Extended Data Fig. 9 |. Stable isotope probing of Mn(II)-oxidising co-culture measured using nanoSIMS.

a, Summary of stable isotope probing analysis of cells dissolved from Mn oxide nodules, either with paraformaldehyde fixation and FISH, or without (to avoid dilution with natural abundance isotopes). Cells of Species A and Species B were either identified by FISH or by elemental composition (Species B cells were observed to have higher ¹⁴N/¹⁵N ratios), and their isotopic compositions were obtained via nanoSIMS (n=the total number of cell regions of interest analysed in the nanoSIMS images). For FISH-nanoSIMS analyses, a total of 2 and 5 nanoSIMS images from single cultures incubated with either MnCO₃ or Mn¹³CO₃, respectively, were examined. For nanoSIMS analyses without paraformaldehyde fixation and FISH, a total of 3 and 17 nanoSIMS images from single cultures incubated with either MnCO₃ or Mn¹³CO₃, respectively, were examined. b–u, Individual secondary ion images from nanoSIMS showing incorporation of inorganic ¹³C and ¹⁵N into the cells of both species (dissolved from Mn oxide nodules grown in the presence of b–k, MnCO₃ and ¹⁵NO₃⁻, or l–u, Mn¹³CO₃ and ¹⁵NO₃⁻.), and Species B cells could have higher ¹⁴N content than Species A. Secondary ions 12C2 (mass 24 for ¹²C), 13C12C (mass 25 for ¹³C), 14N12C (mass 26 for ¹⁴N), 15N12C (mass 27 for ¹⁵N), 32S (mass 32 for ³²S) were simultaneously measured. The counts of the secondary ions are shown in brackets [minimum maximum] and displayed using the colour scale shown on the right of the images. b–f and l–p correspond to the top and bottom panels in Fig. 4, respectively. White arrows indicate Species B cells identified in FISH showing high ¹⁴N in nanoSIMS. v, NanoSIMS measurement of residual Mn associated with cells grown with Mn¹³CO₃ and ¹⁵NO₃⁻, after dissolving from Mn oxide nodules. Same nanoSIMS image area was analysed as l–p, except 55Mn16O (mass 71 for ⁵⁵Mn) was measured (n=1 nanoSIMS image) in addition to other secondary ions. Negligible amount of Mn was found in the biomass, indicating that any remaining Mn¹³CO₃ substrate had been completely dissolved away during sample preparation, and thus did not interfere with the ¹³C analyses.