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

ABSTRACT

Nitrous oxide (N ₂ O) 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 N ₂ O 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 NO ₃ ⁻ –N (initial ~100 mg N/L) within 12 h. In monoculture, CT accumulated more N ₂ O (4.11 µg L ⁻¹ N ₂ O–N) and less residual nitrite, whereas AJ showed the opposite pattern (0.28 µg L ⁻¹ N ₂ O–N). The 1:1 consortium reconciled both liabilities, limiting nitrite build‐up and holding N ₂ O–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 N ₂ O 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 N ₂ . 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 N ₂ O, suggesting a community‐engineering principle for low‐N ₂ O nitrogen removal.