Adaptation and development of novel molecular-microbial methods for investigating ultra-oligotrophic alpine karstic ground water ecosystems and drinking water resources

Abstract

Etwa 25% der globalen Bevölkerung werden, zum größten Teil oder zur Gänze, mit Karstgrundwasser versorgt. Quellen in alpinen Lagen spielen dabei eine wichtige Rolle in der Bereitstellung von qualitativ hochwertigem Rohwasser. Beispielsweise erfolgt die österreichische Wasserversorgung zu über 50% aus alpinen and montanen Karstgebirgen. Bis Dato beschäftigten sich die meisten Untersuchungen mit der hydrologischen Situation oder der Vegetation in den Quelleinzugsgebieten. Da bei der Beurteilung der Wasserqualität, neben den bekannten hygienische Parametern, zunehmend mikrobielle Aspekte eine große Rolle spielen (Biostabilität des Wassers, Wiederverkeimung, Biofilmeintrag, Resistenzen) ergeben sich neue Fragestellungen. In den letzten Jahren wurde versucht erstmals neue mikrobiologische- und molekularbiologische Methoden in diesen oligotrophen Grundwassersystemen zu etablieren, um einen Einblick in die mikrobielle Gemeinschaft zu bekommen, die diese Ökosysteme bewohnt und beeinflusst. Es wurde der Beweis erbracht, dass es eine stabile autochthone mikrobielle endokarst Gemeinschaft (AMEC, autochthonous microbial endokarst community) gibt, die im Karstkörper (Höhlen, Schächte) angesiedelt ist. Neben der Bestimmung der Zellzahl, bakterieller Biomasse und bakterieller Produktion wurde auch eine nähere Charakterisierung der Gemeinschaft mit Hilfe von V3 16S-rRNA-DGGE Profilen und CARD-FISH vorgenommen. Die neu gewonnenen mikrobiologischen Erkenntnisse wurden zusammen mit hydrologischen, biochemischen und physikalischen Daten analysiert, um einen ersten Einblick in die Funktionsweise eines Karstökosystems zu bekommen und damit die Bedeutung der autochthonen Mikroorganismen für die Stabilität dieses Habitats und damit für die Wasserqualität zu verstehen. Die Etablierung dieser Methoden in solchen Habitaten ist für Trinkwasserversorger weltweit von Bedeutung. Die Existenz von Archaea etwa wurde nicht nur in Karstsystemen, sondern in verschiedenen Grundwässern aus ganz Europa nachgewiesen.<br />

Spring water of two alpine karst aquifers differing in hydrogeology but of nearby catchments were investigated for their bacterial population dynamics. DKAS 1 represents a dolomitic-limestone karst aquifer spring showing high average water residence time and relative constant flow. LKAS 2 constitutes a typical limestone karst aquifer spring with a dynamic hydrological regime and discharge. DKAS 1 yielded constantly lower cell counts and biomasses (median of 15×106 cells L-1 and 0.22 µg C L-1) as the LKAS 2 (median of 63×106 cells L-1 and 1.1 µg 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).<br />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.<br />In order to compare the applicability of FISH versus CARD-FISH in oligotrophic groundwater systems, prokaryotic population abundance in ground water samples from four different springs of ultra-oligotrophic alpine karst aquifers were investigated. Probe EUB338, a combined EUB338mix (EUB338, EUB338-II, EUB338-III) specific for Bacteria and two archaeal probes targeting Euryarchaea (EURY806) and Crenarchaea (CREN537) were selected. Average total detection efficiencies, normalized to DAPI counts, were remarkably higher for CARD-FISH (83%) as compared to FISH (15%). An evaluation concerning the permeabilization step for probe EUB338-II, designed for Planctomycetales, when using CARD-FISH was performed. Finally twelve bottled natural mineral waters originating from different geological groundwater habitats were analysed by CARD-FISH. A mean of 78% was identified as Bacteria and an average fraction of 11%, ranging from 1% to 22%, was allocated as Archaea, resulting in a total hybridization efficiency of 89% (mean). To investigate in Situ bacterial growth, production rates and its respective controlling factors in alpine karstic spring water, DKAS 1 and LKAS 2 were studied during two annual cycles. Bacterial production, as determined by 3H-leucine incorporation (3H-LI), varied more than 100 fold throughout the year, giving ranges of 1.5 - 164 pgC l-1h-1 in DKAS 1 and 12 - 1800 pgC l-1h-1 in LKAS 2, respectively. Microautoradiography combined with catalyzed reporter deposition - fluorescence in situ hybridization (MAR-CARD-FISH) revealed that only about 7% of the planktonic community took up 3H-leucine. Bacteria accounted for most of the prokaryotic activity, whereas the contribution of Euryarchaea and Crenarchaea was rather small. Based on 3H-LI and MAR-active cells, bacterial generation times were in the range of 2 to 7 weeks. Principal component analysis, including hydrological, chemical and biological key parameters, indicated different controlling mechanisms on bacterial communities in both spring waters. Bacterial activity in the dynamic LKAS 2 was strongly governed by a dynamic-hydrological component, whereas spring water from the more constant DKAS 1 environment revealed a complex relationship to intrinsic-metabolism components. Extremely low bacterial production in spring water from alpine karstic aquifers, especially during base flow conditions, points to a high bio-stability, which is important regarding its use for water supply.