Testing Different Membrane Filters for 16S rRNA Gene-Based Metabarcoding in Karstic Springs

Introduction: Karstic springs are used worldwide by rural communities as sources of fresh water for humans and livestock. In Romania, one-third of the population has no direct access to a public water supply. The present study is part of a country-wide project to develop simple, quick and cheap methods for seasonal environmental and microbiological monitoring of karstic springs used as drinking water by rural populations. Critical steps for monitoring workflow consist of evaluating water quality and selecting suitable membrane filters to efficiently capture environmental DNA for further microbial diversity estimation using 16S rRNA gene-based metabarcoding. Methods: Several commercial membrane filters of different compositions and pore sizes were tested on the water sampled from three karstic springs in Romania, followed by water chemistry and whole community 16S rRNA gene-based metabarcoding analysis. Results: We found that different types of applied membrane filters provide varying recovery in diversity and abundance of both overall and pathogenic bacteria. Conclusions: The result of the experiment with different filters shows that mixed cellulose ester, cellulose acetate, and nitrate membranes of 0.20 and 0.22 mu m are the best for amplicon-based metabarcoding monitoring of karst springs.