Metabolic redirection and capacity expansion in heterotroph-reinforced microalgae-methanotroph systems under high ammonia stress.

The microalgae-methanotroph binary system can capture dissolved methane and nutrients from anaerobic digestion (AD) effluent in situ, but its nitrogen removal relies solely on cellular assimilation, which is capacity-limited and sensitive to high ammonia. Here, a heterotroph-reinforced microecosystem was enriched on the organic carbon already present in AD effluent to probe its stress response and carbon-nitrogen flows. Elevated ammonia inhibited soluble methane monooxygenase and constrained microalgal assimilation, confining the binary system to a narrow nitrogen-removal range of 33.6-38.7 mg/L. In contrast, the heterotroph-reinforced system reached 81.9 mg/L total nitrogen removal at 150 mg/L NH4+-N, more than double the binary peak. Driven by that effluent-derived organic carbon, heterotrophs such as Ralstonia became dominant and, by the nitrogen mass balance, revealed a nitrogen flux that assimilation cannot account for, amounting to 49.8 mg/L or 61 % of the nitrogen removed. A gaseous route mediated by heterotrophic nitrification and aerobic denitrification is the most plausible explanation. This carbon-driven sink relieved ammonia toxicity on core enzymes and pushed removal beyond the assimilation ceiling. Multi-trophic cooperation driven by the organic carbon already present in AD effluent therefore offers a carbon-self-sufficient route to stable carbon and nitrogen co-capture under high ammonia loads.