Ectoine modulates mixotrophic denitrification pathway partitioning to sustain stable nitrogen and phenol removal under hypersaline stress

Hypersaline wastewater containing phenolic compounds imposes coupled osmotic and cytotoxic stresses that severely disrupts biological treatment processes. While compatible solutes are known to enhance cellular osmoprotection, their capacity to regulate microbial metabolic, particularly the balance between autotrophic and heterotrophic denitrification pathways under combined salinity stress remain poorly understood. This study reveals that the compatible solute ectoine modulates pathway partitioning in mixotrophic denitrification systems, enabling efficient nitrogen and phenol removal under 4% salinity. The ectoine amended reactor maintained nitrogen removal above 95% and phenol degradation above 80%, whereas the unprotected control collapsed to 34% and 33% respectively. Multi-scale mechanistic investigations revealed a coordinated protection cascade. First, ectoine enhanced cellular resilience by suppressing reactive oxygen species (ROS) by 88.2%, maintaining ATP level and electron transport activity, thereby preserving bioenergetic integrity. Second, structural fortification was achieved through intensified extracellular polymeric substance (EPS) production. The protein-to-polysaccharide ratio increased from 0.70 to 1.51 creating a protective matrix that stabilized membrane permeability and preserved catalytic enzymes, with nitrate reductase and nitrite reductase activities increasing 2.16- and 2.93-fold. Third, metagenomic profiling revealed community reconfiguration, with selective enrichment of halotolerant heterotrophs (Halomonas, Marinobacter) to 49% relative abundance. Aromatic‑degradation genes (catA, benB) rose by 7‑ and 48‑fold, while nitrogen‑metabolism genes (nasA, norC) remained high representation. This restructuring reversed pathway contributions from 81% sulfur-autotrophic dominance to 82% heterotrophic dominance. Ectoine thus functions as a metabolic modulator that links cellular stress alleviation and community-level functional potential to pathway repartitioning, offering a feasible strategy for the biotreatment of saline phenolic wastewater.