Nitrogen-source responses of Spirogyra across defined media and authentic wastewater: Control-aware process accounting, ¹⁵N tracing, and transcriptomics

The percentage-removal metric cannot distinguish algal nutrient capture from abiotic loss or ensure that performance in a defined medium transfer to authentic wastewater. We addressed both limitations for the filamentous green alga Spirogyra sp. FACHB-1990 using a control-aware process inventory, ¹⁵N tracing, and time-matched transcriptomics. In a defined municipal-wastewater medium, NH₄⁺-N and NO₃⁻-N declined by 53% and 84%, respectively, over 96 h. A nitrogen inventory closing at 93.7% recovery attributed only approximately 38% of the filtered TDN decline to biomass N. Recovery-corrected NH₃ trapping accounted for approximately 16% of apparent ammonium loss, and a pH-trajectory-approximated algae-free control lost 46.5% of filtered TDP. Against a batch-matched synthetic reference, one authentic primary-effluent batch yielded 96 h TN transfer coefficients of 0.758 unfiltered (95% CI 0.616-0.896) and 0.921 following 0.22 µm filtration (0.762-1.097). Parallel ¹⁵N tracing at equal 13.5 mg N L⁻¹ pools gave excess-¹⁵N biomass-inventory ratios of 18.69 at 6 h (15.02-23.41) and 5.78 at 72 h (4.56-7.23). The labeled pools were designed to be nominally near-matched at approximately 10 atom %, but substrate-pool enrichment was not assayed. These ratios are therefore semi-quantitative cumulative contrasts rather than fluxes. Time-matched RNA-seq showed lower NRT2.1 abundance under NH₄⁺ than NO₃⁻ at 6 h (log₂FC = -3.90). Percentage-removal figures for filamentous-algae systems therefore require a closed nutrient inventory and an authentic-wastewater benchmark before they can inform process design.