Molecular tracking of the in-situ bioaugmentation process of Pseudomonas sp. BO3-4 using key degradation genes.

In-situ bioaugmentation is often constrained by unclear environmental factors and the difficulty of tracking microbial activity, limiting the efficiency of groundwater remediation. This study developed a functional gene-based approach to track microbial degradation activity during field-scale bioaugmentation of toluene-contaminated groundwater. Based on transcriptomic analysis, todC1 (dioxygenase) and todE (ring-cleavage enzyme) were selected as molecular markers due to their critical roles in toluene degradation and high upregulation (>700-fold) in Pseudomonas sp. BO3-4. Following bioaugmentation with intermittent aeration, the absolute abundances of these genes increased to 2.3-2.5 log copies/mL, closely correlating with the proliferation of strain BO3-4 (relative abundance 67.93%-71.13%). Toluene removal reached 91.3%-94.6%, representing a five-to six-fold enhancement compared to the preceding aeration-only phase and its positive correlation with functional gene abundance validated these markers as robust, quantitative indicators of bioremediation efficacy. The accumulation and subsequent degradation of intermediates (benzyl alcohol, o-cresol, m-cresol) were observed, and microbial communities recovered as contamination diminished, indicating minimal ecological disturbance. Dissolved oxygen (DO) declined from 3.09 to 3.12 mg/L to 1.37-2.00 mg/L after bioaugmentation, and redundancy analysis identified DO as a key factor regulating degradation activity. This study validates functional gene markers and identifies DO as key factors for tracking and optimizing in-situ bioaugmentation in groundwater.