Denitrification and N₂O Emission in Estuarine Sediments in Response to Ocean Acidification: From Process to Mechanism

Global estuarine ecosystems are experiencing severe nitrogen pollution and ocean acidification (OA) simultaneously. Sedimentary denitrification is an important way of reactive nitrogen removal but at the same time leads to the emission of large amounts of nitrous oxide (N₂O), a potent greenhouse gas. It is known that OA in estuarine regions could impact denitrification and N₂O production; however, the underlying mechanism is still underexplored. Here, sediment incubation and pure culture experiments were conducted to explore the OA impacts on microbial denitrification and the associated N₂O emissions in estuarine sediments. Under neutral (in situ) conditions, fungal N₂O emission dominated in the sediment, while the bacterial and fungal sources had a similar role under acidification. This indicated that acidification decreased the sedimentary fungal denitrification and likely inhibited the activity of fungal denitrifiers. To explore molecular mechanisms, a denitrifying fungal strain of Penicillium janthinellum was isolated from the sediments. By using deuterium-labeled single-cell Raman spectroscopy and isobaric tags for relative and absolute quantitation proteomics, we found that acidification inhibited electron transfers in P. janthinellum and downregulated expressions of the proteins related to energy production and conservation. Two collaborative pathways of energy generation in the P. janthinellum were further revealed, that is, aerobic oxidative phosphorylation and TCA cycle and anoxic pyruvate fermentation. This indicated a distinct energy supply strategy from bacterial denitrification. Our study provides insights into fungi-mediated nitrogen cycle in acidifying aquatic ecosystems.