 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 [pH7.5]). The liquid part was recovered for the second step, which was adding another 5 microliters of denaturation buffer, heating at 70 °C for 5 min, and adding 5 μL of neutralization buffer, followed by recovery of the liquid part and pooling with the first extract. The third step was adding an additional 5 μL of denaturation buffer, heating at 70 °C for 10 min, and adding 5 μL of neutralization buffer, followed by recovery of the liquid part. Heating was done with an Applied Biosystems Veriti Thermal Cycler (Thermo Fisher Scientific). The lysis of cells was confirmed by checking processed membrane with fluorescence microscope after staining with SYBR Green I (Thermo Fisher Scientific). Negative control extractions (8 separate extractions) were performed by using polycarbonate membrane without sorted cells. The resultant DNA extract was then used directly for first-round PCR amplification with universal primers targeting the V4 region of the 16S rRNA gene (U515F: 5′-TGY CAG CMG CCG CGG TAA-3′, U806R: 5′-GGA CTA CHV GGG TWT CTA AT-3′). Also, PCR No-Template-Control reactions (PCR-NTCs) run without template DNA (6 separate reactions) were performed. First-round PCR amplicons were quantified and purified via gel electrophoresis and then indexed and barcoded in the second PCR. The DNA sequencing was performed on a MiSeq Illumina platform following the methods outlined by Hoshino and Inagaki53. The obtained sequence reads were quality trimmed and filtered by FASTQ Toolkit in Basespase (Illumina). Resultant paired-end reads were processed using mothur54 following the Mothur software package (v.1.35.0) of Illumina MiSeq Standard Operating Procedure55. All sequence reads from negative control extractions and PCR-NTCs, along with sequences from all samples were used to generate operational taxonomic units (OTUs) at 97% similarity using usearch 10.0 program56. To remove sequences that may be of exogenous origin57, the most conservative way to remove contaminant sequences considers all taxa that are identified in the laborato