Unveiling bisphenol A-degrading bacteria in activated sludge through plating and 13C isotope labeled single-cell Raman spectroscopy.

Bacteria play a crucial role in biodegradation of recalcitrant endocrine-disrupting compounds (EDCs), such as bisphenol A (BPA). However, in-situ identification of BPA-degrading bacteria remains technically challenging. Herein, we employed a conventional plating isolation (PI) and a new single cell Raman spectroscopy coupled with stable isotope probing (Raman-SIP) approach to enrich and identify BPA-degrading bacteria from activated sludge (AS). AS-inhabitant bacteria were exposed to either 12C-BPA or 13C-BPA as sole carbon source over three consecutive generations. While PI relies on colony proliferation on agar media, Raman-SIP enables identification of in situ BPA-degrading bacteria in a culture-independent way. The results showed that BPA dissipation correlated with increased bacterial growth. The uptake of 13C-BPA by single cells was verified by Raman spectra, suggesting occurrence of both metabolic and biosynthesis processes. This direct tracking of the fate of 13C-BPA within cells highlights the advantages of Raman-SIP over PI technique. PI isolated four BPA-degrading bacterial strains belonging to Comamonas, Pseudomonas, and Herbaspirillum genera. Meanwhile, Raman-SIP identified labeled cells belonging to Comamonas and Pseudomonas genera. Metagenomics of labeled cells revealed the presence of fifteen genes associated with benzene ring cleavage. This study provides a novel Raman-SIP approach for detecting and characterizing BPA-assimilating bacteria at a single cell level.