Heterotrophic ammonium assimilation (HAA) represents an emerging approach to conventional biological processes by enabling simultaneous removal and recovery of reactive nitrogen and organic matter from municipal wastewater through assimilation as microbial biomass, with the potential advantage of limiting dissimilatory pathways associated with gaseous nitrogen loss and N2O emissions. This study investigated strategies to maximize HAA by activated sludge through the manipulation of process parameters such as dissolved oxygen (DO) concentration and C:N ratio, using volatile fatty acids (VFAs) as external carbon source. Batch experiments were conducted under strict aerobic conditions to determine NH4+-N and COD removal via HAA. At a DO > 6 mg O2/L, the addition of VFAs yielded NH4+ assimilation efficiencies up to 67 and 99 % for C:N 10 and 15, respectively. Based on these results, the process long-term feasibility was evaluated with acetate as carbon source in a bench-scale continuously stirred tank reactor (CSTR), operated for 141 days. At C:N ratio 15 and an average DO of 2.3 ± 1.2 mg O2/ L, nearly complete NH4+-N assimilation (99.1 ± 0.3 %) was achieved with effluent inorganic nitrogen (NH4+-N, NO2–-N, NO3–-N) and soluble COD concentrations of 1.1 ± 0.4 mg N/L and 53.4 ± 19.5 mg/L, respectively. Microbial community analysis revealed the enrichment of Comamonas, (up to 88.6 %) suggesting a potential role in nitrogen assimilation. Overall, these results demonstrate that effective NH4+ removal can be achieved through HAA by regulating process parameters and supplying VFAs, laying the foundation for circular nutrient management in municipal wastewater treatment.
Industrial dye discharge into aquatic ecosystems poses persistent environmental and health risks, requiring sustainable and scalable remediation strategies. Laccases are versatile multicopper oxidases capable of degrading a wide spectrum of intractable dyes; however, their industrial application is often constrained by production costs and downstream processing requirements. Harnessing phototrophic microorganisms as self-sustaining enzyme production platforms offers a promising alternative. In this study, we engineered the model cyanobacterium Synechocystis sp. PCC 6803 to secrete its native laccase, SLR1573, using two secretion tags: the type IV pilus-associated PilA signal peptide and a novel C-terminal repeat-in-toxin (RTX) fusion tag. Both designs reported extracellular laccase activity and decolorization of the anthraquinone dye Remazol Brilliant Blue R. The RTX-tagged laccase exhibited the highest extracellular activity (370 U/L) reported in cyanobacteria, and remarkable thermal stability, maintaining activity at temperatures up to 80 °C. Under acidic conditions, extracellular SLR1573 achieved 80 % decolorization of Remazol Brilliant Blue R within 1 h without the addition of redox mediators. This is the first report of mediator-free decolorization of an anthraquinone dye by a cyanobacterial laccase acting directly in the culture medium. The combination of phototrophic, low-cost cultivation and continuous extracellular enzyme production without biomass disruption could represent a promising step towards a scalable and economically attractive alternative for textile dye remediation. These findings demonstrate the feasibility of coupling enzyme secretion with dye decolorization in a single microbial chassis. This work advances cyanobacteria as cost-effective and sustainable biocatalytic hosts for industrial wastewater remediation.
