Abstract
Getting insights into the quantitative and qualitative contribution of different DNA states, i.e., extracellular (exDNA) and intracellular DNA (iDNA), to the total environmental DNA (eDNA) pool requires reliable methods for their separation. Even though a multitude of respective extraction protocols has been published, their validation is often missing. Here, we selected four protocols for the state‐specific extraction of eDNA and traced the separation of exDNA and iDNA within natural environments using previously designed spike‐and‐recovery controls. Besides accounting for the different eDNA states, the spike‐ins also distinguished different bacterial origins (gram‐positive, gram‐negative). Following their quantification by digital PCR, the recovery of exDNA and iDNA spike‐ins in both the target as well as nontarget eDNA states differed among the selected extraction protocols and environmental matrices, albeit the effect of the former was far more decisive. While the recovery of exDNA spike‐ins was mainly affected by the chemical composition of the washing buffer and the duration of each washing step, the lysis method determined the recovery of spiked iDNA. These aspects were further combined within an optimised protocol, providing a valuable step towards a more concise understanding of factors governing the state‐specific extraction of eDNA and hence their relevance in molecular microbial ecology.