A Synthetic Consortium of Comamonas Testosteroni and Acinetobacter Johnsonii Suppresses Nitrous Oxide Emission

Nitrous oxide (N₂O) released from wastewater treatment plants (WWTPs) is largely governed by the metabolism of dominant resident microorganisms, yet how interactions among co-occurring denitrifiers shape the net fate of N₂O remains poorly resolved. Here, a defined two-member consortium was assembled, comprising Acinetobacter johnsonii (AJ), a frequently recovered species in wastewater treatment plants, and Comamonas testosteroni (CT); its denitrification behaviour was dissected using time-resolved performance assays, a nitrogen mass balance, 16S rRNA community tracking, and quantitative proteomics. All cultures removed more than 99% of influent NO₃ ^--N (initial ~100 mg N/L) within 12 h. In monoculture, CT accumulated more N₂O (4.11 µg L^-1 N₂O-N) and less residual nitrite, whereas AJ showed the opposite pattern (0.28 µg L^-1 N₂O-N). The 1:1 consortium reconciled both liabilities, limiting nitrite build-up and holding N₂O-N to 0.37 µg L^-1, approximately one order of magnitude below CT. CT expressed a canonical periplasmic denitrification module (periplasmic nitrate reductase NapAB and norB, a cbb3-type cytochrome-c oxidase related to nitric oxide reductase) but lacked detectable nitrous oxide reductase (NosZ), consistent with partial denitrification and N₂O accumulation. AJ instead expressed an assimilatory nitrate-reduction repertoire (nitrate transporter F946_02194 and NAD(P)H-dependent nitrite reductase subunits F946_02190/02191), consistent with nitrate assimilation into biomass rather than complete denitrification to N₂. We propose thcD-derived electron cross-feeding from CT to AJ supports this assimilatory route, consistent with redirecting nitrogen away from gaseous intermediates. The consortium raised intracellular nitrogen to 25.3% of the initial nitrogen pool while minimising N₂O, suggesting a community-engineering principle for low-N₂O nitrogen removal.