Development of a quantitative PMA-16S rRNA gene sequencing workflow for absolute abundance measurements of seawater microbial communities

Background Ecological risk assessments rarely consider the impacts of environmental stress on microbial communities. The incorporation of microbial community responses into these evaluations requires establishing sensitivity thresholds based on the absolute abundance of viable taxa. While essential for describing microbial community dynamics, sequencing-based analyses are generally limited to assessing relative proportions and fail to reveal the magnitude or directionality of abundance shifts. To address this, we developed and validated a workflow combining propidium monoazide (PMA) treatment, 16S rRNA gene amplicon sequencing, and quantitative microbiome profiling (QMP) to determine the absolute abundance of viable taxa in seawater microbiomes. Results Using natural seawater, microbial load estimates from droplet digital PCR (ddPCR) and flow cytometry (FC) correlated strongly for total and intact cell counts, confirming the suitability of both methods for normalising 16S rRNA gene amplicon sequencing data. Additionally, we demonstrated that PMA at concentrations of 2.5–15 µM effectively inhibited PCR amplification of DNA from membrane-compromised cells, reducing 16S RNA gene copies by 24–44% relative to untreated samples. Samples with known proportions of intact cells were generated by mixing heat-killed and natural seawater, enabling abundance assessments by normalising 16S rRNA gene amplicon sequencing data to intact cell loads estimated via ddPCR and FC. This approach facilitated detailed comparisons of the effects of QMP versus relative microbiome profiling (RMP) on alpha and beta diversity metrics and on relative and absolute amplicon sequence variant (ASV) abundance profiles. Unlike RMP, QMP captured significant shifts in the microbial community composition across samples with decreasing proportions of intact cells. Furthermore, RMP failed to detect abundance changes at ASV-level, while QMP revealed consistent abundance declines. Conclusion The PMA-16S rRNA sequencing workflow, coupled with QMP, enhanced the accuracy in representing microbial community dynamics by addressing key limitations of RMP such as the inclusion of damaged cells or extracellular DNA and the misleading proportions of identified taxa. This workflow is particularly suited for quantifying the magnitude and direction of changes in taxa abundance following stress exposure, making it directly applicable to stress-response modelling and supporting the integration of microbial sensitivity thresholds into future regulatory guidelines.