Diazotrophic cyanobacteria N2 fixation analysis

Two types of heterocyst-forming diazotrophic cyanobacteria i.e. Anabaena sp. and R. raciborskii were used in this study. A model strain Anabaena sp. PCC 7120 was obtained from the Freshwater Algae Culture Collection of the Institute of Hydrobiology, China. The bloom-forming cyanobacterium of R. raciborskii XM1 was isolated in a Xiamen reservoir during a summer bloom in 2018 (33 (link)). To further confirm the N₂ fixation potential of these strains, dinitrogenase reductase genes (nifH) were amplified and visualized on agarose gels. Briefly, extracted cyanobacterial DNAs were polymerase chain reaction amplified with primers nifH1 (5′-TGYGAYCCNAARGCNGA-3′) and nifH2 (5′-ADNGCCATCATYTCNCC-3′). The appearance of nifH at around 359 bp in agarose gel electrophoresis is shown in Fig. S2a. These two cyanobacterial strains were cultured in BG11 or N-free BG11 (BG11₀) medium at ∼28°C and 90 rpm under constant light condition. They were cultured in BG11₀ to ensure heterocyst formation before isotope labeling. N₂-fixing A. chroococcum (ACCC10096) and E. coli MG1655 were purchased from the Guangdong Culture Collection Center of Microbiology, China, and cultured in N-free azotobacter media and Luria−Bertani broth, respectively before co-culture. All culture medium recipes are provided in Supplementary information.
The chemicals used for stable isotope incubation included ¹⁵N-NaNO₃ (98 atom% ¹⁵N, Sigma−Aldrich, United Kingdom), ¹³C-NaHCO₃ (99 atom%; Cambridge Isotope Laboratories, Inc., UK), and ¹⁵N₂ (99 atom%; Aladdin, China). Unless otherwise stated, all chemicals were purchased from Sinopharm Chemical Reagent Co., China.