Nitrous Oxide (N₂O) production in axenic Chlorella vulgaris microalgae cultures: evidence, putative pathways, and potential environmental impacts

Abstract. Using antibiotic assays and genomic analysis, this study demonstrates nitrous oxide (N₂O) is generated from axenic Chlorella vulgaris cultures. In batch assays, this production is magnified under conditions favouring intracellular nitrite accumulation, but repressed when nitrate reductase (NR) activity is inhibited. These observations suggest N₂O formation in C. vulgaris might proceed via NR-mediated nitrite reduction into nitric oxide (NO) acting as N₂O precursor via a pathway similar to N₂O formation in bacterial denitrifiers, although NO reduction to N₂O under oxia remains unproven in plant cells. Alternatively, NR may reduce nitrite to nitroxyl (HNO), the latter being known to dimerize to N₂O under oxia. Regardless of the precursor considered, an NR-mediated nitrite reduction pathway provides a unifying explanation for correlations reported between N₂O emissions from algae-based ecosystems and NR activity, nitrate concentration, nitrite concentration, and photosynthesis repression. Moreover, these results indicate microalgae-mediated N₂O formation might significantly contribute to N₂O emissions in algae-based ecosystems (e.g. 1.38–10.1 kg N₂O-N ha⁻¹ yr⁻¹ in a 0.25 m deep raceway pond operated under Mediterranean climatic conditions). These findings have profound implications for the life cycle analysis of algae biotechnologies and our understanding of the global biogeochemical nitrogen cycle.