The study of microbial communities based on the total environmental DNA (eDNA) is influenced by the presence of different eDNA states, i.e., intracellular (iDNA) and extracellular DNA (exDNA), and the choice of the DNA extraction method. Although the use of spike-and-recovery controls facilitates the diagnosis of such issues, appropriate experimental setups simultaneously accounting for the different eDNA states and their bacterial origins are missing. Here, we used two single-gene deletion mutants of both Escherichia coli and Bacillus subtilis to trace exDNA and iDNA spike-ins of each selected model organism within environmental samples. Unique primer/probe sets were developed for each strain, allowing their absolute quantification using multiplex digital PCR assays. The proposed spike-and-recovery controls were successfully applied to various environments including soil, sediment, sludge and compost. While the percent recovery of spiked iDNA differed significantly between E. coli and B. subtilis, results were similar for both model organisms in the case of spiked exDNA, emphasising that the fate of DNA molecules in the environment is similar irrespective of their bacterial origin. Hence, future studies may benefit from the proposed approach to better understand methodological ambiguities related to the eDNA extraction in general as well as the separation of the different eDNA states.
