Ecological significance of dark carbon fixation driven by ammonia oxidation in estuarine waters

Microbial-mediated dark carbon fixation (DCF) is increasingly recognized as an important process for primary production in global oceanic ecosystems, with ammonia oxidation (AO) providing a major energy source for DCF in oxygenated waters. However, a significant research gap remains in integrating the DCF and AO processes mediated by ammonia-oxidizing archaea (AOA) and bacteria (AOB) into a systematic framework within estuarine ecosystems. In this study, we investigated the DCF and AO rates in water samples from the Yangtze Estuary through incubation experiments with ¹⁴C and ¹⁵N isotope probing techniques. The measured DCF and AO rates ranged from 17.58 to 168.70 nM C/h and from 4.78 to 499.63 nM N/h, respectively. Our results revealed significant positive correlations among DCF rates, AO rates, and ammonium concentrations. Amplicon sequencing revealed that chemoautotrophs utilizing the Calvin-Benson-Bassham (CBB) cycle were predominantly affiliated with Burkholderiales, while those utilizing the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle were primarily associated with Nitrosopumilaceae. Temperature played a crucial role in shaping the composition of chemoautotrophic communities harboring the CBB cycle, whereas salinity was a key factor modulating the composition of chemoautotrophic communities harboring the 3-HP/4-HB cycle. Niche differentiation within nitrifier communities enables their effective adaptation to the frequent environmental fluctuations characteristic of estuarine ecosystems. This study highlights the ecological significance of DCF driven by ammonia oxidation in estuarine waters and emphasizes the need to integrate DCF into blue carbon assessments in coastal ecosystems.