Optimizing viable bacteria detection in ballast tank sediments: Addressing PCR inhibitors and relic DNA

The significant ecological risks posed by microbial communities in ballast water and sediments highlight the necessity for accurate species monitoring. However, the complex physicochemical conditions within ballast tanks limit the effectiveness of conventional environmental DNA (eDNA) metabarcoding approaches. This study focuses on two key challenges in monitoring viable microorganisms in ballast tank sediments: interference from relic DNA and PCR inhibition. For eDNA samples from the ballast tanks of three vessels, we paired PMA (propidium monoazide)-treated and untreated samples; each extract was either further processed to remove PCR inhibitors or left untreated. The resulting four sample groups were subjected to qPCR for 16S rRNA gene quantification, 16S rRNA amplicon sequencing, and comparative microbial diversity analyses to assess the impacts of these methodologies. PMA treatment successfully differentiated between intact and relic DNA, reducing total DNA and 16S rRNA gene copies by 9.45 % and 6.93 %, respectively. Subsequent inhibitor removal unmasked the true community abundance, yielding a 16-fold increase in 16S rRNA gene copies and a significant restructuring of the microbial composition. Notably, combining PMA treatment with inhibitor removal decreased 16S rRNA gene copies while increasing ASV richness, suggesting that inhibitors may impair PMA's ability to exclude relic DNA. Our findings emphasize the need to combine PCR inhibitor removal with PMA treatment for quantifying viable microorganisms; however, the optimal protocols for their co-processing require further refinement.