Multi-omics evidence supporting the role of Halomonas enrichment contribution to high-salinity denitrification

High-salinity wastewater poses a significant challenge for nitrogen removal becauase microbial communities must adapt to extreme osmotic stress while maintaining their functional efficiency. Marine bacteria, naturally adapted to saline environments, offer a potential solution through the rapid enrichment of salt-tolerant and halophilic species. This study comparatively evaluated activated sludge (AS) and marine sludge (MS) as inocula for high-salinity denitrification systems. At a salinity of 10 g NaCl/L, both reactors achieved highly efficient denitrification by the 3rd day. However, the MS reactor outperformed the AS reactor in total nitrogen removal efficiency at 30 g NaCl/L, with this advantage becoming more pronounced at higher salinity levels. At low-to-moderate salinities, both reactors contained diverse dominant denitrifying bacteria, whereas Halomonas emerged as the primary genus at high salinity. In the AS reactor, sludge disintegration was observed, followed by a significant re-establishment of the microbial community, which was characterized by the predominance of Halomonas. In the MS reactor, Halomonas, which was present even at low salinity levels, demonstrated enhanced competitiveness under high-salinity conditions, resulting in significant enrichment. To adapt to low-to-moderate salinities, bacteria in the AS reactor accumulated compatible solutes such as glutamate and proline, whereas microorganisms in the MS reactor also employed primary sodium pumps, including oxaloacetate decarboxylase and Na⁺-pumping NADH: ubiquinone oxidoreductase, to extrude Na⁺. At high salinity, Halomonas adapted by synthesizing ectoine as an osmoprotectant. The use of MS as seed sludge resulted in faster nitrogen removal and more efficient enrichment of halophilic denitrifying bacteria. This study provides crucial theoretical support for implementing MS in high-salinity wastewater treatment systems.