Revival of Viable but Non- Culturable (VBNC) Soil Bacteria Using Micrococcus luteus Culture Supernatant with Resuscitation Promoting Factor

Abstract

A major limitation in environmental microbiology is the inability to culture a large fraction of microbial communities due to dormancy and entry into the viable-but-non-culturable (VBNC) state, leading to the great plate count anomaly. Resuscitation-promoting factors (Rpf) have emerged as promising tools to overcome this; however, their functional application in complex soil systems remains underexplored. In this study, supernatant-derived Rpf (SRpf) was extracted at optimal growth phase and functionally validated through protein profiling and peptidoglycan hydrolytic activity assays. The resuscitation potential of SRpf was evaluated using sterile soil bioaugmentation model containing VBNC Micrococcus luteus, and non-sterile soil models. Recovery was assessed using culturability-based methods on R2A and SA media alongside metabolic activity assays to distinguish physiological revival from artefactual growth. SRpf treatment resulted in a significant, time-dependent increase in colony-forming units and metabolic activity across all model systems compared to controls, confirming genuine cellular reactivation. Notably, combined application of SRpf with sublethal concentrations of lysozyme revealed a synergistic enhancement of resuscitation efficiency, highlighting the role of controlled cell wall modulation in microbial revival. Five isolates were obtained as the resuscitated soil bacteria and their identification was carried out.