Improved DNA purification with quality assurance for evaluation of the microbial genetic content of constructed wetlands

Efficient isolation of target DNA is a crucial first step of DNA-based metagenomic analyses of environmental samples. Insufficient quantity and purity of DNA isolated using commercial kits result in missing genetic information, especially for large-diameter substrates in constructed wetlands (CWs). Here, we addressed this problem by devising a cost-effective calcium chloride lysozyme-sodium dodecyl sulfate (SDS) method (CCLS), with key improvements in the steps of humic acid removal and cell lysis. The buffer comprising Tris, EDTA, Na₂O₂P₇ and PVPP (TENP), and skim milk, could reduce adsorption between microorganisms and substrates, and calcium chloride precipitated and removed over 94% of humic acid. This humic acid removal step, when compared to the PowerSoil DNA kit (MO BIO Laboratories Inc.) (MBKIT), significantly enhanced the DNA purity (A260/230) from 0.68 to 1.63 (p < 0.01). When gentle and extended cell lysis in CCLS replaced the short but violent bead-beating in the MBKIT, DNA yield and the amount of lysed bacteria detected by quantitative real-time polymerase chain reaction (qPCR) on average increased by 2 and 4 folds, respectively, compared to that obtained using the MBKIT (p < 0.01). Furthermore, the full-length bacterial 16S rRNA gene and nirK gene from denitrifying microorganisms were successfully amplified from CCLS-generated DNA. Additionally, bacterial diversity indices of richness, Shannon, and evenness examined by denaturing gradient gel electrophoresis (DGGE) increased by 75, 30, and 7%, respectively, by CCLS compared to that using the MBKIT. Hence, the CCLS method enables improved evaluation of microbial density and diversity in CW systems.

     

Assessment of rhizosphere processes for removing water-borne macrolide antibiotics in constructed wetlands

Plant and Soil - Limited information is available on plant rhizosphere processes for removing antibiotics in antibiotic-contaminated waters. This study identifies rhizosphere processes and...