Supplementary Material for: Comparison of DNA Extraction Efficiency and Reproducibility of Different Aeration Diffuser Biofilms Using Bead-Beating Protocol

An existing bead-beating DNA extraction protocol was employed to compare the DNA extraction recovery and fragment quality of 6 different aeration diffuser biofilms. <i>Escherichia coli</i>, <i>Gordonia amarae</i>, and mixed liquor were used as controls. The fraction of total DNA_biofilm decreased monotonically with increasing number of beat beatings (BB) when the amount of DNA present was sufficient (&gt;4 μg_DNA/cm²), excluding the ceramic disk. While controls required only 2 BBs, 3 out of 5 BBs achieved ≥70% of total DNA (70.3 ± 1.7%) for 5 out of 6 biofilms. Quantitative polymerase chain reaction (PCR) analyses of 353 and 1,505 basepair (bp) amplicons from pure culture extracts showed target copy numbers were not degraded for the first 2 BBs, but the third BB decreased amplicon concentrations by 0.65 and 1.12 log for <i>E. coli</i>, and 0.39 and 0.40 log for <i>G. amarae</i>, respectively. The 353 bp fragment amplification from biofilm samples showed minimal degradation for the first 3 BBs. PCR and gel electrophoresis confirmed integrity of amplified 1,505 bp DNA fragments over the 5 BBs, except in the EDPM (75 mm diameter, tube) diffuser biofilm (4.98 ± 0.62 μg_DNA/cm²). Taken together, this study showed type of diffuser membrane biofilms had no effects on extraction efficiency, but low DNA concentrations reduced extraction performance.