Denitrifying synthetic community – Experimental Group 2 (6 h)

On May 18, 2025, the denitrifying synthetic community was resuspended in 7 % salinity, low-carbon 2216E medium to an OD₆₀₀ of approximately 0.8. Sodium nitrate was then added to a final concentration of 1.6 mmol L⁻¹ NO₃⁻–N, and cultures were incubated for 6 h at 25 °C in a biochemical incubator. After incubation, cells were harvested by centrifugation at 4,500 rpm for 10 min, and the resulting pellets were immediately frozen in liquid nitrogen. Samples were stored on dry ice and shipped to Novogene Bioinformatics Institute (Beijing, China), where metatranscriptomic sequencing was performed on a NovaSeq 6000 system (library insert size: 350 bp; paired-end read length: 150 bp), yielding 10 Gb of data per sample.