Development a Laboratory Based Sub-Sampling Methodology for Sea Surface Microlayer Biological Characterization

The Sea Surface Microlayer (SML) is a unique chemical and biological interface between the ocean and the atmosphere, playing a fundamental role in global biogeochemical cycles. Notably, the SML acts as the primary gateway for the emission of marine bacteria from the water column into the atmosphere and therefore serves as a critical interface between the ocean and the atmosphere. Despite its importance, direct biological sampling in the open sea remains technically challenging due to physical disturbances and the inherent fragility of the microlayer. To address these limitations, we developed and validated a dedicated biological sub-sampling methodology designed to facilitate SML collection and analysis in a controlled environment. The technique utilizes laboratory-based SML reformation from surface-water (SW) subsamples. To determine the optimal sampling window, 24-hour incubation experiments and 16S rRNA gene quantification were conducted using field samples and enriched marine bacteria. Results showed that while the SML exhibited compositional instability during the first two hours post-sampling, a consistent steady state in bacterial abundance was achieved between 3 and 6 hours. Consequently, a 4-hour stabilization period was established as the optimal timeframe for representative SML collection. Further results on the stability of the SML microbial composition will be discussed. The methodology was validated via comparative assessments against in situ sampling in the Mediterranean and Red Seas. Statistical analyses confirmed no significant differences in 16S rRNA gene copy numbers between field-collected and laboratory-reformed samples (p > 0.05). Application of this protocol across a Pacific Ocean latitude gradient revealed distinct microbial signatures in SW, the SML, and the atmosphere. Genomic data positioned the SML as a transitional mediator between the ocean and air, while 16S rRNA transcript analysis showed tight clustering in the SML and atmosphere, suggesting selective environmental control over active communities. Collectively, this stabilization approach provides a robust and standardized alternative to traditional sampling, particularly in rough sea conditions. By enabling stable biological characterization of the SML, this research enhances our understanding of the mechanisms controlling marine-atmosphere microbial exchange and their impact on global ecological networks and nutrient cycling.