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

Overview

This study evaluates sampling and sequencing methodologies for picocyanobacteria (Prochlorococcus and Synechococcus) and their associated phages in the Salish Sea estuary. Picocyanobacteria represent model organisms for examining host-pathogen coevolution due to their small genomes, global aquatic distribution, and growth regulation by both viral and abiotic factors. The research addresses technical challenges in capturing these organisms and their viral associates from environmental samples.

Methods and approach

The investigation developed and refined protocols for sampling, filtering, and sequencing cyanobacteria, cyanophages, and associated environmental parameters. Multiple filtration approaches were tested, including fully in-line sequences, in-situ filtration, and combinations of in-line with single vacuum flask filtrations. DNA extraction was performed across different filter fractions using multiple commercial kits. The protocols incorporated size fractionation strategies intended to discriminate cell types and viral particles.

Results

The optimized protocol combining in-line and single vacuum flask filtrations achieved threefold processing time reduction compared to alternative methods in certain cases. DNA extraction yields ranged from approximately 400 to 1200 nanograms across all filter fractions, with inter-kit variation observed. The protocol successfully discriminated large-cell eukaryotic organisms. However, size fractionation of picocyanobacteria was substantially compromised by interference from free DNA, multicellular structures, and abundant tycheposons, limiting the effectiveness of separation by cell size.

Implications

The findings demonstrate that standard size fractionation approaches encounter significant limitations when applied to picocyanobacteria-dominated environmental samples. The presence of extracellular DNA, aggregated cellular structures, and tycheposons—viral particles with specific compositional features—necessitate methodological refinements beyond conventional filtration-based separation. These technical constraints require consideration in future studies employing similar sampling strategies in productive aquatic ecosystems. The work identifies specific procedural improvements in filtration efficiency while simultaneously highlighting fundamental challenges in isolating target organisms from complex microbial assemblages.