Mutual symbiosis of electroactive bacteria (EAB) and denitrifier may be the key for solving the refractory carbon and residual nitrogen in wastewater treatment plant effluent. However, its application is hampered by unclear co-metabolic model and uncertain electron transfer. Here, we achieved 3–5 times increase in refractory carbon degradation, 40 % improvement in denitrification, and 36.0 % decrease in N₂O emission by co-culturing P. aeruginosa strain GWP-1 and G. sulfurreducens. Such an enhancement is obtained by both refractory carbon co-metabolism and interspecies electron transfer (IET) between GWP-1 and G. sulfurreducens. Importantly, IET was quantified via isotopic approach, which revealed that G. sulfureducens supplies more electrons to GWP-1 when the system was fed with cellulose (0.071 mM) than glucose (0.012 mM). This study demonstrates that the residual refractory carbon and nitrogen in treated wastewater could be further converted by mutual symbiosis of EAB and denitrifiers, which paves a synergic way for pollution and carbon reduction.

Background: Denitrification in wastewater treatment is severely limited under low-temperature and low-carbon (“dual-low”) conditions, hindering sustainable nitrogen removal. Biofilm systems, though energy-efficient, suffer from reduced efficiency in such environments due to impaired interspecies electron transfer (IET). Granular activated carbon (GAC), a conductive mediator, offers potential to enhance IET between electroactive microorganisms (EAMs) and denitrifiers, yet its role in dual-low systems remains underexplored. This study investigates GAC’s capacity to optimize biofilm functionality and mitigate greenhouse gas (GHG) emissions under these constraints. Results: Under dual-low conditions (4–6°C, C/N = 4), GAC increased denitrification efficiency by 19.4–21.9% and reduced N₂O emissions by 10.6–22.9%. Metatranscriptomes revealed upregulation of denitrifying genes (e.g., nosZ) and electron transport pathways (e.g., omcB in Geobacter). FISH/SEM confirmed GAC-driven coacervates of EAMs and denitrifiers, linked by nanowires, enhancing direct electron transfer. Microbial diversity decreased, but functional redundancy improved, with Pseudomonas fluorescens and Geobacter sulfurreducens dominating. TOC removal rose under low temperatures, indicating enhanced carbon utilization. Conclusions: GAC fosters synergistic EAM-denitrifier partnerships, enabling efficient denitrification and GHG mitigation in cold and carbon-limited (“dual-low”) biofilm systems, advancing sustainable wastewater management.

Constructed wetlands (CWs) have been proven to effectively immobilize plastic particles. However, little is known about the differences in the impact of varying sized plastic particles on nitrous oxide (N₂O) release, as well as the intervention mechanisms in CWs. Here, we built a lab-scale wetland model and introduced plastic particles of macro-, micro-, and nano-size at 100 μg/L for 370 days. The results showed that plastic particles of all sizes reduced N₂O release in CWs, with the degrees being the strongest for the Nano group, followed by Micro and Macro groups. Meanwhile, ¹⁵N- and ¹⁸O-tracing experiment revealed that the ammoxidation process contributed the most N₂O production, followed by denitrification. While for every N₂O-releasing process, the contributing proportion of N₂O in nitrification-coupled denitrification were most significantly cut down under exposing to macro-sized plastics and had an obvious increase in nitrifier denitrification in all groups, respectively. Finally, we revealed the three mechanism pathways of N₂O release reduction with macro-, micro-, and nano-sized plastics by impacting carbon assimilation (RubisCO activity), ammonia oxidation (gene amo abundance and HAO activity), and N-ion transmembrane and reductase activities, respectively. Our findings thus provided novel insights into the potential effects of plastic particles in CWs as an eco-technology.

Ammonia-oxidizing microorganisms (AOMs, archaea (AOA) and bacteria (AOB)) are primarily responsible for the ammoxidation in constructed wetlands (CWs). However, little is known about evaluating the response of AOA and AOB to engineered nanomaterials (ENMs) and quantifying the shift of their contribution to ammoxidation. Herein, we operated a series of CWs exposing to silver nanoparticles (Ag-NPs), single-walled carbon nanotubes (SWCNTs), and polystyrene nano-sized plastics (PS-NPs) with the wastewater-accumulating concentration of ENMs for 180 days. The results showed that the abundance of AOA amoA gene in situ was far lower than that of AOB, while the abundance ratio of AOA to AOB increased by 15 folds after 180-day experiment. Using DNA stable isotope probing (DNA-SIP) experiment, we found that the active AOB microbiota varied substantially but the AOA was more stable across different groups. Furthermore, the co-occurrence analysis proved that ENMs stress increased the negative coexistence pattern of AOA and AOB; predictive functional profiling showed that the ENMs enhanced the functional advantage of AOA by inhibiting AOB (mainly hydroxylamine oxidation process). Finally, the contribution of AOA increased under exposing to SWCNTs (18.35%), PS-NPs (24.92%), and Ag-NPs (32.14%) compared with control group (0.03%) for 180 days. Despite this, AOB was still the primary executant of ammoxidation in CWs. Overall, in our study, the differences in activities and contributions of AOMs were quantified in CWs, and a significantly negative coexistence relationship between AOA and AOB was revealed when exposed to emerging nanomaterials.