Nah13co3

The intracellular concentration of CoA, acetyl-CoA, and butanoyl-CoA was determined using ¹³C-isotope labelling experiment following the protocol used in the previous study with minor modifications [18 (link)]. Fully ¹³C-labeled cell extracts of BUOHSE and DC7 strains were prepared. Cells were cultivated as described previously except that NaH¹³CO₃ (> 98 atom % ¹³C, Cambridge Isotope Laboratories, Inc., USA) was used instead of NaHCO₃. Sampling was performed 3 days after IPTG induction in the same way as described above except that pre-cooled deionized water was used as washing solvent instead of NH₄CO₃. Cells were extracted as described above except without the freeze-drying step. Extraction was repeatedly conducted for four times. After samples were concentrated for 2 h using centrifugal concentration, all samples were combined in a 15 mL centrifuge tube. This ¹³C-labelled cell extract was used as an internal standard. Six point calibration curve was constructed for each metabolite using peak area ratios of U-¹²C to U-¹³C, as described previously [18 (link)]. The detailed description of calibration curve for each metabolite is listed in Additional file 1: Table S1. Analysis mode was multiple reaction monitoring (MRM) mode in IP-RP-LC/QqQ-MS system (Additional file 1: Table S4).

All experiments were conducted using a clone of Daphnia magna (DK-35-9, hereafter Daphnia), initially grown and maintained on Acutodesmus sp. which was obtained from the Institute of Zoology, University of Basel. We also used Cryptomonas erosa (CPCC 466) as high quality diet control in our experiments. Acutodesmus sp. was cultured using modified WC solution supplemented with biotin and cyanocobalamin (B₁₂)66 . Cryptomonas erosa was cultured using AF6 media67 . In addition, we cultured some more phytoplankton strains listed in Table 1. Each strain was cultured in a medium specific to that strain (Table 1) and were grown at 20 °C under a 14 h:10 h light:dark cycle with light intensity of 30–70 μmol m⁻² s⁻¹. To obtain differences in carbon isotope signals between the diets, Acutodesmus sp. cultures were enriched with ¹³C, 3% of the NaHCO₃ in the MWC media consisted of NaH¹³CO₃ (99%), Cambridge Isotope Laboratories].

An axenic strain of marine diatom Thalassiosira pseudonana CCMP1335 was cultured at 18 ^oC in three replicate 15-L polycarbonate bottles containing 10 L of L1 medium [35 (link)] in which NaH¹³CO₃ (Cambridge Isotope Laboratories, CLM-441) was the source of inorganic carbon (labeling efficiency, 78% of C atoms). The light cycle consisted of 16 h light, during which light intensity varied gradually between 0 and 150 µmol photon m⁻² s⁻¹ with a maximum intensity at noon, followed by 8 h of dark. Axenic T. pseudonana cultures were pre-incubated for 6 days to achieve cell numbers required for metabolite analysis. The cultures were inoculated with bacterial strain Ruegeria pomeroyi DSS-3 that was grown overnight at 30^oC on ½ YTSS liquid medium and washed three times in L1 medium (final concentration, 10⁶ bacterial cells mL⁻¹). Co-cultures (n = 3) were pre-incubated for two days, after which samples were collected every 6 h over the next 48 h for bacterial mRNA sequencing, phytoplankton and bacterial cell counts, and phytoplankton endometabolome analysis.

Euglena gracilis NIES-48 was cultured in culture flasks (working volume: 20 mL) in a 14 h:10 h light:dark cycle with illumination approximately 150 μmol photons m⁻² s⁻¹ at 25 °C using AF-6 medium. Before cultivation with ¹³C-stable isotope media, Euglena gracilis cells were grown in normal AF-6 medium for at least 3 days as preculture. The cells in the preculture were transferred to AF-6 − N medium including 20 mM of NaH¹³CO₃ (¹³C: 99%, Cambridge Isotope Laboratories) or NaH¹²CO₃ (Wako Pure Chemical) for induction of ¹³C- or ¹²C-paramylon, respectively. After purging of air with filtered (0.22 μm) nitrogen gas and enclosing in a capped culture flask (working volume: 20 mL), cells were incubated in static conditions under continuous light illumination (~150 μmol photons m⁻² s⁻¹) at 28 °C for 2 days prior to cell sorting. For the evaluation of the sorting performance, the two isotope-incorporated cells were fluorescently labeled 1 h before applying them to the RIACS using different nuclear staining dyes [¹³C: 20 mM of Hoechst 33342 (62249, Thermo Fisher), ¹²C: 5 µM of SYTO 82 (S11363, Thermo Fisher)].

Thalassiosira weissflogii is a cosmopolitan marine diatom and belongs to the genus Thalassiosira which is part of the phytoplankton community in the Arabian Sea off the coast of India (Sawant and Madhupratap, 1996 ). Prior to the cruise, an axenic clone of T. weissflogii (CCMP, Bigelow Laboratories for Ocean Science, USA) was cultured in artificial seawater and L1 culture medium. The medium was enriched with 99%—¹³C-bicarbonate (NaH¹³CO₃, Cambridge Isotope Laboratories, Inc., USA) and 50%—¹⁵N-sodium nitrate (Na¹⁵NO₃, Cambridge Isotope Laboratories, Inc., USA). Algae were cultured at 16°C for 28 days (light: dark = 16:8; 35 PSU), harvested by centrifugation (500 G; 30 min), sonicated (2000 Hz; 5 min), and rinsed three times in ultrapure water to remove inorganic salts and dissolved organic carbon. Finally harvested algae were lyophilized (−60°C; −0.0001 mbar; 24 h). The produced phytodetritus contained 27.75 atom%¹³C and 33.70 atom%¹⁵N and displayed a C/N ratio (C:N) of 4.06 (Hunter et al., 2012a (link)).

Chemoautotrophy rate measurements were started by injecting 100 µl of 20 mM NaH¹³CO₃ (99% ¹³C; Cambridge Isotope Laboratories, Andover, MA, USA) horizontally into the sediment cores at 0.5 cm depth intervals by using the line-injection method [2] . The ¹³C-label was dissolved in artificial seawater lacking calcium or magnesium in order to avoid precipitation [21] . The label was made oxygen free by bubbling with nitrogen gas shortly before injection. Sediment cores were incubated in the dark within 2°C of the in-situ temperature (see Table 1) for various periods of up to 4 days, and were ventilated daily by removing the top stopper for one minute (ZK) or incubated without top stoppers (RK) to circumvent the development of suboxic condition in the headspace. After incubation, sediment cores were sliced to a depth of 5 cm and sediment slices were quickly centrifuged (4500 rpm, 5 min) to collect porewater for concentration and ¹³C analysis of dissolved inorganic carbon (DIC). Sediments were subsequently frozen at −20°C and lyophilized before further analysis. Unlabelled, control cores were also processed.

In situ microcosms were performed to assess the potential for inorganic carbon uptake through the addition of NaH¹³CO₃. Sample collection and the microcosm set-up followed previously described methods [39 –41 ]. In summary, samples (biofilms, filaments, etc.) were place in sterile serum vials, overlaid with spring water, capped, and amended with NaH¹³CO₃ (100 μM final concentration) (Cambridge Isotope Laboratories, Inc., Andover, MA, USA). All assays were performed in triplicate between 10 AM and 2 PM. To assess the potential for photoautotrophic NaH¹³CO₃ uptake, microcosms were incubated in the light. In contrast, microcosms were wrapped in foil to assess the potential for chemoautotrophic (dark) NaH¹³CO₃ uptake. At the emergence, both green and white filaments were present. We performed assays on each by sampling predominantly green filaments and predominantly white filaments. We assumed the majority of uptake in the green biomass would be due to photoautotrophy and thus performed one set of microcosms on the bulk filaments/biofilm (green + white) in the light and then separated the biofilm types (green or white) for microcosms performed in the dark. Following incubation, vials were flash frozen on dry ice and stored at − 80 °C until processed (described below). Calculation of assimilation rates is described below.