separate 13C− from the 12CH− and 12C15N− from 13C14N−. Samples were measured using a 1–2 pA Cs+ primary beam that was rastered over 25 × 25 µm field of a 256 × 256 pixels with a counting time of 5 ms per pixel. Recorded images and data were processed using CAMECA WinImage software and OpenMIMS plugin42 in ImageJ43 distribution of Fiji44. Different scans of each image were aligned to correct image drift during acquisition. Final images were created by adding the secondary ion counts of each recorded secondary ion from each pixel over all scans. Intracellular carbon and nitrogen uptake from stable isotope-labeled substrates was calculated by drawing regions of interest (ROI) on CN− images (recognizing cells in the images) and calculating 13C/12C and the 15N/14N ratio (calculated from the 12C15N/12C14N ratio). Instrumental mass fractionation of NanoSIMS analysis was calibrated by the conversion factor obtained by comparing carbon and nitrogen isotopic ratios of E. coli cells of varying isotopic enrichments measured at single cells with NanoSIMS and at bulk with an elemental analyzer/isotope ratio mass spectrometer (EA/IRMS, FlashEA 1112/DeltaPlus Advantage, Thermo Fisher Scientific). Concentration of bicarbonate (DIC) in the original sample determined on board7 was used to calculate substrate incorporation ratio (atom %) for bicarbonate in single cells.
Biomass and isotope calculations
Isotope incorporation data analysis and display as violin plots of the kernel density function were done using R45 with the “ggplot2”46, “cowplot”47, “ggsci”48, “scales”49 packages. The “active” cell ROIs, those incorporated 13C- and/or 15N-labeled substrates, were determined by their isotopic ratio exceeding the 99.7% confidence interval of background carbon and nitrogen isotopic abundance of polycarbonate membranes (1.24 atom % for 13C and 0.446 atom % for 15N). As measures for the rate of microbial biomass synthesis, we calculated the biomass-based specific growth rate and the substrate incorporation-based biomass generation rates. The biomass-based specific growth rates (µB, Eq. (1)) were calculated from the abundance of cells in the sediment samples at the start of incubation X0 and the abundance at the incubation period t (Xt). The substrate incorporation-based biomass generation rates14,50 (CµS and NµS, Eqs. (2) and (3), for carbon and nitrogen substrates, respectively) were calculated from the fractional abundance of isotope label in cellular biomass, where μS is the biomass generation rate (encompassing both cell maintenance and generation of new cells), t is the length of the incubation, Flabel is the labeling strength, Ft is the single-cell NanoSIMS measurement, and Fnat is the natural abundance. For the calculation of CµS and NµS, a conservative approach was used by only including ROIs where either 13C or 15N ratio was above the 99.7% confidence interval of backgrounds shown above. These rate calculations assumed that the carbon and nitrogen for the biomass generation were all derived from the substrates.
To document the physiological status of microorganisms in the original sediment samples, the fraction of microbes that originally existed in the sediment samples (f0) was calculated from the observed active ROI fractional ratio (ft), a factor of biomass increase (A) by following equation (Eqs. (4)–(6)).
If the number of ROIs was not enough to give enough sampling depth for fulfilling the Eq. (5), the f0 could not be calculated and was shown as N.A.
DNA extraction and sequencing
Microbial cells on non-coated polycarbonate membrane were lysed using a protocol modified after Morono et al.51 at ISO Class 1 clean air environment in super-clean room of JAMSTEC-KOCHI52. Sorted cells on the membrane were lysed by three-step alkaline treatments to minimize fragmentation of extracted DNA. First, five microliters of denaturation buffer (200 mM KOH, 20 µM EDTA) was added to the tiny piece of membrane with sorted cells, incubated at room temperature for 5 min, followed by adding 5 μL of neutralization buffer (200 mM HCl, 300 mM Tris-HCl [pH7.