Ice-nucleating Bacteria Enriched in the Sea-Surface Microlayer as Potential Sources of Atmospheric Ice-Nucleating Particles

Abstract. The sea-surface microlayer (SML) forms the ocean’s thin biological and chemical film that mediates air-sea exchange. This interface is enriched in surface-active organic matter and biogenic particles that can act as ice-nucleating particles (INP), yet the microbial sources of marine INPs remain poorly resolved. Here, we isolated and characterized ice-nucleating bacteria from the SML and underlying water (UW) of a semi-enclosed coastal inlet in Japan. Among 92 bacterial isolates, six strains induced freezing above –15 °C, indicating active ice nucleation. These isolates, affiliated with Flavobacteriia and Gammaproteobacteria, were heat-labile, and lost activity after 0.22 μm filtration, consistent with large (>100 kDa) outer-membrane ice-nucleation proteins. Flow-cytometric assays confirmed that heating up to 100 °C did not cause a decline in bacterial cell numbers, indicating that INA loss resulted from denaturation of heat-labile proteins rather than cell lysis. In contrast, Proteinase K treatment caused marked membrane disruption, suggesting that proteolytic inactivation and cell damage jointly contributed to INA reduction. One strain, 4U17, retained high INA after Proteinase K treatment despite extensive cell lysis, suggesting a protease-resistant nucleator that may be non-proteinaceous or shielded within extracellular or cell-derived particles. Amplicon sequencing of environmental samples revealed that taxa related to these ice-nucleating bacteria were consistently enriched in the SML relative to UW. Together, our results identify the marine SML as a reservoir for biogenic ice nucleators that may contribute to the oceanic source of atmospheric INPs.