Quantitative PCR (qPCR) is a common, culture-independent method to estimate gene copies at levels as broad as Kingdom and specific as guild, function or species; however, it is dependent upon several factors that affect the amplification efficiency. Amplification efficiency of an individual qPCR reaction is dependent on template type, qPCR product, and assay optimization including primer design. Although qPCR reactions can be optimized, the design of primers to meet guidelines for relatively short amplicon length (75–150 base pairs) is often unfeasible especially when targeting a broad phylogeny of organisms or functional genes. The addition of exogenous non-target plasmid DNA (0.05 ng µL-1) as a PCR additive significantly increases the amplification efficiency for long target amplicons up to 723 bp. The method is applicable for different template types including circular and linearized plasmids (24–30% increase) and cell-free DNA extracts (up to 26%), as well as different thermocyclers and qPCR products. As an exogenous DNA source, the pUC19 plasmid provides flexibility with a reduced chance of non-target amplification due to the relatively simple, short, and known DNA sequence. Overall, this method enhances amplification efficiency, reduces template bias, and lowers qPCR variability with a strong benefit for long amplicon assays.

Converting pre-existing gas storage facilities to dihydrogen storage raises critical questions about storage quality and dihydrogen consumption by prokaryotes. To investigate biologically driven changes during such transitions, we analyzed data from five dihydrogen pulse experiments conducted in pressurized bioreactors that replicate deep aquifer pressure and temperature conditions. Our goal was to determine whether consistent community-level responses to dihydrogen injection could be identified. We found that dihydrogen exposure consistently led to a decline in fermentative ASVs, likely driven by environmental filtering. Hydrogenotrophic sulfate reducers initially dominated in some experiments, with total sulfate depletion observed in certain cases, followed by the emergence of methanogenic archaea. In some instances, a succession pattern involving Thermodesulfovibrio and Methanothermobacter appeared across taxonomically distinct communities, suggesting deterministic ecological processes. Additionally, we observed potential dispersal limitation and selection pressures, possibly linked to pH shifts caused by autotrophy. These findings underscore the importance of considering microbial dynamics in dihydrogen storage strategies in deep aquifers and suggest that, despite initial variability, predictable ecological succession may occur under specific geochemical conditions.