Community succession and protein enhancement in a mixed methanotroph-microalgae system with stepwise increase of ammonium loading - Inhibition and adaptation

The integration of methanotrophs and microalgae in coculture systems presents a promising approach for sustainable biogas valorization and single-cell protein (SCP) production, offering dual benefits of greenhouse gas mitigation and nutrient recovery from waste streams. However, the resilience and metabolic interplay of these consortia under ammonium stress, common in industrial wastewater, remain poorly understood, limiting their scalability. This study systematically investigated the performance of a microalgae-methanotroph consortium under stepwise ammonium concentrations (130, 200 and 260 mg NH₄⁺-N/L). The system demonstrated remarkable acclimation, achieving stable biogas conversion with 95.8 ± 5.3 mg CO₂-C/(L·day) and 109.0 ± 11.4 mg CH₄-C/(L·day) even at highest ammonium concentration. The SCP content increased from 32 % to over 52 % of cell dry weight with yield peaking at 90 mg/(L·day) under 200 mg N/L. A microbial community shift from Methylosinus to ammonia-tolerant Methylococcus dominance underpinned functional stability. Metagenomic analyses revealed ammonium-driven metabolic adaptations: extracellular substance secretion reprograms under stress, nitrogen assimilation was enhanced via glutamine synthetase, and antioxidant defenses were activated. Network analysis highlighted intensified competition (31 % negative correlations) under stress, yet key synergies within coculture system sustained carbon and nitrogen metabolism. These findings resolve knowledge gaps in ammonium-stressed consortia dynamics and provide insights for engineering systems to advancing the circular bioeconomy.