From single-cell sorting to metabolic pathways of bisphenol A: Cultivation, genome, and biotransformation products

Unlocking the metabolic potential of unculturable microorganisms is important for understanding degradation of emerging contaminants. Single-cell Raman-activated sorting (scRACS) offers a breakthrough for isolating functional bacteria, but cultivating these cells after sorting remains a challenge, which blocks in-depth mechanistic investigations. In this study, a two-step cultivation strategy, including oligotrophic and eutrophic conditions, was developed to recover and proliferate single cell sorted by Raman for investigating biotransformation of bisphenol A (BPA). The cultivation of a BPA-degrading bacterial cell was achieved and identified as Comamonas testosteroni. Bioinformatic analysis showed that this bacterium encompasses genes encoding BPA-degrading enzymes, including monooxygenase, peroxidase, hydratase, and hydroxylase. The presence of tatA, secA, and gspA in the membrane secretion system indicates a transport system for BPA uptake and byproduct expulsion, which aligns with the detection of ¹³C₁₂-BPA inside the cell, proving that scRACS links bacterial genotypes and phenotypes. Ten transformation products (TPs) of BPA were identified using liquid chromatography-high-resolution mass spectrometry and gas chromatography-mass spectrometry in parallel with stable isotope tracing using gas chromatography-combustion isotope-ratio mass spectrometry. Mapping of genes, enzymes, and TPs enabled the proposal of four biodegradation pathways. This study provides foundational insights into the cultivation and inherited functions of bacterial cells using scRACS, highlighting its potential for elucidating the biodegradation of micropollutants, such as BPA.