ballast sediment metagenome and 16s RNA sequence
Relic DNA of biological origin and humic substances of abiotic origin coexist in ballast water sediments, and together they interfere with microbial detection; however, the extent of their respective impacts remains unclear. This study systematically investigated the interactions between humic substances and relic DNA, as well as their responses to different removal sequences, and revealed a key mechanism: humic substances can adsorb relic DNA, thereby significantly influencing detection outcomes under different treatment orders. Using a paired experimental design, combined with PMA-qPCR, 16S rRNA gene amplicon sequencing, and absolute quantification metagenomic sequencing, we comprehensively evaluated the effects of removal sequence on the detection of active microorganisms.The results showed that humic substances are the dominant interfering factor, with concentrations far exceeding the PCR inhibition threshold and exerting a substantially stronger impact than relic DNA. They not only inhibit qPCR efficiency but also alter community structure and interfere with functional expression and pathogen risk assessment. Based on the mechanism by which humic substances adsorb relic DNA, we found that the "humic substances first, relic DNA second" strategy (Pre-treatment) enables the simultaneous removal of both, significantly increasing relic DNA removal efficiency and improving qPCR amplification efficiency by 206.7%. This approach more accurately reflects active microbial communities and their functional potential, and effectively avoids DNA loss and the "artificial inflation of functional diversity" caused by the "relic DNA first, humic substances second" strategy (Post-treatment).This study, for the first time, elucidates the intrinsic mechanism by which removal sequence influences detection outcomes from the perspective of humic substance-relic DNA interactions. On this basis, a standardized pretreatment workflow of "humic substances first, relic DNA second" is established, providing a reliable methodological framework for the accurate detection of active microorganisms in complex samples such as ballast water sediments and for global biosecurity risk assessment in maritime transport.