Assessment of Microbial Community Dynamics in River Bank Filtrate Using High-Throughput Sequencing and Flow Cytometry

Abstract

Surface-groundwater interactions play an important role in microbial community compositions of river bank filtrates. Surface water contaminations deriving from environmental influences are attenuated by biogeochemical processes in the hyporheic zone, which are essential for providing clean and high-quality drinking water in abstraction wells. Characterizing the flow regime of surface water into the groundwater body can provide substantial information on water quality, but complex hydraulic dynamics make predictions difficult. Thus, a bottom up approach using microbial community shifting patterns as an overall outcome of dynamic water characteristics could provide more detailed information on the influences that affect groundwater quality. The combination of high-throughput sequencing data together with flow cytometric measurements of total cell counts reveals absolute abundances among taxa, thus enhancing interpretation of bacterial dynamics. 16S rRNA high-throughput sequencing of 55 samples among six wells in a well field in Austria that is influenced by river bank filtrate within a time period of 3 months has revealed both, clear differences as well as strong similarity in microbiome compositions between wells and dates. A significant community shift from April to May occurred in four of six wells, suggesting that surface water flow regimes do affect these wells stronger than others. Triplicate sampling and subsequent sequencing of wells at different dates proved the method to be reproducible. Flow cytometric measurements of total cells indicate microbial shifts due to increased cell counts and emphasize the rise of allochthonous microorganisms. Typical freshwater bacterial lineages (Verrucomicrobia, Bacteroidetes, Actinobacteria, Cyanobacteria, Armatimonadetes) were identified as most increasing phyla during community shifts. The changes are most likely a result of increased water abstraction in the wells together with constant river water levels rather than rain events. The results provide important knowledge for future implementations of well utilization in dependency of the nearby Danube River water levels and can help drawing conclusions about the influence of surface water in the groundwater such that hygienically save and clean drinking water with a stable microbial community can be provided.

“Every cell counts”—experiences with flow cytometry for Austrian drinking water supply

“…3. Separation of LNA and HNA should rely on the density Details of the methodology for sample handling and flow cytometric measurements used in the case studies are given in Fiedler et al (2018) and . In short, 50 ml of sample were collected in sterile centrifuge tubes (VWR, United States), kept cool at 4 °C and processed within 48 h. For the measurement 297 µl of well mixed sample were either stained with 3 µl of SYBR Green I (Life Technologies, Eugene, OR, United States) diluted 1:100 with DMSO (Fluka, Switzerland) for TCC or with a mixture of SYBR Green I and propidium iodide (6 µM final concentration) diluted 1:100 with 10 mM Tris buffer (pH = 8.1) for ICC.…”

“…The combination of FC data with microbial community analysis based on High-throughput-sequencing (HTS) might provide additional insights that neither of the methods might bring on their own. Community analysis based on DNA-sequencing solely, will always end with relative abundances of the detected microbial community, whereas merging relative abundances with FC data allows for the calculation of estimated absolute abundances (Fiedler et al 2018). Otherwise, sequencing data might shed light onto clusters appearing or changing in FC plots.…”

Advancing Microbial Risk Assessments of Subsurface Water Sources

“…750 mL aliquots of the groundwater samples were filtered through EMD Millipore Sterivex polyethersulfone 0.22 µm syringe filters (Millipore, Massachusetts, USA), stored in dedicated Whirl-Pak TM (Nasco c , Fort Atkinson, Wisconsin, USA) and kept in a -80 • C freezer until DNA extraction. A commercial kit (DNeasy PowerWater Sterivex Kit, Qiagen, MO BIO Laboratories, USA) was then used to extract the DNA (e.g., (Fiedler et al, 2018)). 16S rRNA gene fragments were amplified and labelled with a unique, sample-specific multiplex-identifier ("barcode") in a PCR-based one step barcoding procedure.…”

“…Clustering-independent approaches for determining amplicon sequence variants (ASVs, also known as sub-OTUs (Knight et al, 2018)) have been more recently advocated to replace OTUs given their ability to distinguish subtle but real biological sequence variants (Callahan et al, 2017). While ASVs undoubtedly contribute to increased differentiation of taxa at the species and sub-species levels (which may be fundamentally important in cladistics-based studies), appreciable differences in bacterial community composition related to changing source water quality conditions appear to be captured at higher taxonomic classifications (e.g., class/order) across many studies (Lin et al, 2012;Flynn et al, 2013;Ben Maamar et al, 2015;Gülay et al, 2016;Graham et al, 2017;Pogoda, 2017;Lee et al, 2018;Fiedler et al, 2018). Accordingly, a phenetics-based OTU approach was applied.…”

“…Although there is no evidence that a community endemic to groundwater sources exists or has been described (Griebler and Lueders, 2009), differences in groundwater and surface water bacterial communities may be used to evaluate the vulnerability of subsurface water sources to fecal pathogen intrusion (e.g. Lin et al, 2012;Ben Maamar et al, 2015;Braun et al, 2016;Graham et al, 2017;Pogoda, 2017;Fiedler et al, 2018). Savio et al (2019) observed dynamic response of Flavobacteriia proportions in karst aquifer water bacterial core community during recharge events.…”