Evidence of the Significant Contribution of Heterotrophic Diazotrophs to Nitrogen Fixation in the Eastern Indian Ocean During Pre-Southwest Monsoon Period

Nitrogen fixation continually transforms atmospheric nitrogen (N2) into biologically available nitrogen in the oceans, which actively sustains marine new production, especially in oligotrophic ecosystems. Recently, heterotrophic diazotrophs were found to be widespread and potentially important for nitrogen fixation in the Eastern Indian Ocean (EIO). However, no direct measurement has been taken in this region to explore the contribution of heterotrophic diazotrophs to total nitrogen fixation. To address this gap, we examined size-fractioned nitrogen fixation rates (NFRs) using an isotope 15 N tracer technique, diazotroph community structure analysis based on molecular detection, and primary productivity (PP) measurements using an isotope 14C tracer technique within the euphotic zone of the EIO during the pre-southwest monsoon period. Our results reveal that diazotroph communities are dominated by heterotrophic diazotrophs and filamentous cyanobacteria in the surface waters of the EIO. Size-fractioned data show that the   10 μm fraction exhibits spatial heterogeneity in the surface samples, with significantly higher rates in the Bay of Bengal (BoB) compared to the southeastern Indian Ocean. Correspondingly, the NFRs of the > 10 μm fraction match well with high-throughput sequencing and microscope counts. In comparison, the NFRs of the < 10 μm fraction do not display significant regional differences, which is attributed to the major fixation efficiency by different bacteria. Based on the Redfield ratio (C:N = 6.6:1), depth-integrated NFRs were estimated to contribute 0.3–1.3% of the total PP in the study region. Overall, our study provides baseline NFRs in the EIO and highlights the potential importance of non-cyanobacteria to nitrogen fixation. Our findings have significance in the biogeochemical understanding of the coupling between nitrogen and carbon cycling in the oligotrophic Indian Ocean.