Functional screening and single-cell cultivation of marine CO₂-fixing bacteria via flow-mode Raman-activated cell...

Most marine CO₂-fixing microorganisms remain uncultivated due to strong culture bias and low throughput of conventional approaches, which fail to link in situ function with isolated strains and render slow-growing or low-abundance taxa virtually inaccessible. This study presents an integrated single-cell workflow that incorporates ¹³C-NaHCO₃ labeling, high-throughput flow-mode Raman-activated cell sorting (RACS) and microwell cultivation for the isolation of active CO₂-fixing bacteria from the Yellow Sea. Function-guided sorting was achieved by monitoring the ¹³C-induced Raman shifts of carotenoids (ν₁ band: ∼1507 to ∼ 1503.78 cm^-1 at 24 h). Genomic and physiological analyses identified Paraburkholderia aromaticivorans FR-4 as a novel facultative chemoautotrophic nitrite-oxidizing bacterium (NOB). Its genome encodes complete nitrite oxidation and Calvin cycle pathways, together with key carbon acquisition genes (carbonic anhydrase, bicarbonate transporter). FR-4 grows autotrophically using NO₂^- as the electron donor and CO₂/HCO₃^- as the carbon source, confirming its ability to couple nitrite oxidation with carbon fixation, while retaining metabolic flexibility for heterotrophic growth. By directly linking in situ carbon-fixing activity, genotype, and phenotype, this workflow provides a targeted strategy for exploring elusive marine CO₂-fixing bacteria and overcomes critical limitations of conventional cultivation.