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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: 1Engineering Water > Water, Health, and Sanitation2Science of Water > Water Extremes3Water and Life > Nature of Freshwater Ecosystems.

Vibrio cholerae is a human pathogen and natural inhabitant of aquatic environments. Serogroups O1/O139 have been associated with epidemic cholera, while non-O1/non-O139 serogroups usually cause human disease other than classical cholera. V. cholerae non-O1/non-O139 from the Neusiedler See, a large Central European lake, have caused ear and wound infections, including one case of fatal septicaemia. Recent investigations demonstrated rapid planktonic growth of V. cholerae non-O1/non-O139 and correlation with zooplankton biomass. The aim of this study was to elucidate the interaction of autochthonous V. cholerae with two dominant crustacean zooplankton species in the lake and investigate the influence of the natural bacterial community on this interaction. An existing data set was evaluated for statistical relationships between zooplankton species and V. cholerae and co-culture experiments were performed in the laboratory. A new fluorescence in situ hybridisation protocol was applied for quantification of V. cholerae non-O1/non-O139 cells, which significantly reduced analysis time. The experiments clearly demonstrated a significant relationship of autochthonous V. cholerae non-O1/non-O139 with cladocerans by promoting growth of V. cholerae non-O1/non-O139 in the water and on the surfaces of the cladocerans. In contrast, copepods had a negative effect on the growth of V. cholerae non-O1/non-O139 via competing bacteria from their surfaces. Thus, beside other known factors, biofilm formation by V. cholerae on crustacean zooplankton appears to be zooplankton taxon specific and may be controlled by the natural bacterial community. Electronic supplementary material The online version of this article (doi:10.1007/s00248-010-9764-9) contains supplementary material, which is available to authorized users.

Aims: We compared the applicability of catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and FISH to enumerate prokaryotic populations in ultra-oligotrophic alpine groundwaters and bottled mineral water Methods and Results: Fluorescent oligonucleotide probes EUB338 and EUB338mix (EUB338 ⁄ EUB338-II ⁄ EUB338-III) were used to enumerate bacteria and probes EURY806 and CREN537 for Euryarchaea and Crenarchaea, respectively. Improved detection of Planctomycetales by probe EUB338-II was tested using a different permeabilization step (proteinase K instead of lysozyme). Total detection efficiency of cells in spring water of four different alpine karst aquifers was on average 83% for CARD-FISH and only 15% for FISH. Applying CARD-FISH on bottled natural mineral waters resulted in an average total hybridization efficiency of 89%, with 78% (range 77–96%) bacteria and 11% (range 3–22%) identified as Archaea. Conclusions: CARD-FISH resulted in substantially higher recovery efficiency than FISH. Hence, CARD-FISH appears very suitable for the enumeration of specific prokaryotic groups in ground- and drinking water. Significance and Impact of the Study: This study represents the first evaluation of CARD-FISH on ultra-oligotrophic ground- and drinking water. Results are relevant for basic research and drinking water distributors. Archaea can comprise a significant fraction of the prokaryotic community in bottled mineral water.

Metaproteomics and its potential applications are very promising to study microbial activity in environmental samples and to obtain a deeper understanding of microbial interactions. However, due to the complexity of soil samples the exhaustive extraction of proteins is a major challenge. We compared soil protein extraction protocols in terms of their protein extraction efficiency for two different soil types. Four different protein extraction procedures were applied based on (a) SDS extraction without phenol, (b) NaOH and subsequent phenol extraction, (c) SDS–phenol extraction and (d) SDS–phenol extraction with prior washing steps. To assess the suitability of these methods for the functional analysis of the soil metaproteome, they were applied to a potting soil high in organic matter and a forest soil. Proteins were analyzed by two-dimensional liquid chromatography/tandem mass spectrometry (2D-LC–MS/MS) and the number of unique spectra as well as the number of assigned proteins for each of the respective protocols was compared. In both soil types, extraction with SDS–phenol (c) resulted in “high” numbers of proteins. Moreover, a spiking experiment was conducted to evaluate protein recovery. To this end sterilized forest soil was amended with proteins from pure cultures of Pectobacterium carotovorum and Aspergillus nidulans. The protein recovery in the spiking experiment was almost 50%. Our study demonstrates that a critical evaluation of the extraction protocol is crucial for the quality of the metaproteomics data, especially in highly complex samples like natural soils., Highlights ► Comparison of protein recovery from natural soil samples with different protocols. ► Direct soil protein extraction with gel-free separation techniques (2D-LC–MS/MS). ► Unique sets of proteins were observed for individual extraction approaches. ► Only small overlap of unique spectra between four different extraction protocols.

Bacterial abundance and community composition were investigated along trophic gra- dients in the barrier reef lagoon of Noumea, New Caledonia. Bacterial abundance and the percent- age of high nucleic acid (%HNA) bacteria (a potential indicator for bacterial production) increased from offshore waters towards the head of the bays. 16S rRNA gene PCR and denaturing gradient gel electrophoresis (DGGE) were used as genetic fingerprints for assessing differences in bacterial com- munity composition. Sequences of DGGE bands were assigned to (1) the genera Rugeria and Roseobacter (Rhodobacteriaceae), (2) the SAR11 cluster, (3) other Alphaproteobacteria, and (4) the genus Alteromonas. Removal of the operationally defined attached bacteria by prefiltration did not affect community profiles in offshore waters but had a strong influence in the bays, probably due to the much higher particle load and thus, attached bacteria in the bays. For the free-living community, the number of bands decreased linearly with increasing water residence time, chlorophyll a concen- tration, and viral abundance. Specific bands were found for offshore waters and the 2 investigated semi-enclosed bays, whereas the lagoon showed no specific bands. A similarity analysis showed spe- cific clusters for offshore water, the lagoon, and the bays. A principle component analysis together with cluster and correlation analysis indicated that water residence time, viruses, and a complex top- down cascading effect of ciliate grazers on flagellates influenced community composition. Also, data from fingerprints of the total and free-living communities suggest that the free-living and the attached community are controlled by different mechanisms.

Spring waters from alpine karst aquifers are important drinking water resources. To investigate in situ prokaryotic heterotrophic production (HP) and its controlling factors, two alpine karst springs of contrasting hydrogeology but of nearby catchments were studied over two annual cycles. Heterotrophic production in spring water, as determined by [3H]leucine incorporation, was low but revealed strong seasonal variations ranging from 0.06 to 6.83 pmol C l−1 h−1 (DKAS1, dolomitic karst-spring) and from 0.50 to 75.6 pmol C l−1 h−1 (LKAS2, limestone karst-spring). Microautoradiography combined with catalyzed reporter deposition - fluorescence in situ hybridization (MAR-CARD-FISH) showed that only about 7 % of the picoplankton community took up [3H]leucine resulting in generation times of 3 to 684 days. Principal component analysis, applying hydrological, chemical and biological parameters demonstrated that planktonic heterotrophic production in LKAS2 was strongly governed by hydrogeographical components (e.g. discharge), whereas variations in DKAS1 are also strongly influenced by changes within the aquifer itself. Measurements in sediments recovered from LKAS2, DKAS1 and similar alpine karst aquifers (n=12) revealed an 106-fold higher heterotrophic production (average 19 μmol C dm−3 h−1) with significantly lower generation times as compared to the planktonic fraction, highlighting the metabolic potential of surface associated endokarst communities to add to self-purification processes. Estimates of microbially mediated CO2 in this compartment indicated a possible contribution to karstification.

Spring water of two alpine karst aquifers differing in hydrogeology but of nearby catchments were investigated for their bacterial population dynamics. Dolomite karst aquifer spring 1 (DKAS 1) represents a dolomitic-limestone karst aquifer spring showing high average water residence time and relative constant flow. Limestone karst aquifer spring 2 (LKAS 2) constitutes a typical limestone karst aquifer spring with a dynamic hydrological regime and discharge. Dolomite karst aquifer spring 1 yielded constantly lower cell counts and biomasses (median of 15 x 10(6) cells l(-1) and 0.22 microg C l(-1)) as the LKAS 2 (median of 63 x 10(6) cells l(-1) and 1.1 microg C l(-1)) and distribution of morphotypes and mean cell volumes was also different between the considered systems, indicating the influence of hydrogeology on microbial spring water quality. Molecular bacterial V3 16S-rDNA profiles revealed remarkable constancy within each spring water throughout the investigation period. Time course analysis of a flood event in LKAS 2 further supported the trend of the temporal constancy of the microbial community. Except for one case, retrieval of partial and full length 16S rDNA gene sequences from the relative constant DKAS 1 revealed similarities to presently known sequences between 80% to 96%, supporting the discreteness of the microbial populations. The gathered results provide first evidence for the presence of autochthonous microbial endokarst communities (AMEC). Recovery of AMEC may be considered of relevance for the understanding of alpine karst aquifer biogeochemistry and ecology, which is of interest as many alpine and mountainous karst springs are important water resources throughout the world.