How the ammonium-to-nitrate ratio shaped system performance: The role of keystone taxa and network stability in microalgal-bacterial symbiotic system.

The microalgal-bacterial symbiotic system (MBSS) represents a promising sustainable technology for wastewater treatment, and its performance is tightly linked to operational parameters. Nevertheless, the systematic understanding of how the ammonium-to-nitrate ratio drives MBSS performance remains largely insufficient. Here MBSS was operated under a constant total nitrogen concentration with varying ammonium-to-nitrate ratios (4: 0, 3: 1, 2: 2, 1: 3, 0: 4). By integrating physicochemical analysis, amplicon sequencing, nitrogen cycling functional prediction, and multivariate statistics, it was found that the ammonium-to-nitrate ratio significantly regulated MBSS performance and bacterial community dynamics. Specifically, high-ammonium conditions (4: 0 ratio) promoted microalgal growth, total phosphorus (TP) removal, and chemical oxygen demand (COD) removal, whereas high-nitrate conditions (0: 4 ratio) enhanced total inorganic nitrogen (TIN) removal. Functional prediction revealed distinct nitrogen metabolic pathways under different conditions. Under high-ammonium conditions, bacteria preferentially employed nitrogen-removal pathways (e.g., amoA → hao → narB → nirS → norB → nosZ) that operated in concert with microalgae. Conversely, under high-nitrate conditions, bacteria competed with microalgae for nitrogen, employing pathways such as napA/nasA → nirK → norB. Co-occurrence network analysis indicated that high-ammonium conditions promoted a tightly connected, stable community. Furthermore, five keystone taxa (Pseudoxanthomonas, Cloacibacterium, Dyella, Paraburkholderia, and Pandoraea) were identified, collectively explaining 74.2% of system performance variation. Comprehensive assessment via microalgal-bacterial multifunctionality index, developed by combining key data on pollutant removal efficiency and bacterial community characteristics, confirmed that MBSS achieved optimal overall performance when ammonium served as the sole nitrogen source. This work clarified the regulatory mechanism of the ammonium-to-nitrate ratio in MBSS, providing a theoretical basis for optimizing nitrogen-containing wastewater treatment.