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

Oana Teodora Moldovan,Edina Szekeres,Horia Leonard Banciu,Marius Kenesz,Erika Andrea Levei,Marial Alexandra Hoaghia,Andreea Baricz,Ruxandra Năstase-Bucur,Iulia Chiciudean,Traian Brad,Ionuț Cornel Mirea

doi:10.3390/w12123400

Abstract

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 µm are the best for amplicon-based metabarcoding monitoring of karst springs.

Highlights

Karstic springs are used worldwide by rural communities as sources of fresh water for humans and livestock
We aimed to evaluate the most suitable membrane filter type and filtration strategy to efficiently recover environmental DNA for further fast and accurate whole community diversity assessment using 16S rRNA gene-based metabarcoding
Nine different types of membrane filters with different pore sizes were tested for their efficiency in retaining biomass that sourced for Environmental DNA (eDNA) from karstic spring water (Table 1)

Summary

Introduction

Karstic springs are used worldwide by rural communities as sources of fresh water for humans and livestock. 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 μm are the best for amplicon-based metabarcoding monitoring of karst springs. The 2006–2017 Eurostat survey (published in 2020, [5]) shows that 5 out of 12 European countries have less than 90% of households connected to a public water supply, with Romania ranked at the lowest percentage of 67.5%. One cause of this shortcoming might be the sparse distribution of houses in most rural areas that makes access to a public water supply problematic if not impossible. Small rural populations use local sources of water that are not under the monitoring programs of water agencies

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Discussion

Conclusion