Optimizing Viable Bacteria Detection in Ballast Tank Sediments: Addressing PCR Inhibitors and Relic DNA

Accurate monitoring of viable microorganisms in ballast tank sediments is critical for effective ballast water management. This study tackles two key challenges in viable microorganism monitoring in ballast tank sediments: interference from PCR inhibitors and the persistence of relic DNA. We employed a PCR inhibitor removal kit and propidium monoazide (PMA) treatment to address these limitations, respectively, followed by qPCR and 16S rRNA gene amplicon sequencing. Removing PCR inhibitors resulted in a dramatic 1600% average increase in 16S rRNA gene copy numbers and significantly altered the observed microbial community composition. Interestingly, when PMA treatment preceded inhibitor removal, we observed a decrease in 16S rRNA gene copies coupled with an increase in ASV richness, suggesting that PCR inhibitors can compromise PMA's ability to exclude relic DNA. These findings underscore the critical importance of PCR inhibitor removal in sediment monitoring, while highlighting the need to optimize PMA protocols when quantifying viable microorganisms in sediments.