Suitability and setup of next-generation sequencing-based method for taxonomic characterization of aquatic microbial biofilm

Abstract

A robust and widely applicable method for sampling of aquatic microbial biofilm and further sample processing is presented. The method is based on next-generation sequencing of V4-V5 variable regions of 16S rRNA gene and further statistical analysis of sequencing data, which could be useful not only to investigate taxonomic composition of biofilm bacterial consortia but also to assess aquatic ecosystem health. Five artificial materials commonly used for biofilm growth (glass, stainless steel, aluminum, polypropylene, polyethylene) were tested to determine the one giving most robust and reproducible results. The effect of used sampler material on total microbial composition was not statistically significant; however, the non-plastic materials (glass, metal) gave more stable outputs without irregularities among sample parallels. The bias of the method is assessed with respect to the employment of a non-quantitative step (PCR amplification) to obtain quantitative results (relative abundance of identified taxa). This aspect is often overlooked in ecological and medical studies. We document that sequencing of a mixture of three merged primary PCR reactions for each sample and further evaluation of median values from three technical replicates for each sample enables to overcome this bias and gives robust and repeatable results well distinguishing among sampling localities and seasons.

Marine biofilms on different fouling control coating types reveal differences in microbial community composition and abundance

“…The V4‐V5 region of the 16S rRNA gene has been one of the most broadly used variable regions in studies examining environmental biofilms on artificial surfaces (e.g., Bakal et al, 2018 ; Li et al, 2017 ; Pereira et al, 2017 ), while 515F/926R has been suggested as a primer set that increases percentage detection of various prokaryotic taxa (Pollet et al, 2018 ) as well as been the most effective region in minimizing overestimation due to intragenomic heterogeneity (Sun et al, 2013 ).…”

Marine biofilms on different fouling control coating types reveal differences in microbial community composition and abundance

“…The V4-V5 region of the 16S rRNA gene has been one of the most broadly used variable regions in studies examining environmental biofilms on artificial surfaces (e.g. Li et al ., 2017; Pereira et al ., 2017; Bakal et al ., 2018), while 515F/926R has been suggested as a primer set that increases percentage detection of various prokaryotic taxa (Pollet et al ., 2018) as well as been the most effective region in minimising overestimation due to intragenomic heterogeneity (Sun et al ., 2013).…”

A Modular Coupling System for Efficient Treatment of Rural Domestic Wastewater

“…Submerged plants are planted in the submerged oxygen pond, which can use the photosynthesis and respiration of submerged plants to build an ecological food chain system and realize the deep purification of water quality. Pebbles are preferably paved in the multi-stage falling water oxygenation ecological ditch, and they lay in a single ecological ditch facilitate the propagation and growth of biofilm on pebbles and promote the occurrence of nitrification reaction [25][26][27]. Aquatic plants are planted at the junction of the two ecological ditches and where the water flow is slow to form a multi-functional water drop oxygenation and ecological ditch, strengthen the purification effect, beautify the environment and increase economic benefits.…”