The contribution of oyster microbiomes to denitrification and nitrous oxide emissions in estuarine and coastal ecosystems

Oysters are important nitrogen transformers in estuaries. Studies have shown that benthic-pelagic coupling by oysters stimulates sedimentary denitrification. The microbiomes living in and on oysters may also have high denitrification activity, generating N₂O and N₂ as end products. In order to evaluate the importance of oyster microbiome denitrification and its contribution to N₂O emission in estuaries, continuous flow experiments with ¹⁵NO₃^- addition were conducted with oyster reef adjacent sediments, live oysters, live oysters with biofilm removed, and oyster shells. Denitrification rates were highest in live oysters, followed by scrubbed oysters, oyster shells, and finally by sediments, with the lowest activity. N₂O production followed a similar trend, but with scrubbed oysters and shells being similar. We also found a significant linear correlation between N₂ and N₂O production, which indicates that denitrification is the source of N₂O from oyster and oyster shells. Molecular analysis of microbiomes in oyster shells, guts and gills will be conducted using next generation sequencing of 16S rRNA gene and quantitative PCR assays targeting the functional genes in denitrification. We expect that within live oysters, the anaerobic gut microbiome will have higher abundances of denitrifiers than the shell biofilms or sediments, corresponding to higher denitrification activities in live oysters. This study will demonstrate that enhanced denitrification activity in oysters, in conjunction with sedimentary denitrification stimulated by oyster biodeposits, leads to oyster reefs being nitrogen removal hotspots and potential emission sources of N₂O in estuarine and coastal ecosystems. In addition, the importance of microbiome structures in relation to oyster denitrification will be revealed.