WIREs Water - 2018 - Savio - Opening The Black Box of Spring Water Microbiology From Alpine Karst Aquifers To Support

Received: 25 April 2017 Revised: 25 January 2018 Accepted: 26 January 2018
DOI: 10.1002/wat2.1282
OVERVIEW
Opening the black box of spring water microbiology from alpine

karst aquifers to support proactive drinking water resource
management
Domenico Savio1,2 | Philipp Stadler2,3 | Georg H. Reischer4,5 | Alexander K.T. Kirschner5,6 |
Katalin Demeter2,4 | Rita Linke4,5 | Alfred P. Blaschke2,5,7 | Regina Sommer5,6 |
Ulrich Szewzyk8 | Inés C. Wilhartitz9 | Robert L. Mach4 | Hermann Stadler10* |
Andreas H. Farnleitner1,4,5
1
Division Water Quality and Health, Department
Pharmacology, Physiology and Microbiology, Over the past 15 years, pioneering interdisciplinary research has been performed on
Karl Landsteiner University of Health Sciences, the microbiology of hydrogeologically well-defined alpine karst springs located in
Krems a. d. Donau, Austria
the Northern Calcareous Alps (NCA) of Austria. This article gives an overview on
2
Centre for Water Resource Systems, Technische
these activities and links them to other relevant research. Results from the NCA
Universität Wien, Vienna, Austria
3
springs and comparable sites revealed that spring water harbors abundant natural
Institute for Water Quality, Resource and Waste
Management, Technische Universität Wien, microbial communities even in aquifers with high water residence times and the
Vienna, Austria absence of immediate surface influence. Apparently, hydrogeology has a strong
4
Institute of Chemical, Environmental & impact on the concentration and size of the observed microbes, and total cell counts
Bioscience Engineering, Research Group (TCC) were suggested as a useful means for spring type classification. Measurement
Environmental Microbiology and Molecular
Diagnostics, 166/5/3, Technische Universität of microbial activities at the NCA springs revealed extremely low microbial growth
Wien, Vienna, Austria rates in the base flow component of the studied spring waters and indicated the
5
Interuniversity Cooperation Centre for Water and importance of biofilm-associated microbial activities in sediments and on rock sur-
Health, www.waterandhealth.at faces. Based on genetic analysis, the autochthonous microbial endokarst community
6
Unit Water Hygiene, Institute for Hygiene and (AMEC) versus transient microbial endokarst community (TMEC) concept was pro-
Applied Immunology, Medical University of
Vienna, Vienna, Austria posed for the NCA springs, and further details within this overview article are given
7
Institute of Hydraulic Engineering and Water to prompt its future evaluation. In this regard, it is well known that during high-
Resources Management, Technische Universität discharge situations, surface-associated microbes and nutrients such as from soil
Wien, Vienna, Austria habitats or human settlements—potentially containing fecal-associated pathogens as
8
Department of Environmental Technology, the most critical water-quality hazard—may be rapidly flushed into vulnerable karst
Technical University of Berlin, Berlin, Germany
9
aquifers. In this context, a framework for the comprehensive analysis of microbial
Department of Environmental Microbiology,
Eawag, Swiss Federal Institute of Aquatic Science
pollution has been proposed for the NCA springs to support the sustainable manage-
and Technology, Dübendorf, Switzerland ment of drinking water safety in accordance with recent World Health Organization
10
Department for Water Resources Management guidelines. Near-real-time online water quality monitoring, microbial source track-
and Environmental Analytics, Institute for Water, ing (MST) and MST-guided quantitative microbial-risk assessment (QMRA) are
Energy and Sustainability, Joanneum Research,
examples of the proposed analytical tools. In this context, this overview article also
Graz, Austria
*This article is dedicated to Hermann Stadler, the founder of the field of integrated karst hydrology in Austria. He died too young and against any expecta-
tions in 2016.
Abbreviations: AMEC, Autochthonous microbial endokarst community; DKAS, Dolomite karst aquifer spring; DNA, Deoxyribonucleic acid; EM, Epifluor-
escence microscopy; FCM, Flow cytometry; FISH, Fluorescence in situ hybridization; HPC, Heterotrophic plate counts; HTS, High-throughput sequencing;
LKAS, Limestone karst aquifer spring; MST, Microbial source tracking; NCA, Northern Calcareous Alps; PSP, Pollution source profiles; QMRA, Quantita-
tive microbial-risk assessment; qPCR, Quantitative real-time polymerase chain reaction; RNA, Ribonucleic acids; SFIB, Standard fecal indicator bacteria;
TCC, Total cell counts/concentrations; TMEC, Transient microbial endokarst community; VBNC, viable but not cultivable
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any
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© 2018 The Authors. WIREs Water published by Wiley Periodicals, Inc.
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2 of 20 SAVIO ET AL.
Correspondence
Andreas H. Farnleitner, Division Water Quality provides a short introduction to recently emerging methodologies in microbiological
and Health, Department Pharmacology, diagnostics to support reading for the practitioner. Finally, the article highlights
Physiology and Microbiology, Karl Landsteiner
future research and development needs.
University of Health Sciences, Dr.-Karl-Dorrek-
Straße 30, 3500 Krems a. d. Donau. This article is categorized under:
Email: andreas.farnleitner@kl.ac.at
Engineering Water > Water, Health, and Sanitation
Funding information
GWRS-Vienna research project (Vienna Water,
Science of Water > Water Extremes
MA31); Austrian Science Fund (FWF): project P Water and Life > Nature of Freshwater Ecosystems
23900 & project W 1219 (Vienna Doctoral
Programme on Water Resource Systems) KEYWORDS
alpine karst aquifers, spring water microbiology, natural spring water microbes,
external fecal pollution, drinking water resource protection, water abstraction
management, online water quality monitoring, fecal indicator, microbial fecal
source tracking, microbial risk assessment, future challenges
1 | INTRODUCTION
Alpine karst aquifers play a vital role in the drinking water supply in many regions throughout the world (Ford & Williams,
2007a). For example, at least 95% of the drinking water demand of the City of Vienna is provided by such ground water
resources, and more than 1.6 million Viennese inhabitants appreciate the high-quality drinking water from the nearby mountain
regions that is directly delivered to their households (Griebler & Avramov, 2015; ten Brink et al., 2011). Maintaining the highest
quality standards requires coordinated and information-driven efforts, including the sustainable protection of the catchments, opti-
mized spring-abstraction management, and sufficient final treatment. For example, it is well known that during precipitation
events, karst springs can be very rapidly influenced by microbes originating from fecal pollution from the surface (Bucci, Petrella,
Naclerio, Allocca, & Celico, 2015; Pronk, Goldscheider, & Zopfi, 2006; Stadler et al., 2008). Thus, an adequate understanding of
the potential factors affecting microbiological water quality is of utmost importance to guide a target-oriented and sustainable
water resource management. Until recently, however, in particular the information on the microbiological water quality of alpine
karst springs has mainly been sourced from a fragmentary puzzle of routinely performed surveillance activities. In-depth knowl-
edge on the different aspects of microbiological water quality was largely missing for alpine karst springs. Thus, intensive
research activities on the microbiology of alpine karst springs were prompted at the Northern Calcareous Alps (NCA) of Austria
at the start of the new millennium. Thereby, a detailed knowledge on the hydrogeological background of the studied alpine karst
model systems was regarded as a basic and essential requirement for the interdisciplinary research efforts.
The karst springs in focus of this overview article are typical springs of the alpine karst regions (cf. Figure 1). The term
alpine karst, as used in this article, refers to karst formations formed in areas of high altitude and relief (Field, 1999). The
definition of alpine karst is therefore not geographically restricted to the karst landscapes of the Alps (Ford, 1971; Günay,
2010; Ozyurt & Bayari, 2008), it generally relates to karst systems of regions determined by mountainous topography and
high altitude climate (Figure 1). In large parts, alpine karst systems are vulnerable to fecal contamination, where vegetation
and soil covers are thin or not abundant. Point recharge in respective areas may be enhanced due to overlaying geological
layers, as well as thrust and fold structures (Goldscheider, 2005, 2010; White, 1969). Distinct hydrograph peaks during early
summer months caused by snow melt in the hydrogeological catchment area are symptomatic for karst springs draining
alpine karst systems of temperate latitudes (Stadler et al., 2008, 2010).
The aim of this article is to give an overview on the developed methodologies and the established knowledge on the NCA
spring water microbiology that has been studied over more than 15 years. The results will also be set into context of literature
from other relevant areas and future research requirements. Special emphasis is also given to components with importance for the
water quality management of drinking water supplies. It has to be mentioned that this work is not designed as a conventional in-
depth review on general karst microbiology. To keep this work digestible for a practitioner in the field of water resource manage-
ment, also a short introduction to recently emerging methodologies in the field of microbiological diagnostics will be given.
2 | E S T AB L I S H E D S T AN D A R D A N D E M E RG I N G M E T H O D S I N M I C R O B I O L O G I C A L
D IA GN O S T I CS
At a basic level, microorganisms are defined as organisms that are invisible to the naked eye (<150 μm). The first
methods—based on cultivation on artificial growth media—were introduced in the second half of the 19th century (Koch,
SAVIO ET AL. 3 of 20
FIGURE 1 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
1882). Thereby, the detection is based on cell division, which results in the formation of colonies that are large enough to be
detected by the naked eye (Figure 2a). Even today, most standardized detection methods for health-related water quality test-
ing such as for the detection of fecal indicator bacteria are based on this highly sensitive and straightforward enumeration
principle (International Organisation for Standardisation, 1999, 2000a, 2000b; Reasoner & Geldreich, 1985). However, most
microbes occurring in aquatic habitats cannot be grown on standard cultivation media as they require very specialized and
often unknown growth conditions (Staley & Konopka, 1985). Besides missing knowledge on the optimal growth conditions
of most bacteria, additional challenges in the cultivation may arise from bacteria being in a dormant or so-called viable but
not cultivable (VBNC)-state (Königs, Flemming, & Wingender, 2015; Li, Mendis, Trigui, Oliver, & Faucher, 2014; Oliver,
(a) Cultivation-based (b) Direct cell (c) Nucleic acid

detectiion detectiion detection
Multiplication by Double-stranded
cell division (ds) DNA
Total cell
count
Principle
t0: 20 cells t1: 21cells t2: 22 cells
Target-
specific FISH
t3: 23 cells t4: 24 cells t5: 25 cells
Cultivation on defined
growth medium Optical detection DNA Profiling/
Amplification
systems sequencing Fingerprinting
Methods
Microscopy PCR Sanger DGGE

Flow cytometry qPCR HTS T-RFLP
Solid phase microarray
cytometry (Chip)
FIGURE 2 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
2010; Xu et al., 1982). In the field of microbiological water quality monitoring, the difficulties to cultivate or detect par-
ticular microbes of interest (e.g., intestinal pathogens) resulted in the development of the so-called indicator bacteria con-
cept (Bonde, 1966), where alternative organisms are grown and used as a proxy for the microorganism of interest.
In contrast to cultivation-based methods, direct cell-based investigation methods such as the traditional microscopy-
based approach overcome the necessity of growth, using optical systems to detect or quantify cells directly in the water
phase or on a filter surface after filtration or enrichment (Figure 2b). The latest developments of automated optical sys-
tems such as flow cytometry (FCM) or solid-phase cytometry even support the automated enumeration of the total num-
ber of microbial cells (total cell counts, TCC) in water samples (Besmer et al., 2014, 2016; Hammes et al., 2008;
Hoefel, Grooby, Monis, Andrews, & Saint, 2003; Page et al., 2017; Riepl et al., 2011). In a very recent review, Van
Nevel et al. (2017) even initiated a discussion on whether FCM could replace a standard cultivation-based method (het-
erotrophic plate counts, HPC) in routine water quality monitoring. One major challenge when working with environmen-
tal microorganisms is that their variability in terms of morphological appearance and shape is highly restricted (Young,
2007). Consequently, with exceptions, different groups or species cannot be reliably differentiated by conventional
cell-based detection methods such as FCM or traditional epifluorescence microscopy (EM). To distinguish or identify
particular microbes of interest, microscopy, or FCM has to act in concert with specific cell-labeling methods (DeLong,
Wickham, & Pace, 1989; Pickup, 1991; Schloter, Aßmus, & Hartmann, 1995). Many labeling techniques such as fluo-
rescence in situ hybridization (FISH; Amann, Krumholz, & Stahl, 1990; Pernthaler, Pernthaler, & Amann, 2002;
Wagner & Haider, 2012) are based on the power of targeting specific intracellular nucleic acid molecules (Figure 2b,
see the following paragraph).
As the most essential biomolecule in all organisms, DNA (deoxyribonucleic acid) contains the genetic information
(“construction plan”) of every single cell. Its informational content is determined by the sequential order of the building
blocks (nucleotides) Guanine (G), Adenine (A), Cytosine (C), and Thymine (T), forming a four-letter-based “code” com-
monly referred to as a “DNA sequence” (Figure 2c). A typical bacterial genome, for example, is comprised of 106–107
nucleotides, which equals approximately 1–10 MB when translated into computer language. This quantity highlights the
enormous informational capacity stored in a tiny molecule of approximately 2 nm in diameter and approximately 1.4 mm
in length. This encoded information is used for the specific identification and analysis of microbes by nucleic acid-
targeting technologies. Potential target nucleic acids include not only DNA but also RNA—a second type of nucleic acid.
In contrast to the above-mentioned labeling techniques, short fragments of DNA/RNA molecules (up to a maximum of
several hundred base pairs in length) can also be specifically detected by nucleic acid-based amplification techniques. For
example, one of the commonly applied approaches to quantify microbes based on their characteristic DNA fragments is
the so-called quantitative real-time polymerase chain reaction (qPCR; Figure 2c). This method is also commonly applied
in so-called microbial source tracking (MST), where pollution source-associated bacteria (such as bacteria occurring in par-
ticular animals like ruminants or pigs) are targeted to investigate their host’s contribution to fecal pollution. For a compre-
hensive review of currently available MST tools targeting bacteria of the order Bacteroidales, see the work by Ahmed,
Hughes, and Harwood (2016). In contrast to the quantification of known DNA targets, the revolutionary invention of
nucleic acid sequencing allows to “read” the informative content stored within the four-letter code of previously unknown
DNA/RNA (Sanger, Nicklen, & Coulson, 1977). This capability to read the informative content of DNA has soon also
been utilized in combination with so called “community profiling” or “fingerprinting” methods, which until recently were
commonly applied to characterize microbial populations in environmental samples (Figure 2c). These methods generate
characteristic patterns from bacterial communities based on the differences in nucleotide composition and/or the length of
the investigated nucleic acid fragments, and further enable their isolation and individual analysis by DNA sequencing
(Burtscher, Zibuschka, Mach, Lindner, & Farnleitner, 2009; Farnleitner et al., 2000, 2004; Lee, Zo, & Kim, 1996; Liu,
Marsh, Cheng, & Forney, 1997; Muyzer, de Waal, & Uitterlinden, 1993; Muyzer & Smalla, 1998). Another very recent
and ground-breaking invention was the rise of novel high-throughput sequencing (HTS) techniques, which once again rev-
olutionized the entire field of bioscience by providing the capability to analyze millions of nucleic acid sequences in paral-
lel within a few hours (Rothberg & Leamon, 2008). As a consequence, nucleic acid sequence data analysis based on
bioinformatical tools emerged as a new subdiscipline in microbiology, and the required know-how and processing time to
handle the huge amounts of generated data are increasingly considered a “bottleneck” of large field investigations (Carlos,
Tang, & Pei, 2012; Scholz, Lo, & Chain, 2012). The application of this novel methodology to water samples of all kinds
enables an in-depth characterization of their microbial communities’ composition, and has recently also been applied in
combination with FCM to study the temporal community dynamics in the context of the biostability2 of water (Prest
et al., 2014).
Besides these cell-based and molecular biological methods, other available methods increasingly applied in water quality
monitoring such as adenosine triphosphate (ATP) detection (Vang, Corfitzen, Smith, & Albrechtsen, 2014) make a trade-off
between reduced specificity but faster detection/higher time resolution (Højris, Christensen, Albrechtsen, Smith, & Dahlqvist,
2016; Lopez-Roldan, Tusell, Cortina, Courtois, & Cortina, 2013).
3 | THE G EN ER AL CHA RA CT ERIST IC S O F K AR ST SYST EMS
Karst denominates a specific kind of landscape with soluble rocks in the underground, such as carbonates (e.g., limestone,
dolomite), that developed an extensive underground water system containing complex cavity- and cave structures (Ford &
Williams, 2007b; cf. Figure 1). Karst comprises terrains with distinct hydrology and landforms, characterized by sinking
streams, caves, enclosed depressions (e.g., dolines), fluted rock outcrops and large springs (Ford & Williams, 2007b; Kre-
sic & Stevanovic, 2010). Karst generation is determined not only by the rock solubility, but rock structures, such as thrusts
and folds and stratigraphy, as well as leveling of the receiving water bodies are also significant (Ford, 1971; Ford & Wil-
liams, 2007b; White, 1969). Karst ground water systems evolve over time, distinguishing it from other groundwater systems,
as karst water flow becomes increasingly turbulent due to the progressive solutional enlargement of void space (Ford & Wil-
liams, 2007b). Consequently, equations that can be used to describe laminar water flow in porous aquifers become inapplica-
ble to karst (Ford & Williams, 2007b). As karst landforms develop, the karst water level is striving for the level of the
receiving water body. It is therefore that a vertical sequence of caves and springs can often be found on hill slopes of karst
landscapes. Lower situated springs draining the phreatic zone are constantly discharging (Figure 1). During hydrological
events, higher situated springs act as the karst system’s overflow and are draining the epiphreatic zone (cf. Figure 1). Such
springs fall dry during base flow conditions. The complex and hydraulically connected network of underground voids, cavi-
ties and caves determines the hydrogeological catchment. The hydrogeological catchment of a karst spring is not determined
by surface topography and can therefore exceed the orographical catchment area significantly (Goldscheider, 2005; Stadler
et al., 2008). Karst systems often show a prompt and direct hydrological response to precipitation events, resulting in high-
discharge dynamics of springs (Kresic & Bonacci, 2010; Stadler et al., 2010). Thin or absent soil covers in combination with
extended rock cavities prevent an effective natural purification of infiltrating water. Surface-associated pollution can be intro-
duced almost unhindered into the groundwater (Eckhardt, 2010; Goldscheider, 2010; Kresic, 2010). Therefore, such karst
landscapes are highly vulnerable systems in which surface-associated contamination has rapid and direct impact on the water
quality (Farnleitner et al., 2005; Sinreich, Pronk, & Kozel, 2014).
4 | SEL ECT ED MODE L C ATC H MEN TS, HYD R O G E O L O G Y , A N D H Y D R O L O G Y - G U I D E D

I N V E S T IG A T I ON S
The five selected alpine karst springs in focus of this overview article are situated at different locations in the eastern part of
the NCA of Austria, with catchment sizes ranging from 4 to 60 km2 (Farnleitner et al., 2005, 2011; Reischer et al., 2011).
Catchments reach a maximum altitude of approximately 2,300 m above sea level (m.a.s.l.), with wide plateaus and steep
slopes. Springs are situated close to their recipients at altitudes between 500 and 800 m.a.s.l. Alpine pastures, krummholz
areas and alpine forests are the main land-use features (Dirnböck, Dullinger, Gottfried, & Grabherr, 1999). Tourism activities
(hiking and mountaineering), agriculture based on summer pastures (mainly cattle), and wildlife represent the potential
(fecal) pollution sources (Reischer et al., 2011). The lithology of the Triassic sediments ranges from different limestones
(Wettersteinkalk and Dachsteinkalk) to dolomite (mainly Wettersteindolomit; Bryda, n.d.). Although the investigated areas
feature intensive karstification, direct access to the karst aquifer and its water table is not provided. Caves are widely scat-
tered, but spacious caves in direct connection with the investigated springs are not present (Plan, Hartmann, & Hart-
mann, 2016).
The four limestone karst aquifer springs (LKAS2, 4, 6, 8) and one dolomite karst aquifer spring (DKAS1) in the NCA
region were investigated in detail for several years (for a schematic cross section, see Figure 1). Infield online sensors
installed at all selected spring sites for the continuous measurement of discharge and other physicochemical parameters (tem-
perature, pH, electrical conductivity, turbidity, spectroscopic absorption at 254 nm, etc.) enabled a detailed hydrological
characterization of the springs (Stadler et al., 2008, 2009, 2010). The mean discharges of the studied springs varied according
to the hydrogeology, catchment size, and altitude, and ranged from 250 to 5,100 L per second during the investigation
period. The estimated mean water residence time of the LKAS systems ranged from 0.5 to 1.5 years, whereas the residence
time for DKAS1 was on the order of 22 years (Stadler & Strobl, 1997). Hydrology-guided microbiological investigations
were supported by automated sampling using data communication via low-earth-orbit (LEO) satellites (Stadler et al., 2008).
A nested sampling design was developed to representatively cover the base flow conditions as well as periods of increased
FIGURE 3 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
precipitation during the annual investigations. In addition, detailed precipitation event-triggered sampling efforts during
shorter time periods complemented the nested sampling design (Farnleitner et al., 2011).
5 | OC C UR RE N C E O F M I C RO B I AL CO M M U N IT IE S I N S P RI NG W A T E R O F AL P IN E K A RS T
A QU IF E R S
Microbes are essential for energy and matter flux in the environment (e.g., degradation of organic matter). Their ubiquitous
occurrence is only limited by a few environmental factors such as extremely alkaline/acidic conditions (pH >12.5 and <0.5)
or temperature (>121 C; Hendry, 2006). Thus, microbes are expected to inhabit any accessible habitat on earth within these
limits of life. However, in contrast to many other habitats (Whitman, Coleman, & Wiebe, 1998), until recently, hardly any
information was available on the occurrence of microbes in spring water from (alpine) karst aquifers (Griebler & Lueders,
2009). The only exceptions were a few cultivation-based studies (Menne, 1999; Pavuza, 1994).
5.1 | Abundance and variability of total microbial cell counts

A first study investigated the occurrence and dynamics of total prokaryotic1 cell counts (Figure 3a,b) by EM in the NCA
alpine spring water from two hydrogeologically contrasting but closely located karst aquifers over several years (Farnleitner
et al., 2005; Wilhartitz et al., 2009). Spring DKAS1 represents a dolomite–limestone karst aquifer featuring a high average
water residence time and a relatively constant flow (Qmin/Qmax discharge ratio of 1:1.6), whereas spring LKAS2 drains a typ-
ical limestone karst aquifer with a very dynamic hydrological regime and discharge (Qmin/Qmax discharge ratio of 1:40; see
section 4 ‘selected model catchments’ for more details). The detected levels of total cell counts (TCC) and its variations in
the spring water reflected the different hydrogeological situation of the systems. DKAS1 showed a relatively stable TCC that
only ranged from 8 to 20 × 103 cells per mL (n = 74) during the almost 5-year study period (from year 2001 to 2005,
Table 1). In 2005, EM counts were paralleled by FCM counts. TCC obtained by FCM and EM followed the same pattern
throughout the whole year and averaged to 1.4 × 104 cells per mL for both methods (Wilhartitz et al., 2013). The TCC did
not reveal any relationship to the determined hydrological and physicochemical parameters (e.g., discharge, turbidity, electri-
cal conductivity, and spectroscopic absorption at 254 nm). Principal component analysis indicated a biological component in
the DKAS1 aquifer (e.g., comprising bacterial biomass and activity), which was largely independent from the prevailing dis-
charge regime (Wilhartitz et al., 2009). In contrast, the TCC from the dynamic LKAS2 showed close association to the pre-
vailing hydrological conditions and measured water quality characteristics (Farnleitner et al., 2005; Wilhartitz et al., 2009,
2013). The TCC ranged from 26 to 107 × 103 cells per mL (n = 74) during the investigation period, with the lowest concen-
trations during the winter season and the peak values connected to high-discharge events during summer season. In addition,
SAVIO
ET AL.
TABLE 1 A summary table on total prokaryotic cell concentrations (TCC) in water from available studies on distinct karst aquifers in Europe
Total cell counts (TCC)

[103 cells/mL]
Geographic
Study Mean/ Study Method location
site Geology Altitude mediana Range period Season n value (dye) (country) Reference
Spring Limestone Alpine 63.0a 44–107 1 year All seasons n = 15 EM (AO) Northern Farnleitner et al. (2005)
karst (LKAS) Calcareous
44.4a 27–70 2 years All seasons n = 19–25 EM (AO) Wilhartitz et al. (2009)
Alps (Austria)
50.6a 26–85 3 years All seasons n = 40 EM (AO) Wilhartitz et al. (2013)
a
Dolomite 14.8 13–20 1 year All seasons n = 15 EM (AO) Farnleitner et al. (2005)
karst (DKAS)
13.1a 11.2–19 2 years All seasons n = 19–25 EM (AO) Wilhartitz et al. (2009)
13.2a 8–19 3 years All seasons n = 40 EM (AO) EM (AO)
Limestone 27.0 21–34 1 year Winter to n = 56 EM (DAPI) Wilhartitz et al. (2007)
and dolomite summer
karst springs
Spring Karst Subalpine 3–500 1 year Autumn and n = 14 FCM (SG I) Jura Mountains Sinreich et al. (2014)
hydrogeological and alpine spring and Alpine
settings Chain
(Switzerland)
Spring Limestone karst Low mountain 19–650 1 year Spring and NA FCM (SG I) North western Page et al. (2017)
(highly range autumn (every 45 min) Switzerland
21–389
karstified) (Switzerland)
15–339
Ground Limestone karst Lowland karst 11–94 1 year Spring to n=8 EM (SG II) Hainich national Opitz et al. (2014)
water (German (upper aquifer) autumn park (Germany)
Muschelkalk)
Lowland karst 2.7–380
(lower aquifer)
12–370
Pool Limestone karst Epiphreatic 270–520 1 year Autumn and n=8 EM (DAPI) Bärenschacht cave Shabarova and
subsurface winter in Bernese Pernthaler (2010)
karst (950 m) Oberland
100–290 n=6 FCM (SG I) Shabarova, et al.
(Switzerland)
(2013)
Note. FCM = flow cytometry; EM = epifluorescence microscopy (used fluorescence dye in the respective study); NA = information not available; DAPI = 40 ,6-diamidino-2-phenylindole; SG I = SYBR® Green I (Invitrogen); SG II =
SYBR® Green II (Invitrogen); AO = acridine orange.
a
Indicates statement of “median” instead of “mean” concentrations.
7 of 20
a detailed investigation of a high-discharge event at LKAS2 in summer 2008 revealed TCC up to 387 × 103 cells per mL
(data not published). Similarly to DKAS1, EM counts gave similar results as FCM counts at base flow conditions (discharge
<4,500 L s−1) in 2005. However, during rainfall events absolute numbers diverged, with FCM counts being higher. As a
result, averaged TCC in LKAS2 were 4.4 × 104 cells per mL for EM and 7.8 × 104 for FCM (Wilhartitz et al., 2013). In
agreement with the TCC results, also prokaryotic cell volumes and sizes, cell biomasses and cell shapes (morphotypes)
reflected the hydrogeological differences between the DKAS1 and the LKAS2 site (Farnleitner et al., 2005; Wilhartitz et al.,
2009, 2013). Compared with LKAS2, DKAS1 revealed smaller cell volumes and increased proportions of coccoid cells
(Figure 3a,b). It could be shown that bacterial cells represented the dominant fraction in the spring water of the LKAS2,
LKAS4, LKAS8, and the DKAS1, whereas archaeal cells contributed only a small fraction (≤12%) of the total prokaryotic
cell counts (Wilhartitz et al., 2007). A few years later, the observations from LKAS and DKAS1 were also supported by a
study investigating the TCC in karst springs within the Swiss Jura Mountains and the Alpine Chain (National Groundwater
Monitoring programme of Switzerland) based on FCM analysis (Sinreich et al., 2014). There, the TCC ranged from 3 to
500 × 103 cells per mL of spring water, well reflecting the range of various other investigated hydrogeological systems
(Table 1). From this result, Sinreich et al. (2014) concluded that the TCC represents a valuable intrinsic parameter for karst
aquifer characterization. Moreover, they suggested a hydrogeological-microbiological classification system of karst springs
based on the observed TCC ranges (i.e., 103–104, 104–105, 105–106 cells per mL). This system is also supported by TCC
measurements from other available investigations of alpine karst springs or cave pools that fall within the discussed range of
reported cell numbers (Table 1). In this context, it has to be considered that TCC results in different studies may differ
depending on the method used. As mentioned above, for DKAS1 and LKAS2, EM and FCM results were similar under
baseflow conditions, but higher results were obtained with FCM during rainfall events. TCC results obtained by EM also
depend on the used fluorochrome (Seo, Ahn, & Zo, 2010). It is known that, for example, 40 ,6-diamidino-2-phenylindole
(DAPI) staining tends to underestimate TCC in comparison to acridine orange (Seo et al., 2010). In the discussed studies
from the NCA region (Wilhartitz et al., 2009, 2013), acridine orange (Merck, Darmstadt, Germany) was used, while SYBR
Green (Invitrogen, Waltham, MA, USA) was used for FCM. For an alluvial groundwater aquifer system, we compared
EM SYBR Gold staining (instead of Acridin orange) with FCM SYBR Green and found FCM values being on average 26%
lower than the values obtained by EM, with a highly significant correlation between both methods (r = 0.91, n = 138,
p < 0.001; unpublished data). Besides, it has to be highlighted that the spring water of alpine karst aquifers also contains a
substantial number of bacteriophages (i.e., viruses specifically infecting prokaryotic cells) as well as protozoan organisms,
although the latter could be observed only in very low concentrations (Wilhartitz et al., 2013; cf. Figure 3c,d). Apart from
the information given by Wilhartitz et al. (2013), there is currently hardly any information available on the intrinsic phage or
protozoan community composition in alpine karst spring water.
5.2 | Microbial activity, suspended versus attached growth and biogeochemical significance
Other important questions relate to the potential impact of the microbiota on the “self-purification” of spring water quality
and on carbonate geochemistry (Gray & Engel, 2013). In this respect, prokaryotic activity measurements performed in NCA
spring water from DKAS1 and LKAS2 based on sensitive analytical isotope approaches revealed extremely low heterotro-
phic production rates (i.e., 0.72 to 82 pg carbon L−1 hr−1 for DKAS1, and 6 to 900 pg carbon L−1 hr−1 for LKAS2) during
two annual cycles of investigation (Wilhartitz et al., 2009); these measured production rates were among the lowest values
ever measured for aquatic habitats (Eiler et al., 2003; Kirschner & Farnleitner, 2005; Laybourn-Parry, Quayle, Henshaw,
Ruddell, & Marchant, 2001). The combination of activity measurements, microscopy, and specific cell-labeling techniques
(i.e., catalyzed reporter deposition-FISH-microautoradiography) further demonstrated that, on average, only 7% (range
3–14%) of the observed TCC in the spring water were active, with very long generation times of up to 684 days (Wilhartitz
et al., 2009). These extremely low prokaryotic activity rates in the suspended compartment of spring water from the base
flow component of DKAS1 and LKAS2 also suggest a potentially high biostability2 for water supply purposes. However, it
has to be emphasized that biostability is defined in an operational context and all possible changes during water abstraction,
treatment, disinfection, and supply in the distribution net must be considered (Chowdhury, 2012; Liu, Verberk, & Van Dijk,
2013; Van Der Kooij, 2000; Zhang, Oh, & Liu, 2017). The biostability strongly depends on the type, characteristics, and
management of the water supply system and thus has to be determined for the specific situation.
In contrast to the extremely low growth rates of cells suspended in native spring water, the activity measurements of sediments
recovered from DKAS1, LKAS2, LKAS4, and LKAS8 on average revealed a 1-million-fold (106) higher heterotrophic production
rate per volume when compared with measurements in the overlying spring water. These observations at the NCA springs are in
agreement with those of previous studies of porous aquifers, which often reported that groundwater samples do not accurately
reflect the aquifer microbiology due to a high ratio between the rock and sediment attached and the suspended cells in the water
column (Alfreider, Krössbacher, & Psenner, 1997; Lehman, 2007). Hence, the activity rates determined by Wilhartitz et al. (2009)
FIGURE 4 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
highlight the importance of rock or sediment-surface-associated microbial processes within karst aquifers (e.g., at sediment parti-
cles, fractures, and conduits) and suggest their role on self-purification and geochemical processes. In situ colonization experiments
with natural limestone and dolomite rocks from LKAS/DKAS placed directly in the spring (Figure 4) first indicated that microbial
colonization is indeed supported within the sediment and rock structures of alpine karst aquifers (Figure 5). However, more inves-
tigations are required to substantiate these initial findings at NCA spring water and to better understand the processes associated
with rock and sediment surfaces in alpine karst aquifers.
5.3 | Elucidating the microbial community structure by genetic analysis

To further characterize the potential function of the observed bacterial communities in alpine spring water, basic information
on their composition and structure is of interest. Therefore, PCR-dependent fingerprinting based on a ribosomal gene
(i.e., PCR-DGGE profiling of the bacterial V3-16S-rRNA gene) was performed with NCA spring water from DKAS1 and
LKAS2 during 2000 and 2001. The bacterial community profiles revealed remarkable stability for each spring water type,
being easily classifiable as either DKAS1 or LKAS2 DGGE-profile types (Farnleitner et al., 2005). Another study that
applied a comparable methodology to spring water from a catchment in the Swiss Jura Mountains supported the high tempo-
ral stability of bacterial populations in the base flow component of alpine karst spring water (Pronk et al., 2006; Pronk, Gold-
scheider, & Zopfi, 2009). Because of its high average water residence time (~20 years) and extremely low vulnerability to
microbial pollution from surface soil layers, the spring water of DKAS1 was also subjected to ribosomal gene sequencing.
The retrieved nucleic acid sequences revealed low similarities when compared with sequences in available databases
(Pruesse, Peplies, & Glöckner, 2012), indicating the existence of unique bacterial communities at the DKAS1 habitat
(Farnleitner et al., 2005). Some of these sequences also indicated the existence of bacterial populations with chemolithoauto-
trophic activities. For example, the sequences sharing the highest similarity with the most abundant sequence were affiliated
with the family Nitrospiraceae, which is a physiologically highly diverse family that includes nitrite or ferrous iron oxidizers
(Daims, 2014). A few years later, Nitrospiraceae-like sequences were also reported in several other studies on the bacterial
community composition in pristine karstic or granitic alpine aquifers or cave systems (Herrmann et al., 2015; Konno et al.,
2013; Kostanjšek, Pašić, Daims, & Sket, 2013; Pleše et al., 2016; Pronk et al., 2009; Wu, Xing, & Zhou, 2010).
5.4 | Defining the AMEC versus TMEC concept

By bringing together all the results recovered from the NCA LKAS/DKAS sites, the so-called autochthonous microbial endo-
karst community (AMEC) versus transient microbial endokarst community (TMEC) concept was formulated for alpine
spring water (Farnleitner et al., 2005). The aim of this concept is to predict the proportion of AMEC versus TMEC and its
typical range of variation in spring water according to the catchment and the hydrogeology of the investigated system. To
facilitate its correct understanding and interpretation (Brannen-Donnelly & Engel, 2015; Wilhartitz & Farnleitner, 2010), the
concept is described in more detail in the following section. Microbes that are unable to proliferate and unable to form stable
populations within the karst aquifer are considered TMEC. Their occurrence in spring water is considered to be controlled
by two factors: (a) the extent of microbial cell input from external environments (e.g., direct input by swallow holes, surface
runoff during precipitation-triggered events, seepage from the soil–plant–vegetation compartment) and (b) by the governing
physical and biological factors of microbial cell transport through the aquifer toward the spring (e.g., convection, dispersion,
dilution, attachment, straining, persistence, and grazing). In contrast, the AMEC is considered to be a collection of microbes
FIGURE 5 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 4. 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
that possess the physiological capabilities to build sustainable populations by growth and reproduction under the prevalent
conditions in the karst aquifer. AMEC cells may grow in the water phase (suspended mode) or attached to rock or sediment-
surfaces (biofilm mode; Figure 6a). During situations of increased discharge, sediment and rock-attached cells are thought to
be increasingly detached and mobilized and show up in the suspended fraction (Figure 6b). At this point, we want to empha-
size that the definition of the AMEC is not linked to an endemic evolutionary origin in karst systems. Instead, these microbes
are likely also occurring in other habitats such as alpine soils, streams, or lakes. The question whether endemic AMECs do
exist in karst aquifers (in a sense that they evolved and occur only within this habitat) is still speculative and subject to fur-
ther research (Griebler & Lueders, 2009).
Investigations applying cutting edge RNA/DNA HTS techniques to microbial communities (cf. Figure 2c) are needed to
further evaluate and develop the AMEC/TMEC concept. Although not directly comparable, recent information from HTS
FIGURE 6 Schematic illustration of a cross-sectional view into a conduit in the phreatic zone of a limestone karst aquifer (cf. Figure 1) 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
investigations of stagnant cave pools in the epiphreatic zone of a karst system (Shabarova et al., 2013, 2014; Shabarova &
Pernthaler, 2010) and an epigenic cave stream (Brannen-Donnelly & Engel, 2015) indeed indicated the presence of stable
and transient bacterial populations. For example, Shabarova et al. (2014) defined a bacterial “core” assemblage as the subset
of those bacterial genotypes that were present in all samples during their temporal study of the bacterial community develop-
ment in stagnant rock pools. On the contrary, these authors presumed that nonpersistent genotypes might have been intro-
duced from interconnected environments and from other (e.g., anoxic) endokarst habitats, which supports the idea of a
TMEC (Shabarova et al., 2014).
6 | NE W ST R AT E G I E S TO M ON IT O R A ND M A NA GE M I CR O B I AL F E C AL PO L L UT I O N
During precipitation events and therewith associated high-discharge situations, microbes from soil, sediment, plant, animal and
human habitats (cf. Figure 1) may be flushed into alpine karst aquifers via the overlaying soil surface layers. Depending on the
input load, fate, and mobility (see also AMEC/TMEC hypothesis definition above), these cells may finally also be detected in
the spring water, as also reflected in an increase of copiotrophic microbes3 measurable by HPC techniques (Figure 6; Farnleit-
ner et al., 2005). If this water is used for water supply, nutrients from catchment surface-associated habitats can also decrease
the biostability of the spring water, potentially resulting in undesirable changes of water quality related to taste, odor, or turbid-
ity of the water (Chowdhury, 2012; Van der Kooij et al., 2013). As intestinal pathogens can occur in extremely high concentra-
tions in human and animal excreta, microbial fecal pollution represents the most critical water quality hazard in alpine spring
water (Hrudey & Hrudey, 2004; Kralik, 2001). Depending on the hydrogeology and the hydrological situation of the consid-
ered aquifer and spring systems, fecal pollution can occur extremely quickly, and the corresponding pollution levels can
increase by more than several orders of magnitude during an precipitation-triggered high-discharge event situation (Sinreich
et al., 2014; Stadler et al., 2008). Despite its high relevance for water supply and public health, until recently, microbial fecal
pollution in spring water was considered a “black box” phenomenon. Consequently, this lack of knowledge also hindered
target-oriented water quality management.
Fecal pollution
Management
Water resource Tools
measures
MST-guided
Treatment and Health-risk
Step 3
QMRA
disinfection assessment of
requirement fecal contamination Scenario
modeling
Catchment
Catchment
Step 2
survey (PSP)
Characterization of
management and
fecal contamination Genetic MST
pollution control
markers
Standard fecal
Step 1
indicators (SFIB)
Spring-abstraction Monitoring of
management fecal contamination "On-line"
monitoring
FIGURE 7 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
6.1 | The framework for integrated fecal pollution analysis and management
To promote advanced spring water quality management for the 21st century, a new strategy was recently proposed based on
the NCA LKAS and DKAS research; this strategy is the so-called “framework for integrated fecal pollution analysis and
management” (Farnleitner et al., 2018; Stalder et al., 2011a). Three interacting levels (“three-step approach”) characterize the
backbone of the concept (Figure 7), with relevance to the following issues: (a) is there a problem with fecal pollution? (b) if
yes, what is the reason for it? and (c) what is the actual health risk related to the fecal source(s) that contribute to the
observed pollution? The suggested framework can also be referred to as a “bottom-up approach” because it starts at the most
general level (i.e., general pollution monitoring, including all types of fecal pollution sources from humans, life stock, and
wildlife) and becomes more specific as it proceeds from the bottom to the top of the diagram (i.e., looking for the responsible
pollution source(s) and the associated health risks for the consumer of the drinking water). The three steps will be explained
in detail below.
Step 1: Detection and online monitoring of microbial fecal pollution
Microbiological water quality monitoring has been based on the detection of standard fecal indicator bacteria (SFIB) for
more than 100 years. SFIB can be easily detected by cultivation-based standard procedures (e.g., ISO 9308-1 for Escherichia
coli, E. coli, and ISO 7899-2 for intestinal enterococci; International Organisation for Standardisation, 2000a, 2000b). The
most basic requirements of sensitive fecal indicators are the occurrence of these bacteria in high concentrations in the excreta
of humans and warm-blooded animals and their inability to replicate outside of the intestinal environment. However, espe-
cially regarding the latter, the capacity of SFIB to indicate fecal pollution in water resources has been increasingly questioned
during the past decade (Ishii & Sadowsky, 2008). The reason for this debate is the suggested existence of “naturalized popu-
lations” of commonly used SFIB, which are thought to persist and proliferate outside the intestinal environments, including
in sediments and soils (Brennan, Abram, Chinalia, Richards, & O'Flaherty, 2010; Byappanahalli, Nevers, Korajkic, Staley, &
Harwood, 2012; Derry & Attwater, 2014; Ishii & Sadowsky, 2008). Thus, the SFIB were rigorously evaluated for the
LKAS/DKAS systems, as there was hardly any available information on their performance characteristics in alpine catch-
ments. These investigations clearly demonstrated that SFIB can indeed be reliably used for sensitive fecal pollution monitor-
ing at alpine karst water resources (Reischer et al., 2008; Stadler et al., 2010). Moreover, for the LKAS2 system, the
microbial fecal pollution dynamics could also be elucidated in a high-resolution mode using automated high-discharge event-
triggered sampling for SFIB and other microbial indicators, combined with online monitoring of discharge and physicochem-
ical parameters (Stadler et al., 2008).
FIGURE 8 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)
Rapid online monitoring of fecal pollution events in spring water would be highly desirable as an extension to traditional
SFIB detection, as cultivation usually takes more than one working day. For this reason, automated enzymatic detection,
using β-D-glucuronidase (GLUC) activity, was evaluated as a tool for near-real-time online fecal pollution monitoring.
GLUC activity was previously suggested as a rapid biochemical indicator of fecal pollution in rivers and estuaries
(Farnleitner et al., 2001; Farnleitner, Hocke, Beiwl, Kavka, & Mach, 2002; Tryland & Fiksdal, 1998) but has not been
applied in groundwater habitats. A two-year investigation at the LKAS2 site demonstrated that robust automated online
determination of enzymatic microbial activity rates directly at alpine spring water locations can be successfully implemented
based on the currently available technology (Ryzinska-Paier et al., 2014; Stadler et al., 2016). However, contrary to expecta-
tions, GLUC activity did not qualify as a rapid surrogate parameter for cultivation-based SFIB pollution. Thus, further inves-
tigations are needed to clarify the actual indication capacity of GLUC (and other enzymatic alternatives) as a rapid means for
fecal pollution monitoring in such habitats (Ender, Goeppert, Grimmeisen, & Goldscheider, 2017; Ryzinska-Paier et al.,
2014). As an alternative to complex biochemical online detection, physicochemical online measurements may also support
the real-time detection of surface-associated fecal pollution influence in spring water. Indeed, the spectral absorption coeffi-
cient at 254 nm (SAC254) could be identified as a real-time early-warning proxy for fecal pollution in the LKAS systems
(Figure 8). This finding was based on a statistical time-series analysis using an autosampling setup that generated “high-reso-
lution” data series for the LKAS2, LKAS4, and LKAS6 sites during high-discharge events occurring in summers of
2005–2008 (Stadler et al., 2008). Interestingly, the SAC254 also increased between 3 and 6 hr earlier (so called “lead-time”)
than the potential onset of fecal pollution, irrespective of the studied high-discharge event (Stadler et al., 2010). The physical
online monitoring parameter turbidity, as a rapid proxy for surface-associated fecal pollution, was demonstrated on a spring
in the Swiss Jura Mountains. Pronk et al. (2006) and Pronk, Goldscheider, and Zopfi (2007) applied the continuous detection
of particle-size distributions and showed that a relative increase of finer particles (0.9–10 μm) were associated with surface
influence and the potential contamination with fecal indicator bacteria. It is important to note that the ability to predict the
occurrence of fecal pollution by a proxy parameter (or a combination of several parameters) is closely linked to the character-
istics of the habitat. A proxy parameter must not be transferred to other situations without rigorous evaluation, whether such
an application is justified.
Step 2: Characterization and identification of fecal pollution sources
The detection of SFIB indicates and quantifies fecal pollution in spring water. However, SFIB enumeration does not hold
any information about the sources responsible for fecal pollution (i.e., human, life stock, or wildlife). Thus, a combination of
pollution source profiles (PSP; i.e., generating information on potential pollution sources within a specific catchment) and
host-associated genetic fecal markers (i.e., specific DNA fragments of host-associated gut microbes) were developed and
evaluated for MST (i.e., generating information on the origin of fecal pollution directly from the spring water) in the LKAS
catchments. The quantitative assessment of potential fecal pollution sources in the respective catchment—also referred to as
PSP (Figure 7)—facilitated the formation of hypotheses regarding the suspected pollution sources of spring water contamina-
tion (Farnleitner et al., 2011; Reischer et al., 2011, 2013). For example, as much as 99.9% of intestinal E. coli populations
daily produced and deposited in the LKAS6 environment could be allocated to wildlife (42.4%) or livestock ruminant
(57.6%) fecal excreta emissions. In contrast, the availability of human fecal pollution sources was estimated to be negligible
within the same catchment (Farnleitner et al., 2011).
To test the fecal pollution source hypothesis suggested by the PSP, host-associated genetic fecal markers were developed,
evaluated, and subsequently applied directly to the spring (Farnleitner et al., 2011). Based on intestinal Bacteroidetes
populations4, qPCR approaches could be successfully developed for the sensitive detection of a ruminant-associated genetic
fecal marker (i.e., the BacR approach [Reischer, Kasper, Steinborn, Mach, & Farnleitner, 2006]) and a human-associated
genetic fecal marker (i.e., the BacH approach [Reischer, Kasper, Steinborn, Farnleitner, & Mach, 2007]). For example, the
BacR qPCR approach detects, on average, 4.1 × 109 marker molecules per gram of ruminant feces, permitting the ultrasensi-
tive detection of approximately 2 ng of ruminant feces per analyzed water sample (Reischer et al., 2006). Using hydrology-
guided sampling and multiparametric analysis (so called “nested sampling” design), the developed BacR/BacH marker
allowed the determination of the relevant fecal pollution sources of spring water contamination (Farnleitner et al., 2011;
Reischer et al., 2008, 2011). For example, the hypothesis that ruminant animals were the dominant sources of fecal pollution
in the LKAS6 catchment was clearly confirmed. In addition, E. coli contamination could be predicted based on the measured
concentrations of the BacR marker at the considered spring (Farnleitner et al., 2011).
In general, the quantification of host-associated fecal markers in water samples by PCR methodologies is a very recent
development, and many further applications are likely to follow in the near future. At the time the NCA LKAS/DKAS pro-
ject started, just a few PCR assays were available for the qualitative detection of genetic fecal markers (Bernhard & Field,
2000a, 2000b), and qPCR approaches had yet to be developed (Reischer et al., 2006, 2007; Stricker, Wilhartitz, Farnleit-
ner, & Mach, 2008). Meanwhile, a vast number of new qPCR MST marker approaches has been developed during the last
decade for various fecal pollution sources throughout the world (Wuertz, Wang, Reischer, & Farnleitner, 2011). Of these
approaches, many are currently being evaluated for their application predominantly at surface water resources (Boehm et al.,
2013; Hagedorn, Blanch, & Harwood, 2011; Mayer et al., 2016; Reischer et al., 2013). For (karst) ground water systems,
however, just a few studies have been realized since the pioneering LKAS/DKAS work (Bucci, Petrella, Celico, & Naclerio,
2017; Diston, Robbi, Baumgartner, & Felleisen, 2017; Diston, Sinreich, Zimmermann, Baumgartner, & Felleisen, 2015;
Ohad et al., 2015; Zhang, Kelly, Panno, & Liu, 2014). However, these studies already indicate the high potential of identify-
ing host-associated genetic qPCR markers to support traditional fecal pollution monitoring and to guide proactive and target-
oriented management in such systems.
Step 3: Health-risk assessment of microbial fecal pollution
In most cases, water supplies require adequate primary disinfection to produce safe drinking water from karst aquifers
(World Health Organization, 2017). The quantitative microbial-risk assessment (QMRA) approach can help to determine the
health risks associated with fecal pollution (Haas, Rose, & Gerba, 1999, 2014). The QMRA approach also supports the esti-
mation of the required extent of pathogen reduction during disinfection to ensure the required safety level for water con-
sumption (Schijven, Derx, de Roda Husman, Blaschke, & Farnleitner, 2015; Schijven, Teunis, Rutjes, Bouwknegt, & de
Roda Husman, 2011). The QMRA relies on the appropriate choice and quantification of reference pathogens (RP; Haas
et al., 1999). Commonly used RP include representative pathogens from enteric bacteria (e.g., Campylobacter sp.), enteric
parasites (e.g., Giardia sp.), and human enteric viruses (e.g., enteroviruses). As recently demonstrated, many more zoonotic
agents, potentially occurring in mountainous alpine catchment areas, can be relevant to human health (Stalder et al., 2011a,
2011b).
The suggested “bottom-up” approach (Figure 7) uses information from Step 2 to guide the selection of appropriate RP
for Step 3 by integrating results from the PSP and MST. For example, this approach enabled researchers to focus on zoonotic
pathogens in the LKAS2 system, specifically evaluating the quantitative occurrence of Cryptosporidium sp. and
Giardia sp. directly in the fecal pollution sources at the catchment and in the spring water during high-discharge events
(Farnleitner et al., 2018).
7 | CON CLU SION —S T ATU S Q U O A N D F U T U RE RE S E A RC H NE E DS
The presented studies from the NCA area and from studies performed by other groups abroad Austria shed considerable light
on the microbiology in the spring water from alpine karst aquifers. It could be demonstrated that the hydrogeology and catch-
ment situation of a given system has a strong impact on the microbiological characteristics of the spring. According to their
hydrogeological settings, alpine karst springs behave as “individuals” and thus require specific adaptation of their manage-
ment when used for water supply.
Investigations demonstrated that even spring water with a high water residence time and absence of immediate surface
influence is far from being sterile (cf. DKAS2) and harbors characteristic natural (intrinsic) microbial communities. Observed
TCC, sizes, volumes and morphotypes are strongly impacted by the hydrogeological situation of the aquifer. In this regard,
spring water has to be defined in terms of basic physical (e.g., temperature), chemical (e.g., inorganic ion composition), and
biological (e.g., numbers of TCC) water quality characteristics in future concepts. To highlight the ecosystem character of
alpine karst springs, the AMEC/TMEC hypothesis has been suggested for the investigated NCA DKAS and LKAS systems.
It should be noted that further research activities are needed to evaluate this concept at various locations and in different situ-
ations. To date, only very little molecular biological information is available. However, the application of HTS (cf. Figure 2)
TABLE 2 Overview on open research questions and future development goals to fully realize the suggested framework for microbial pollution analysis and
management
Twenty three important research questions and development goals for spring water analysis and management
Sampling
• What is the best technical approach to support microbiological autosampling to realize hydrological-guided monitoring?
• What is the best approach to realize a combined sampling strategy for fecal pollution and biostability2 monitoring?
• What is the best approach for the enrichment of indicators and pathogens from large sampling volumes?
Fecal pollution monitoring
• What are the best complementing bacterial indicators to Escherichia coli (E. coli) and enterococci, supporting cultivation-independent direct detection of total
fecal pollution?
• What is the best suitable bacteriophage to indicate viral fecal pollution?
• What is the best combination of fecal indicators to indicate possible pollution with enteropathogenic bacteria, viruses, or protozoa?
Microbial fecal source tracking (MST)
• What is the persistence of the currently applied genetic fecal markers in alpine spring water?
• Do differences in the persistence between MST markers and standard fecal indicators occur?
• What is the degree of mobility of the currently applied genetic fecal markers in the catchment and the karst aquifer?
• How can the available genetic MST marker assays be extended and refined to differentiate all important fecal pollution sources (e.g., cattle versus sheep versus
chamois versus deer)?
Spring water-abstraction management
• What is the most suitable candidate parameter to facilitate sensitive and robust online monitoring of total fecal pollution?
• What is the most suitable candidate parameter to facilitate human-associated (sewage) fecal pollution monitoring?
• What is the best approach to detect and differentiate recent fecal pollution versus aged fecal pollution at the spring water?
• What is the best approach to combine fecal pollution and biostability2 monitoring?
Quantitative microbial-risk assessment (QMRA)
• What are the most suitable reference pathogens for QMRA?
• What is the significance of wild life versus livestock in terms of water contamination and health risk?
• What is the importance of zoonotic pathogens in terms of water contamination and health risk?
• What is the significance of soil biota in terms of water contamination and health risk?
• What is the most suitable modeling approach to link the catchment situation, spring water quality and QMRA?
• What are the required reduction levels for pathogens in spring water taking into account catchment management, hydrological characteristics, and spring water
abstraction, to comply with World Health Organization water quality standards?
• What is the best approach to evaluate contamination- and health-risk scenarios to support sustainable planning of water safety management infrastructure?
Disinfection
• What is the effect of Ultraviolet (UV) irradiation on the natural spring water bacterial community?
• What is the effect of UV irradiation on the nutrient availability and biostability2 in spring water?
is likely to provide an adequate tool to elucidate this question and to foster further research activities on the microbiome of
spring water of alpine karst aquifers. In the long run, HTS and bioinformatics may use the total amount of biological infor-
mation stored in the analyzed water samples for a more holistic water quality monitoring approach, with potential relevance
for ecology (e.g., analyzing the ecological status), hydrology (e.g., using microbial spring communities as natural tracers),
and public health and water supply (i.e., monitoring biohazard and biostability issues).
Managing the surface-related microbial pollution component

Depending on the aquifer system, a unique selection of measures such as catchment protection, water-abstraction manage-
ment (using the water for drinking water production only when it shows appropriate raw water quality) and sufficient treat-
ment (including disinfection) must be combined to guarantee a high-quality drinking water supply. In this context, the
suggested framework for microbial (fecal) pollution analysis and management (Figure 7) provides a conceptual basis to select
optimal management solutions for specific spring systems and water supply situations that are in accordance with the health-
based water quality safety criteria of the World Health Organization (World Health Organization, 2017), as to ensure a maxi-
mum tolerable infection risk per person and time during drinking water consumption (Farnleitner et al., 2018). The frame-
work also highlights that target-oriented catchment protection, and optimized spring water-abstraction management helps to
minimize the required treatment and disinfection efforts to keep the final drinking water close to natural conditions for a safe
and high-quality drinking water supply. Spring-abstraction management also helps to reduce the potential input of nutrients
and copiotrophic microbes, optimizing the biostability of the water during treatment and distribution. The philosophy of the
suggested framework is in accordance with the guidelines of the Austrian Codex Alimentarius, which states that the greatest
effort should be put into the protection and abstraction of raw water in order to keep the required treatment and disinfection
as low as possible (Federal Ministry of Health and Women's Affairs, 2017).
Online water quality monitoring, MST based on genetic fecal marker enumeration, and MST-guided QMRA are sug-
gested key analytical tools to realize the proposed framework for the spring environment. Near-real-time detection of fecal
pollution needs to be fast and sensitive and able to follow rapidly changing water quality characteristics. However, as demon-
strated for the GLUC approach, such techniques still demand further research effort and development. Whether the recently
emerging online detection systems for TCC enumeration (Besmer et al., 2014, 2016; Hammes et al., 2008; Hammes, Berger,
Köster, & Egli, 2010; Van Nevel et al., 2017) also add significant value to this approach has yet to be evaluated. Irrespective
of the applicability of currently emerging technologies, the general importance of microbiological online monitoring and
automation (e.g., precipitation event-triggered automated sampling) is likely to increase in the near future (Besmer et al.,
2016; Højris et al., 2016; Ryzinska-Paier et al., 2014; Stadler et al., 2016). The application of genetic MST markers also
holds great promise for future water quality testing. Nevertheless, further research is indispensable. For example, the release
of microbes from excreta and the mobility and persistence of fecal markers in the catchment and the aquifer are largely
unknown. Furthermore, not all potential fecal sources in alpine areas are covered yet (e.g., alpine soil fauna). Moreover, to
robustly guide reference pathogen selection for the QMRA (Farnleitner et al., 2018), the required sensitivity and specificity
levels of the genetic fecal markers for specific source groups have to be evaluated in more detail. The QMRA is the least
developed discipline within the presented framework. For example, there is a need for effective approaches to enrich micro-
bial indicators and pathogens from large water volumes and to develop direct detection methods which differentiate between
dead, viable or infectious pathogens. Last, but not least, modeling approaches linking hydrology, microbiology and infection
and disease risks, as previously mainly developed for other systems (Epting, Page, Auckenthaler, & Huggenberger, 2018;
Schijven et al., 2015), are increasingly needed. Such simulation tools will not only describe the status quo, but will also
allow the evaluation of scenarios to design quality management strategies for a sustainable water supply from alpine karst
aquifer springs of the future (Derx et al., 2016). An overview on open research questions and future development goals to
fully realize the suggested framework for microbial pollution analysis and management is given in Table 2. As mentioned
before, such a framework is not restricted to the fecal pollution component only, but considers the overall microbial quality
of the spring water also including biostability aspects.
ACKNOWLEDGMENTS
The work of this paper was supported by the Austrian Science Fund (FWF): project P 23900 (granted to AHF) & project W
1219 (Vienna Doctoral Programme on Water Resource Systems). Further support came from the GWRS-Vienna research
project (Vienna Water, MA31). Gerhard Bryda is acknowledged for providing freshly prepared rock disks for colonization
experiments. We thank also Wolfgang Zerobin, Gerhard Kuschnig, Harald Kromp, Hans Tobler, Rigler Christoph, Ernst
Formann, and Hermann Kain from MA31 for essential support and cooperation. This was a joint study effort of the ICC
Water & Health (www.waterandhealth.at).
CONFLICT OF INTEREST
The authors declare no conflict of interest.
NOTES
1
Prokaryotic cells include bacterial and archaeal cell types. Besides eukaryotes (e.g. plants, animals) and bacteria, archaea
represent the third kingdom of life on earth. Archaea show very similar cell shapes as compared to bacterial cells and thus
cannot be distinguished by the TCC parameter.
2
The concept of biostability describes the ability to distribute and store water in a distribution system without detrimental
quality changes by microbial growth based on the available substrates.
3
Copiotrophic microbes occur and grow in environments which are rich in nutrients and organic carbon. In contrast, oligotro-
phic microbes, such as postulated for AMEC, survive and grow at much lower nutrient and carbon concentrations.
4
Bacteroidetes are dominating members of the intestinal bacterial flora in humans and many animals. Some of the popula-
tions show also a strong host-association (i.e. they predominately occur in a certain type of a host).
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How to cite this article: Savio D, Stadler P, Reischer GH, et al. Opening the black box of spring water microbiology
from alpine karst aquifers to support proactive drinking water resource management. WIREs Water. 2018;5:e1282.
https://doi.org/10.1002/wat2.1282

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Received: 25 April 2017 Revised: 25 January 2018 Accepted: 26 January 2018

Opening the black box of spring water microbiology from alpine

FIGURE 1 Schematic illustration of alpine karst systems with differing

(a) Cultivation-based (b) Direct cell (c) Nucleic acid

t0: 20 cells t1: 21cells t2: 22 cells

Microscopy PCR Sanger DGGE

3 | THE G EN ER AL CHA RA CT ERIST IC S O F K AR ST SYST EMS

4 | SEL ECT ED MODE L C ATC H MEN TS, HYD R O G E O L O G Y , A N D H Y D R O L O G Y - G U I D E D

FIGURE 3 Epifluorescence microscope

5.1 | Abundance and variability of total microbial cell counts

Total cell counts (TCC)

FIGURE 4 Hardware setup for the investigation of microbial colonization.

5.3 | Elucidating the microbial community structure by genetic analysis

5.4 | Defining the AMEC versus TMEC concept

FIGURE 5 Previously unpublished scanning electron microscope (SEM)

FIGURE 8 Installation of an optical s::can probe (blue device; scan

7 | CON CLU SION —S T ATU S Q U O A N D F U T U RE RE S E A RC H NE E DS

Managing the surface-related microbial pollution component

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