O sinks. However, practical, scalable strategies to enhance soil N₂O-reducing capacity are lacking. The study hypothesizes that anaerobic digestate from biogas production can serve as both a substrate and vector to enrich and deliver N₂O-respiring bacteria (NRB) to soils via existing fertilization practices. The objectives are to (1) selectively enrich digestate-adapted NRB under N₂O, (2) isolate and characterize key NRB and their regulatory traits, and (3) test whether NRB-enriched digestates reduce soil N₂O emissions under controlled conditions.

Background work highlights: (1) Global and agricultural contributions to N₂O emissions and the limitations of agronomic measures alone to reduce emissions; (2) Denitrification pathways (nar/nap, nir, nor, nosZ) and regulation by oxygen, with emphasis on organisms with truncated pathways acting as N₂O sources or sinks; (3) Prior demonstrations that inoculating soils with nosZ-only bacteria reduces N₂O emissions but is impractical at scale; (4) Anaerobic digestion (AD) as a growing technology producing nutrient-rich digestates potentially suitable for microbiome engineering; (5) Evidence that pH impairs synthesis of functional Nos but not its activity once formed; (6) Mixed findings on the comparative kinetics of NosZ clade I vs clade II organisms, suggesting both ecological and kinetic determinants of N₂O reduction efficiency.

Digestate sources: Mesophilic (37 °C) and thermophilic (52 °C) anaerobic digesters processing precipitated municipal wastewater sludge (organic matter 5.6% w/w). Digestates contained ~2.1% organic matter, 1.8–1.9 g NH₄⁺-N L⁻¹, 16–32 meq VFA L⁻¹, pH 7.6–8.2. Digestates were transported and used within 3–6 h. Gas-kinetics platform: Robotized incubation system with 30 parallel stirred 120 mL serum vial batches monitored for headspace O₂, N₂, N₂O, NO, CO₂, CH₄. Sampling losses corrected to compute gas production/consumption rates. Enrichment culturing: 50 mL digestate per vial, headspace flushed with He and supplemented with N₂O; N₂O replenished upon depletion. Cultures stirred (300 rpm) at 20 °C. Liquid samples taken for metagenomics, metaproteomics, VFA and 16S rRNA gene quantification. Growth of NRB modeled from N₂O-to-N₂ kinetics. Comparative incubations: Parallel digestate incubations with O₂ or NO₃⁻ as electron acceptors to assess impacts on methanogenesis and to estimate abundances of O₂-, NO₃⁻-, and N₂O-respiring populations. Meta-omics: Illumina HiSeq4000 sequencing; assembly and binning yielded 278 MAGs (149 with completeness >50% and contamination <20%). Proteins extracted and analyzed by nanoLC-MS/MS; metaproteome searches against MAGs database. NosZ and other denitrification proteins tracked over time. Isolation of NRB: Enrichment culture dilutions plated on varied media, incubated anaerobically with N₂O. Colonies re-streaked and cultured aerobically; 16S sequencing. Three isolates selected: PS (Pseudomonas sp.), AS (Azospira sp.), AN (Azonexus sp.). Genome sequencing and phenotyping of isolates: Whole-genome sequencing; ANI comparisons to MAGs; Biolog PM1/PM2 carbon utilization profiles; denitrification phenotyping via batch cultures tracking O₂, NO₂⁻, NO, N₂O, N₂ kinetics through oxic-anoxic transition. Proteomic quantification of reductases to assess bet-hedging (distribution of Nap/Nar, Nir, Nos across cells). Soil mitigation experiments: Microcosms (10 g soil) amended with 3 mL digestate treatments: (i) live digestate, (ii) digestate heated 70 °C for 2 h, (iii) autoclaved digestate with aerobically grown isolates (PS, AS, AN), (iv) PS-grown digestate subsequently heated (PS_70 °C control), and (v) digestate enriched anaerobically with N₂O reducers. Incubated with He + 0.5% O₂, monitoring O₂, NO, N₂O, N₂; two soils differing in pH (pHCaCl2 5.5 and 6.6). Calculated N₂O-index (IN2O) up to 40% and 100% recovery of NO₃⁻-N as N gases. Additional tests assessed persistence after 70 h aerobic storage.

- Indigenous NRB in digestates were selectively enriched under N₂O at 20 °C. Modeled growing population increased from ~2.5×10³ to 1.6×10⁸ cells mL⁻¹ by 110 h, reaching ~3×10⁹ cells mL⁻¹ by 215 h; specific growth rate µ ≈ 0.1 h⁻¹. Cell-specific electron flow during exponential growth was ~5 fmol e⁻ cell⁻¹ h⁻¹, declining as populations increased. - Methanogenesis was inhibited by N₂O and recovered during N₂O depletion; O₂ partially inhibited methanogenesis and NO₃⁻ fully inhibited it. Early enrichment phases showed transient accumulation of H₂ and VFAs consistent with methanogenesis inhibition and subsequent consumption by NRB. - Meta-omics identified 149 quality MAGs; most remained stable, with MAG260 (Dechloromonas-like) and MAG268 increasing markedly. NosZ proteins detected from five nosZ-containing MAGs; only Nos was detected among denitrification enzymes during enrichment. MAG260 accounted for ~92% of detectable Nos at final time point and encoded Nap (not Nar), suggesting preferential electron flow to N₂O over NO₃⁻. - Three isolates obtained: PS (Pseudomonas; nosZ clade I; Nar), AN (Azonexus; nosZ clade II; Nap; 98.2% ANI to MAG260), AS (Azospira; nosZ clade II; Nap). PS had broad carbon utilization; AN and AS specialized on small VFAs and TCA intermediates. - Denitrification phenotypes indicated bet-hedging: PS resembled Paracoccus denitrificans with Nos expression across cells and Nir in a minority, maintaining very low N₂O even with NO₂⁻ present. AN and AS preferentially reduced N₂O over NO₃⁻ (linked to Nap) but not over NO₂⁻; proteomics of AN showed high Nos/Nap ratio (~25) early in denitrification. - Soil microcosms: Digestate enriched with NRB under N₂O produced extremely low IN2O values in both acidic (pH 5.5) and near-neutral (pH 6.6) soils, indicating strong N₂O sink activity carried over from enrichment. Treatments with isolates (PS, AN, AS) significantly reduced IN2O versus the matched heat-killed control (PS_70 °C). PS yielded very low IN2O at both pH levels, indicating robust Nos synthesis at low pH; AN and AS showed higher IN2O in acidic soil, consistent with impaired Nos synthesis at low pH. - Methane monitoring in soil showed CH₄ production when live or N₂O-enriched digestates were used (methanogens intact), but no CH₄ after heat treatment or autoclaving, supporting sanitation to prevent CH₄ emissions. - Effects persisted after 70 h aerobic storage, indicating robustness. Statistical analyses showed significant effects of digestate treatment, soil pH, and their interaction on IN2O (p < 0.001).

The study demonstrates a feasible, scalable route to deploy N₂O-respiring bacteria to soils by leveraging anaerobic digestates as both growth substrates and delivery vectors. Under N₂O, digestate communities supplied fermentation intermediates (VFAs, H₂, acetate) that enabled rapid growth of NRB, while N₂O inhibited methanogenesis and redirected electron donors to NRB. Dominance of a Dechloromonas-like population (MAG260/AN) with Nap suggests a metabolic advantage in prioritizing N₂O reduction over NO₃⁻. Isolate phenotypes revealed regulatory strategies (bet-hedging) that confer strong N₂O sink capacity, particularly in PS, which maintained low N₂O even with NO₂⁻ present. Soil experiments confirmed that NRB-enriched digestates and digestates inoculated with select isolates reduce N₂O emissions across soil pH, with PS performing robustly under acidic conditions. The approach integrates seamlessly with biogas production workflows and could markedly enhance soil N₂O-reducing capacity during denitrification hot moments. Practical considerations include sanitizing digestates to prevent CH₄ emissions and selecting organisms with broad catabolic capacities and pH tolerance for longer-term soil survival and function.

Digestates from anaerobic digestion can be transformed into effective agents for mitigating soil N₂O emissions by enriching or inoculating them with N₂O-respiring bacteria. Meta-omics and gas kinetics showed that NRB exploit fermentation intermediates in digestates under N₂O and that select taxa (e.g., Dechloromonas-like, Pseudomonas sp.) can act as strong N₂O sinks due to metabolic and regulatory traits. Soil microcosm tests confirmed significant reductions in N₂O emission metrics, including under acidic conditions. The strategy is low-cost and scalable as an add-on to existing biogas infrastructure. Future work should refine enrichment protocols, perform long-term storage and field trials, and select consortia with broad substrate utilization, pH resilience, and potential co-benefits such as plant growth promotion or pollutant degradation.

- Field validation is lacking; results are from lab-scale microcosms. - Soil pH can impair de novo Nos synthesis; isolates AN and AS were less effective in acidic soil unless Nos was pre-expressed. - Live digestates can introduce methanogens, leading to CH₄ emissions unless sanitized; sanitation steps are necessary and may affect microbial survival. - nosZ-only organisms did not enrich under the conditions used; enriched NRB carried complete denitrification pathways, potentially producing N₂O under some conditions (e.g., with NO₂⁻ for AN/AS). - Digestate microbial compositions vary with feedstock and AD configuration, leading to variable baseline N₂O effects and competition with indigenous O₂/NO₃⁻ respirers when growing isolates. - Enrichment under N₂O at scale could be resource-intensive; the proposed alternative (aerobic growth of isolates in sanitized digestate) requires careful selection to ensure soil competitiveness and persistence.