Strain specificity and inoculation sequence shape quorum sensing-based biofilm bioaugmentation: Roles of resource...

Quorum sensing (QS)-based bioaugmentation is promising for accelerating biofilm establishment and bioreactor start-up. However, its effectiveness is determined by biotic interactions between exogenous QS bacteria and indigenous microorganisms, which is shaped by the ecological roles of strain-specific traits and inoculation sequence. This study systematically investigated strain-specific biotic interactions between four QS strains (wastewater-derived strain X3, AM7 and AM9, and Pseudomonas aeruginosa PAO1) with forty non-QS strains under three inoculation strategies with different inoculation time (i.e., one-step mixing, pre-inoculation and post-addition). Biotic interactions associated with resource competition and quorum quenching (QQ) activity were distinguished using model strain Escherichia coli with strong substrate uptake capability and Rhodococcus sp. BH4 with QQ activity. The QS-mediated biofilm regulation was jointly shaped by strain-specific characteristics and inoculation sequence. The pre-inoculation strategy enhanced biofilm induction by AM9 and X3, alleviated negative interactions for AM7, but intensified inhibition by PAO1. By contrast, the post-addition strategy strengthened biofilm reduction by AM9, X3 and AM7, while it enhanced biofilm induction by PAO1. Resource competition alone was insufficient to suppress QS-mediated biofilm induction. However, QQ activity induced by BH4 significantly reduced extracellular polymeric substance (EPS) production and QS-mediated biofilm induction, particularly for X3 and AM7, distinguishing as the major ecological constraint. Notably, AM9 with strong QS activity maintained biofilm-promoting capability. This study provides ecological principles for QS strain selection and inoculation optimization, facilitating the improvement of QS-based bioaugmentation for rapid start-up of biofilm-based processes.