Microbial ecological governance of 1,4-dioxane biodegradation under staged nutrient stimulation

Biostimulation through nutrient amendment is a widely used in situ groundwater remediation strategy, yet how nutrient dose and sequence govern microbial community assembly and long-term biodegradation performance remains poorly understood. This study investigated the microbial ecological mechanisms underlying staged ammonium stimulation for 1,4-dioxane biodegradation in the presence of chlorinated solvent co-contaminants (trichloroethylene, 1,1-dichloroethylene, and vinyl chloride). Three treatment trains were compared in soil-groundwater microcosms over 45 days: a low-high-none nutrient sequence (Train 1), a high-none nutrient sequence (Train 2), and a high-none nutrient sequence with bioaugmentation of Mycobacterium austroafricanum JOB-5 (Train 3). Train 1 and Train 3 achieved the highest cumulative 1,4-dioxane removal (∼69% and ∼66%, respectively), while Train 2 remained less effective (∼43%). Community assembly analysis revealed that Train 1 and Train 3 were dominated by deterministic heterogeneous selection, whereas Train 2 reverted toward stochastic drift after nutrient withdrawal. The staged low-high-none nutrient regime in Train 1 functioned as an ecological ratchet, sequentially filtering oligotrophic taxa, enriching copiotrophic degraders (Pseudomonas, Bradyrhizobium, Methylocystis), and guiding the community into a stable, high-function basin of attraction. In Train 3, the bioaugmented strain acted as a keystone catalyst, facilitating cross-feeding and enriching synergistic consortia including methanotrophs (Methylocaldum) and sphingomonads. Ecological dynamic regime analysis confirmed that treatment trains occupied distinct compositional basins while converging toward shared functional enzyme profiles. These findings demonstrate that staged nutrient stimulation, rather than dose alone, critically shapes microbial community trajectories and bioremediation durability in contaminated groundwater.