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Opening the black box of spring water microbiology from alpine karst aquifers to support proactive drinking water resource management

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Over the past 15 years, pioneering interdisciplinary research has been performed on the microbiology of hydrogeologically well‐defined alpine karst springs located in the Northern Calcareous Alps (NCA) of Austria. This article gives an overview on these activities and links them to other relevant research. Results from the NCA springs and comparable sites revealed that spring water harbors abundant natural microbial communities even in aquifers with high water residence times and the absence of immediate surface influence. Apparently, hydrogeology has a strong impact on the concentration and size of the observed microbes, and total cell counts (TCC) were suggested as a useful means for spring type classification. Measurement of microbial activities at the NCA springs revealed extremely low microbial growth rates in the base flow component of the studied spring waters and indicated the importance of biofilm‐associated microbial activities in sediments and on rock surfaces. Based on genetic analysis, the autochthonous microbial endokarst community (AMEC) versus transient microbial endokarst community (TMEC) concept was proposed for the NCA springs, and further details within this overview article are given to prompt its future evaluation. In this regard, it is well known that during high‐discharge situations, surface‐associated microbes and nutrients such as from soil habitats or human settlements—potentially containing fecal‐associated pathogens as the most critical water‐quality hazard—may be rapidly flushed into vulnerable karst aquifers. In this context, a framework for the comprehensive analysis of microbial pollution has been proposed for the NCA springs to support the sustainable management of drinking water safety in accordance with recent World Health Organization guidelines. Near‐real‐time online water quality monitoring, microbial source tracking (MST) and MST‐guided quantitative microbial‐risk assessment (QMRA) are examples of the proposed analytical tools. In this context, this overview article also provides a short introduction to recently emerging methodologies in microbiological diagnostics to support reading for the practitioner. Finally, the article highlights future research and development needs. This article is categorized under: Engineering Water > Water, Health, and Sanitation Science of Water > Water Extremes Water and Life > Nature of Freshwater Ecosystems
Schematic illustration of the suggested “framework for integrated fecal pollution analysis and management” to guide spring water quality management in accordance with the World Health Organization criteria (for details, see main text). Target‐oriented catchment protection and optimized spring water‐abstraction management helps to minimize the required treatment and disinfection efforts and to maximize the biostability of the spring water to provide supply with high‐quality drinking water. The three interacting levels (“three‐step approach”) include (1) the monitoring of potential fecal pollution, (2) the characterization and identification of potential fecal pollution sources in the catchment, and (3) the assessment of the actual health risk from human exposure (drinking), in relation to the fecal source(s). MST, microbial source tracking; QMRA, quantitative microbial‐risk assessment; PSP, pollution source profiling; SFIB, standard fecal indicator bacteria
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Schematic illustration of a cross‐sectional view into a conduit in the phreatic zone of a limestone karst aquifer (cf. Figure ) during base‐flow conditions (a) and under increased discharge conditions due to rapid surface water input during a precipitation event in the catchment (b). The figures indicate the postulated distribution of the most important microbial compartments in these systems, including the suspended fraction in the water column as well as the fraction attached to the conduit walls or alluvial sediments. The proposed differences between the base‐flow and the high‐discharge event situation are (1) an increased mobilization of rock biofilm‐ and sediment‐associated microbes due to the increased shear stress and (2) increased numbers of surface‐associated and transiently occurring microbes (red) infiltrating from the surface (for details, see main text). Red‐colored microbes (bacteria, archaea, protozoa, bacteriophages) represent the transient microbial endokarst communities (TMEC); all other colors (yellow, orange, green, blue) show the autochthonous microbial endokarst community (AMEC), whereas the diversity in colors shall reflect the proposed diversity of these naturally occurring microbes
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Previously unpublished scanning electron microscope (SEM) images from in situ colonization experiments in a spring housing under dark in situ conditions, using freshly sliced natural limestone disks prepared from rock material as found in the DKAS1 catchment (“Wettersteindolomit”) as growth substrates. The hardware setup for the incubation experiment is given in Figure . The pictures show the change of the rock surface during 1 year of incubation in the DKAS1 spring water (1, 6, and 12 months). The increasing amount of particle accumulation or formation on the rock surfaces can be seen. Additionally, the first indications of organic structures became visible (e.g., 12 months). The scale bars indicate the extent of magnification
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Hardware setup for the investigation of microbial colonization. Experiment examining rock surfaces under dark in situ incubation conditions; (1) The water inlet directly from spring DKAS1; (2) the inlets to each of the four inert incubation chambers containing the freshly sliced autochthonous rock disks; and (3) the actual incubation chambers. All parts were built of corrosion‐resistant and inert steel material
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Epifluorescence microscope images of microbial communities in spring water of the limestone karst aquifer 2 (LKAS2) and the dolomite karst aquifer 1 (DKAS1). Panel (a) shows typical prokaryotic cells in the spring water of the DKAS1 system at 1000× magnification, stained with SYBR® Gold nucleic acid gel stain; panel (b) shows prokaryotic communities in the spring water of LKAS2 (large picture), and an example of a detached “biofilm‐floc” (inset, e); the red arrow in panel (c) highlights an example of an heterotrophic nanoflagellate in LKAS2, detected by DAPI fluorescence staining at a magnification of 400×; panel (d) shows an image of bacteriophages, also at 1000× magnification and stained with SYBR® Gold nucleic acid gel stain. The scale bars indicate a width of 5 μm (a, b, and d) and 12.5 μm (c), respectively. The analysis was made with a Nikon Eclipse 80i fluorescence microscope using fluorescence dyes for the detection of microbial cells or phages after they were filtered on filters
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Figure summarizing methods commonly applied for the specific or nonspecific detection or quantification of microorganisms in environmental samples (for details see main text). Panel (a) schematically illustrates the traditional cultivation‐based method commonly applied in water quality monitoring, which works by growing and detecting cultivable microbes on artificial growth media. Panel (b) shows the principle as well as the most commonly used applications for the direct optical detection of microbial cells. The upper scheme illustrates the principle for detecting total cell counts based on nonspecific fluorescence dyes. The graphic below illustrates the specific labeling of particular groups of microbes. Panel (c) summarizes commonly applied methods for the (quantitative) detection of microorganisms by DNA/RNA amplification, DNA/RNA sequencing, and community profiling. The figure shows a schematic illustration of a double‐stranded DNA helix (top) and the corresponding DNA‐sequence (below). DNA, deoxyribonucleic acid; a, adenine; T, thymine; C, cytosine; G, guanine; (q)PCR, (quantitative real‐time) polymerase‐chain‐reaction; HTS, high‐throughput sequencing; DGGE, denaturing gradient gel electrophoresis; T‐RFLP, terminal restriction fragment length polymorphism; and FISH, fluorescence in situ hybridization
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Schematic illustration of alpine karst systems with differing hydrogeological backgrounds, with “LKAS” representing a typical spring of a limestone karst aquifer, and “DKAS” representing a typical spring of a dolomite karst aquifer system. Dashed lines indicate the transition zones between the saturated phreatic, the temporarily flooded epiphreatic, and the unsaturated vadose zones
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Installation of an optical s::can probe (blue device; scan Messtechnik GmbH, Wien, Austria) at a spring environment for near‐real‐time online measurement of turbidity and SAC254 (spectral absorption coefficient at 254 nm)
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Water and Life > Nature of Freshwater Ecosystems
Science of Water > Water Extremes
Engineering Water > Water, Health, and Sanitation

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