Ammonia-oxidizing bacteria promoted heterotrophic biofilm formation in carbon-limited environments: Threshold effect and mechanistic insights.
Abstract
Autotrophic ammonia-oxidizing bacteria (AOB) threaten the biological safety of drinking water distribution systems (DWDSs) by promoting heterotrophic biofilm formation. Given the substantial variability in AOB densities observed across different DWDSs, a quantitative understanding of this interaction is crucial for developing effective mitigation strategies, yet it remains poorly characterized. In this study, a simulated drinking water environment with indigenous bacteria was established to investigate the interaction between Nitrosomonas eutropha (AOB) and Sphingomonas sp. (heterotrophic bacteria) during biofilm formation. Results revealed a concentration threshold effect of AOB, where N. eutropha concentrations exceeding 10³ CFU/mL significantly enhanced Sphingomonas sp. biofilm formation. Quantitatively, approximately 9.26 CFU of N. eutropha supported the growth of 1 CFU of Sphingomonas sp. Mechanistically, high dry mass and lack of flagella in N. eutropha facilitated initial surface colonization, and concentrations above the threshold reduced surface hydrophobicity, thereby promoting Sphingomonas sp. attachment. AOB metabolites subsequently enhanced Sphingomonas sp. activity by reducing reactive oxygen species level, stabilizing biofilm formation. Non-targeted metabolomics analysis identified key metabolites linked to the AOB threshold effect, including pyroglutamic acid, S-adenosylmethionine, and phosphate. These metabolites supported Sphingomonas sp. by enhancing amino acid, energy metabolism, and antioxidation capacity, overcoming the growth barrier in carbon-limited environments. Among them, pyroglutamic acid serves as the primary promoter of Sphingomonas sp. biofilm formation. These findings provide novel insights into the regulatory role of AOB in heterotrophic biofilm formation, and suggest that maintaining AOB below a critical threshold may mitigate biofilm-associated risks under oligotrophic conditions.