<b>Optimizing Viable Bacteria Detection in Ballast Tank Sediments: Addressing PCR Inhibitors and Relic DNA</b>

Accurate monitoring of viable microorganisms in ballast tank sediments is critical for effective management. This study addresses two key challenges in microbial viability assessment: interference from ubiquitous PCR inhibitors in sediments and the persistence of relic DNA. We employed a PCR inhibitor removal kit to eliminate PCR inhibitors and PMA (propidium monoazide) to remove relic DNA, followed by qPCR to detect changes in 16S rRNA gene copy numbers and 16S rRNA gene amplicon sequencing to assess alterations in microbial diversity. The results showed that removing PCR inhibitors increased 16S gene copy numbers by an average of 1600%, and significantly altered ASVs, species diversity indices, and community structure. Notably, prior to inhibitor removal, PMA treatment paradoxically reduced 16S rRNA gene copies while increasing ASV counts, with specific taxa re-emerging (relative abundance: 0.61%–1.20%). This suggests that PCR inhibitor removal compromises PMA's efficacy in suppressing non-viable DNA. These findings underscore the necessity of PCR inhibitor elimination in sediment monitoring while highlighting the need to optimize PMA protocols for accurate viability assessments.