Sampling microbial dynamics in the Salish Sea estuary: evaluating methods to capture cyanobacteria and cyanophage

Abstract

Introduction Picocyanobacteria from the genera Prochlorococcus and Synechococcus thrive across the globe in aquatic environments, have relatively small genomes, and have growth dynamics regulated by both viral interactions and abiotic conditions, making them excellent model organisms for exploring host-pathogencoevolution. Methods We developed and refined methods to sample and sequence cyanobacteria, cyanophages, and measured features of their abiotic environment. Results The protocol described herein can successfully discriminate large-cell eukaryotic organisms, but size fractionation of picocyanobacteria appears to be affected by the presence of free DNA, multicellular structures, and abundant tycheposons. Our preferred final protocol from this exploratory effort included a combination of in-line and single vacuum flask filtrations, which reduced filtration processing time by over threefold in some cases compared to other tested methods, such as a fully in-line sequence or in-site filtrations. We successfully extracted an average of approximately 400–1200 ng for all filter fractions, with some variations between kits. Discussion The protocol described herein can successfully discriminate large-cell eukaryotic organisms, but size fractionation of picocyanobacteria appears to be affected by the presence of free DNA, multicellular structures, and abundant tycheposons.

Key findings

• ▸Size fractionation of picocyanobacteria is compromised by the presence of free DNA, multicellular structures, and abundant tychoplankton.
• ▸The optimal sampling protocol combines in-line filtration with single vacuum flask filtrations.
• ▸The refined method successfully reduces overall filtration processing time compared to previous exploratory efforts.

Keywords

Identifiers

DOI

10.3389/fmars.2026.1769457

Journal

Frontiers in Marine Science

Year

2026