15N2 incubation was performed according to our previous work (26 (link)). Briefly, an aliquot of 20 mL of BG110 medium was filled into a 120-mL crimp-top vial and inoculated with 500 μL of Anabaena sp. PCC 7120 or R. raciborskii XM1 solution. The vials were sealed, and air in the headspace was replaced with Ar gas after 10 min of continual aeration. Then, a gas mixture consisting of N2 and O2 (4:1 v/v) was aerated into the vials to achieve different volume percentages of 15N2 (15N2/(15N2 + 14N2)). Considering that 15N content is 0.36% in natural abundance and 99% in commercial areas, the absolute atmospheric 15N abundances in the incubations were calculated as 0.36, 10.22, 25.02, 49.68, 74.52, and 99.36%. To temporally track N2 fixation and transfer, cyanobacteria were incubated in the prepared 15N2-containing atmosphere and harvested after 6 or 12 h time intervals.
In parallel incubation experiments, to perform 13C labeling, BG110 medium without NaHCO3 was aerated with Ar gas to remove dissolved CO2. Premixed 12C and 13C-NaHCO3 solutions with 13C percentages (13C/(13C + 12C)) of 1.11, 10.90, 25.58, 50.06, 74.53, and 99.00% (calculated after considering the 13C natural abundance) were immediately added to the medium. The vials were sealed, and air in the headspace was replaced with Ar gas after 10 min of continual aeration. Then, a mixture gas consisting of N2 (as a sole source of nitrogen) and O2 (4:1 v/v) was aerated into the vials. After inoculating 500 μL of Anabaena sp. PCC 7120 or R. raciborskii XM1 solution, cyanobacteria were cultured in these media and harvested at 6 or 12 h time intervals.
