In Situ Single-Cell Raman Isotope Analysis Quantifies Cell-to-Cell Metabolic Heterogeneity within Individual Cable...

Cable bacteria are multicellular, electrogenic filamentous microorganisms that oxidize sulfide in anoxic sediments while reducing oxygen near the surface, driving crucial redox transformations in aquatic environments. However, the metabolic diversity and growth dynamics of individual cells within a single filament remain poorly understood. Here, we developed an in situ single-cell Raman microspectroscopy method coupled with dual stable isotope (2H/13C) labeling to quantify cell-to-cell metabolic heterogeneity within individual cable bacteria filaments in intact sediment microcosms. Quantitative analysis of the diagnostic Ni-S Raman bands enabled unambiguous identification of cable bacteria among coexisting non-cable filamentous microbes. Combined D2O and 13C tracers revealed prevalent metabolic activity but pronounced variation in anabolic growth: approximately 48% of suboxic-zone cells displayed concurrent 2H and 13C incorporation, indicative of biomass synthesis, whereas the remaining ∼52% incorporated only deuterium, reflecting maintenance metabolism. Oxic-zone cells exhibited consistent 2H incorporation but negligible 13C labeling, signifying active energy metabolism yet minimal anabolic growth. Suboxic cells within the same filaments showed heterogeneous growth, with actively growing cells directly interspersed with cells performing only maintenance metabolism. These results provide the first quantitative in situ evidence of metabolic heterogeneity among cells across oxic and suboxic zones as well as within the suboxic zone itself in individual cable bacteria filaments.