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Subterranean ecosystems are among the most widespread environments on Earth, yet we still have poor knowledge of their biodiversity. To raise awareness of subterranean ecosystems, the essential services they provide, and their unique conservation challenges, 2021 and 2022 were designated International Years of Caves and Karst. As these ecosystems have traditionally been overlooked in global conservation agendas and multilateral agreements, a quantitative assessment of solution-based approaches to safeguard subterranean biota and associated habitats is timely. This assessment allows researchers and practitioners to understand the progress made and research needs in subterranean ecology and management. We conducted a systematic review of peer-reviewed and grey literature focused on subterranean ecosystems globally (terrestrial, freshwater, and saltwater systems), to quantify the available evidence-base for the effectiveness of conservation interventions. We selected 708 publications from the years 1964 to 2021 that discussed, recommended, or implemented 1,954 conservation interventions in subterranean ecosystems. We noted a steep increase in the number of studies from the 2000s while, surprisingly, the proportion of studies quantifying the impact of conservation interventions has steadily and significantly decreased in recent years. The effectiveness of 31% of conservation interventions has been tested statistically. We further highlight that 64% of the reported research occurred in the Palearctic and Nearctic biogeographic regions. Assessments of the effectiveness of conservation interventions were heavily biased towards indirect measures (monitoring and risk assessment), a limited sample of organisms (mostly arthropods and bats), and more accessible systems (terrestrial caves). Our results indicate that most conservation science in the field of subterranean biology does not apply a rigorous quantitative approach, resulting in sparse evidence for the effectiveness of interventions. This raises the important question of how to make conservation efforts more feasible to implement, cost-effective, and long-lasting. Although there is no single remedy, we propose a suite of potential solutions to focus our efforts better towards increasing statistical testing and stress the importance of standardising study reporting to facilitate metaanalytical exercises. We also provide a database summarising the available literature, which will help to build quantitative knowledge about interventions likely to yield the greatest impacts depending upon the subterranean species and habitats of interest. We view this as a starting point to shift away from the widespread tendency of recommending conservation interventions based on anecdotal and expert-based information rather than scientific evidence, without quantitatively testing their effectiveness., This study is funded by the European Commission via the Marie Sklodowska-Curie Individual Fellowships program (H2020-MSCA-IF-2019; project number 882221), awarded to S.M. Additional support is provided by the PRIN SHOWCAVE “A multidisciplinary research project to study, classify and mitigate the environmental impact in tourist caves” (project number 2017HTXT2R; funded by the Italian Ministry of Education, University and Research). M.B.M. acknowledges support from the Kone Foundation (project number 202007611). T.D., C.F., V.Z., and M.Z. were supported by the Slovenian Research Agency, through core programme P1-0184 and P6-0119. O.T.M. was supported by a grant of the Romanian Ministry of Research, Innovation and Digitization, CNCS/CCCDI— UEFISCDI, project number 2/2019 (DARKFOOD), within PNCDI III. S.I.S. acknowledges funding by MEMOBIC (EU Operational Programme Research, Development and Education No. CZ.02.2.69/0.0/ 0.0/16_027/0008357), and by the Ministry of Education, Youth and Sports of the Czech Republic (grant number CZ.02.1.01/0.0/0.0/16 025/0007417). E.P. is supported by the PON “Research and Innovation” Programme (Axis IV “Education and Research for recovery” – Action IV.6 “Research contracts on Green themes”).

Sediments accommodate the dominating share of groundwater microbiomes, however the processes that govern the assembly and succession of sediment-attached microbial communities in groundwater aquifers are not well understood. To elucidate these processes, we followed the microbial colonization of sterile sediments in in situ microcosms that were exposed to groundwater for almost 1 year at two distant but hydrologically connected sites of a pristine, shallow, porous aquifer. Our results revealed intriguing similarities between the community succession on the newly-colonized sediments and succession patterns previously observed for biofilms in other more dynamic aquatic environments, indicating that the assembly of microbial communities on surfaces may be governed by similar underlying mechanisms across a wide range of different habitats. Null model simulations on spatiotemporally resolved 16S rRNA amplicon sequencing data further indicated selection of specific OTUs rather than random colonization as the main driver of community assembly. A small fraction of persistent OTUs that had established on the sediments during the first 115 days dominated the final communities (68%-85%), suggesting a key role of these early-colonizing organisms, in particular specific genera within the Comamonadaceae and Oxalobacteraceae, for community assembly and succession during the colonization of the sediments. Overall, our study suggests that differences between planktonic and sediment-attached communities often reported for groundwater environments are not the result of purely stochastic events, but that sediment surfaces select for specific groups of microorganisms that assemble over time in a reproducible, non-random way.

Droughts are expected to become more common with climate change resulting in more frequent occurrences of flow intermittency in temperate streams. As intermittency has deleterious effects on fluvial microbial biofilms, there is a need to better understand how droughts affect the microbial functioning and thereby nutrient and organic matter processing in temperate stream ecosystems. Here, the hyporheic zone is of particular importance as it has been shown to be a hot spot for biogeochemical activity under flow intermittence. This study evaluates how drought duration affects microbial biofilm dynamics in the hyporheic zone of intermittent temperate streams. To do so, we used outdoor hyporheic flumes that were subject to periods of drought ranging from 4 to 105 days. Sediment was sampled before and during the drought, and at several occasions after rewetting. Samples were analyzed for extracellular enzymatic activity, bacterial respiration, and bacterial abundances including live to dead cell ratios. The high moisture content remaining in the hyporheic zone of the flumes allowed for the sustained microbial functioning during drought, regardless of drought duration. This can be attributed to cooler temperatures in these climate zones and shading by riparian forests. The high moisture content inhibited the local habitat and community changes that the biofilm might have undergone during more severe desiccation. However, the change in the hyporheic flow regime (flow cessation and resumption) may stimulate microbial processing in these moderate drought conditions. We suggest that the hyporheic zone may act as a buffer against drought and the factors determining this buffer capacity, such as sediment characteristics and climatic regions, need to be analyzed in more detail in future.

In aquatic ecosystems, the biomass, activity and composition of microorganisms are determined to a large extent by local and regional environmental conditions. While karst aquifers are an important source for drinking water, the ecology of microbial communities in karst waters has hardly been studied. This study examined the regional variations and seasonal dynamics of microbial communities in pristine karst spring waters of Slovenia (Central Europe). Fifteen springs distributed across 5 eco-regions exhibiting a strong altitudinal gradient were sampled 4 times a year. Evaluation of the microbial communities included quantification of prokaryotic biomass via total cell counts and microbial activity estimated via measurements of electron transport system activity. The taxonomic structure of the bacterial communities was analysed by terminal restriction fragment length polymorphism fingerprinting. Biological measures were complemented by a set of physico-chemical parameters, including dissolved organic carbon, nutrients, major ions, temperature, electrical conductivity, pH, and dissolved oxygen. Bacterial community structure differed significantly with seasons and eco-regions, with the latter causing greater variation. While the overall power of the environmental variable tested was a moderate factor (15%) in explaining the variability in microbial community structure, catchment altitude was a key determinant. Prokaryotic cell density and microbial activity in spring water decreased with an increase in catchment altitude and were significantly positively correlated. For a better understanding of karst ecosystems and the ecosystem service of water purification, future investigation should address karst microbial communities at a higher phylogenetic and functional resolution.

The science of the total environment 769, 144324 (2021). doi:10.1016/j.scitotenv.2020.144324, Published by Elsevier Science, Amsterdam [u.a.]

The assessment and monitoring of the ecological quality and status of groundwater is a timely issue. At present, various assessment tools have been developed that now await application and validation. One of these, the D‑A‑C index, evaluates the microbiological-ecological quality of groundwater based on of prokaryotic cell counts, microbial activity measurements, and the qualitative characterization of dissolved organic carbon (DOM). The purpose of this paper is to illustrate the different ways of application of the D‑A-(C) index making use of a recently collected data set (n = 61) from the river Mur valley, Austria. First, we present an extension of the D‑A-(C) index by including measurements of dissolved organic matter quality (DOM) derived from fluorescence spectroscopy as additional variables to supplement the analysis of microbial cell density and activity levels. Second, we illustrate how the definition of a reference status for a ‘good’ microbiological-ecological state can improve the analysis and allow for a more sensitive and accurate detection of impacts on groundwater ecosystems. Based on our results, we advocate that the analysis be performed by making use of expert knowledge for the definition of reference sites to which target sites are to be compared.

Groundwaters host vital resources playing a key role in the near future. Subterranean fauna and microbes are crucial in regulating organic cycles in environments characterized by low energy and scarce carbon availability. However, our knowledge about the functioning of groundwater ecosystems is limited, despite being increasingly exposed to anthropic impacts and climate change-related processes. In this work we apply novel biochemical and genetic techniques to investigate the ecological dynamics of an Australian calcrete under two contrasting rainfall periods (LR—low rainfall and HR—high rainfall). Our results indicate that the microbial gut community of copepods and amphipods experienced a shift in taxonomic diversity and predicted organic functional metabolic pathways during HR. The HR regime triggered a cascade effect driven by microbes (OM processors) and exploited by copepods and amphipods (primary and secondary consumers), which was finally transferred to the aquatic beetles (top predators). Our findings highlight that rainfall triggers ecological shifts towards more deterministic dynamics, revealing a complex web of interactions in seemingly simple environmental settings. Here we show how a combined isotopic-molecular approach can untangle the mechanisms shaping a calcrete community. This design will help manage and preserve one of the most vital but underrated ecosystems worldwide.

Understanding microbial community dynamics in the alpine cryosphere is an important step toward assessing climate change impacts on these fragile ecosystems and meltwater-fed environments downstream. In this study, we analyzed microbial community composition, variation in community alpha and beta diversity, and the number of prokaryotic cells and virus-like particles (VLP) in seasonal snowpack from two consecutive years at three high altitude mountain summits along a longitudinal transect across the European Alps. Numbers of prokaryotic cells and VLP both ranged around 104 and 105 per mL of snow meltwater on average, with variation generally within one order of magnitude between sites and years. VLP-to-prokaryotic cell ratios spanned two orders of magnitude, with median values close to 1, and little variation between sites and years in the majority of cases. Estimates of microbial community alpha diversity inferred from Hill numbers revealed low contributions of common and abundant microbial taxa to the total taxon richness, and thus low community evenness. Similar to prokaryotic cell and VLP numbers, differences in alpha diversity between years and sites were generally relatively modest. In contrast, community composition displayed strong variation between sites and especially between years. Analyses of taxonomic and phylogenetic community composition showed that differences between sites within years were mainly characterized by changes in abundances of microbial taxa from similar phylogenetic clades, whereas shifts between years were due to significant phylogenetic turnover. Our findings on the spatiotemporal dynamics and magnitude of variation of microbial abundances, community diversity, and composition in surface snow may help define baseline levels to assess future impacts of climate change on the alpine cryosphere.

Riverbank filtration has gained increasing importance for balancing rising groundwater demands and securing drinking water supplies. While microbial communities are the pillar of vital ecosystem functions in groundwater, the impact of riverbank filtration on these communities has been understudied so far. Here, we followed changes in microbial community composition based on 16S rRNA gene amplicon sequence variants (ASVs) in an initially pristine shallow porous aquifer in response to surface water intrusion during the early stages of induced riverbank filtration over a course of seven weeks. We further analyzed sediment cores for imprints of river-derived ASVs after seven weeks of riverbank filtration. The onset of the surface water intrusion caused loss of taxa and significant changes in community composition, revealing low disturbance resistance of the initial aquifer microbial communities. SourceTracker analysis revealed that proportions of river-derived ASVs in the groundwater were generally

Five decades ago, a landmark paper in Science titled The Cave Environment heralded caves as ideal natural experimental laboratories in which to develop and address general questions in geology, ecology, biogeography, and evolutionary biology. Although the 'caves as laboratory' paradigm has since been advocated by subterranean biologists, there are few examples of studies that successfully translated their results into general principles. The contemporary era of big data, modelling tools, and revolutionary advances in genetics and (meta)genomics provides an opportunity to revisit unresolved questions and challenges, as well as examine promising new avenues of research in subterranean biology. Accordingly, we have developed a roadmap to guide future research endeavours in subterranean biology by adapting a well-established methodology of 'horizon scanning' to identify the highest priority research questions across six subject areas. Based on the expert opinion of 30 scientists from around the globe with complementary expertise and of different academic ages, we assembled an initial list of 258 fundamental questions concentrating on macroecology and microbial ecology, adaptation, evolution, and conservation. Subsequently, through online surveys, 130 subterranean biologists with various backgrounds assisted us in reducing our list to 50 top-priority questions. These research questions are broad in scope and ready to be addressed in the next decade. We believe this exercise will stimulate research towards a deeper understanding of subterranean biology and foster hypothesis-driven studies likely to resonate broadly from the traditional boundaries of this field. S.M acknowledge support by the CAWEB project “Testing macroecological theory using simplified systems”, funded by the European Commission through Horizon 2020 Marie Skłodowska-Curie Actions (MSCA) individual fellowships (Grant no. 882221). I.R.A. was supported by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia, I.P., under the Norma Transitória – DL57/2016/ CP1375/CT0003. D.E. was supported by IFREMER and by the CERES “Climate change and European Aquatic Resources” project funded by European Commission through Horizon 2020 research and innovation programme (Grant no. 678193). C.F., Z.F., and M.Z. were supported by the Slovenian Research Agency (program P1-0184, project N1-0069). J.E.K. was funded by NSF awards DEB1754231 and IOS1933428, and EDGE award 1923372. F.M. was supported by the French National Research Agency projects CONVERGENOMICS (ANR-15-CE32-0005) and EUR H2O’Lyon (ANR-17-EURE-0018). A.M. was supported by the ANCAVE project “Anchialine caves to understand evolutionary processes”, funded by the European Commission through Horizon 2020 Marie Skłodowska-Curie Actions (MSCA) individual fellowships (Grant no. 745530). P.A.V.B. was supported by the project AZORESBIOPORTAL – PORBIOTA (ACORES-01-0145-FEDER-000072), financed by FEDER in 85% and by Azorean Public funds by 15% through Operational Program Azores 2020. A.S.P.S.R. was supported by VILLUM FONDEN (Grant no. 15471) and by a Carlsbergfondet grant (CF19-0609). T.G.P. was supported by Vale S.A. and Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) for individual fellowship (RDP 00092-18). T.P. was supported by the Karst Research Programme P6-0119, LifeWatch ERIC, RI-SI LifeWatch, and EU H2020 project eLTER. info:eu-repo/semantics/publishedVersion

Virtually all heterotrophs incorporate carbon dioxide by anaplerotic fixation. Little explored, however, is the interdependency of pathways and rates of CO2fixation on the concurrent usage of organic substrate(s). Potentially, this could reveal which substrates out of a pool of dissolved organic carbon are utilised by environmental microorganisms. To explore this possibility, Bacillus subtilis W23 was grown in a minimal medium with normalised amounts of either glucose, lactate or malate as only organic substrates, each together with 1 g/L NaH13CO3. Incorporation of H13CO3− was traced by elemental analysis-isotope ratio mass spectrometry of biomass and gas chromatography-mass spectrometry of protein-derived amino acids. Until the late logarithmic phase, 13C incorporation into the tricarboxylic acid cycle increased with time and occurred via [4–13C]oxaloacetate formed by carboxylation of pyruvate. The levels of 13C incorporation were highest for growth on glucose and lowest on malate. Incorporation of 13C into gluconeogenesis products was mainly detected in the lactate and malate experiment, whereas glucose down-regulated this path. A proof-of-principle study with a natural groundwater community confirmed the ability to determine incorporation from H13CO3− by natural communities leading to specific labelling patterns. This underlines the potential of the labelling approach to characterise carbon sources of heterotrophic microorganisms in their natural environments.

Freshwater ecosystems play a key role in shaping the global carbon cycle and maintaining the ecological balance that sustains biodiversity worldwide. Surficial water bodies are often interconnected with groundwater, forming a physical continuum, and their interaction has been reported as a crucial driver for organic matter (OM) inputs in groundwater systems. However, despite the growing concerns related to increasing anthropogenic pressure and effects of global change to groundwater environments, our understanding of the dynamics regulating subterranean carbon flows is still sparse. We traced carbon composition and transformations in an arid zone calcrete aquifer using a novel multidisciplinary approach that combined isotopic analyses of dissolved organic carbon (DOC) and inorganic carbon (DIC) (δ13CDOC, δ13CDIC, 14CDOC and 14CDIC) with fluorescence spectroscopy (Chromophoric Dissolved OM (CDOM) characterisation) and metabarcoding analyses (taxonomic and functional genomics on bacterial 16S rRNA). To compare dynamics linked to potential aquifer recharge processes, water samples were collected from two boreholes under contrasting rainfall: low rainfall ((LR), dry season) and high rainfall ((HR), wet season). Our isotopic results indicate limited changes and dominance of modern terrestrial carbon in the upper part (northeast) of the bore field, but correlation between HR and increased old and 13C-enriched DOC in the lower area (southwest). CDOM results show a shift from terrestrially to microbially derived compounds after rainfall in the same lower field bore, which was also sampled for microbial genetics. Functional genomic results showed increased genes coding for degradative pathways-dominated by those related to aromatic compound metabolisms-during HR. Our results indicate that rainfall leads to different responses in different parts of the bore field, with an increase in old carbon sources and microbial processing in the lower part of the field. We hypothesise that this may be due to increasing salinity, either due to mobilisation of Cl- from the soil, or infiltration from the downstream salt lake during HR. This study is the first to use a multi-technique assessment using stable and radioactive isotopes together with functional genomics to probe the principal organic biogeochemical pathways regulating an arid zone calcrete system. Further investigations involving extensive sampling from diverse groundwater ecosystems will allow better understanding of the microbiological pathways sustaining the ecological functioning of subterranean biota.

Groundwater ecosystems are low productivity environments due to the small amount and poor quality of organic carbon, and nutrient limitation (N, P). However, field data show the overall presence of degradable DOC. Studies exploring the relationship between carbon properties, nutrient availability and microbial production, as well as possible priming in groundwater are missing. In multi-factorial groundwater batch experiments, we investigated the effect of various organic carbon sources (acetate, organic fertilizer, fulvic acids, humic acids) and nutrients (N, P) to DOC degradation, bacterial production and growth efficiency. Focusing on DOC concentrations, total cell counts, ATP, and bacterial carbon production, our study revealed several important findings. The type of organic carbon offered influenced bacterial growth pattern with lower assimilation efficiencies for the labile but energetically least favorable compound (acetate). Growth on acetate was soon P-limited, while more complex DOM (humic acids) sustained slow but long-term growth. Active but non-growing cells maintained continuous DOC turnover at nutrient limitation. The overall bacterial carbon production and growth efficiency ranged from < 1 ng to > 1 mu g C l(-1) h(-1) and < 0.1 to 28%, respectively. None of the experiments revealed evidence for priming in ground water. This study is a first attempt to unravel the multiple limitations and energetic constraints facing microbial communities in oligotrophic groundwater. Future studies should evaluate these findings by including the sediment matrix which carries the major fraction of microbial biomass and by looking in more detail at the structural characteristics and availability of DOM and P species.

Groundwater is not only a vital resource, but also one of the largest terrestrial aquatic ecosystems on Earth. However, to date, ecological criteria are often not considered in routine groundwater monitoring, mainly because of the lack of suitable ecological assessment tools. Prokaryotic microorganisms are ubiquitous in groundwater ecosystems even under the harshest conditions, making them ideal bioindicators for ecological monitoring. We have developed a simple, inexpensive approach that enables ecological groundwater monitoring based on three microbiological parameters that can be easily integrated into existing routine monitoring practices: prokaryotic cell density (D) measured by flow cytometry; activity (A) measured as prokaryotic intracellular ATP concentrations using a simple cell-lysis-luminescence assay; and, as an optional parameter, the bioavailable carbon (C) measured as the concentration of assimilable organic carbon in a simple batch growth assay. We analyzed data for three case studies of different disturbances representing some of the main threats to groundwater ecosystems, i.e. organic contamination with hydrocarbons, surface water intrusion, and agricultural land use. For all three disturbances, disturbed samples could be reliably distinguished from undisturbed samples based on a single index value obtained from multivariate outlier analyses of the microbial variables. We could show that this multivariate approach allowed for a significantly more sensitive and reliable detection of disturbed samples compared to separate univariate outlier analyses of the measured variables. Furthermore, a comparison of non-contaminated aquifers from nine different regions across Germany revealed distinct multivariate signatures along the three microbial variables, which should be considered when applying our approach in practice. In essence, our approach offers a practical tool for the detection of disturbances of groundwater ecosystems based on microbial parameters which can be seamlessly extended in the future by additional parameters for higher sensitivity as well as flexibility.

Aquifers are typically perceived as rather stable habitats, characterized by low biogeochemical and microbial community dynamics. Upon contamination, aquifers shift to a perturbed ecological status, in which specialized populations of contaminant degraders establish and mediate aquifer restoration. However, the ecological controls of such degrader populations, and possible feedbacks between hydraulic and microbial habitat components, remain poorly understood. Here, we provide evidence of such couplings, via 4 years of annual sampling of groundwater and sediments across a high-resolution depth-transect of a hydrocarbon plume. Specialized anaerobic degrader populations are known to be established at the reactive fringes of the plume. Here, we show that fluctuations of the groundwater table were paralleled by pronounced dynamics of biogeochemical processes, pollutant degradation, and plume microbiota. Importantly, a switching in maximal relative abundance between dominant degrader populations within the Desulfobulbaceae and Desulfosporosinus spp. was observed after hydraulic dynamics. Thus, functional redundancy amongst anaerobic hydrocarbon degraders could have been relevant in sustaining biodegradation processes after hydraulic fluctuations. These findings contribute to an improved ecological perspective of contaminant plumes as a dynamic microbial habitat, with implications for both monitoring and remediation strategies in situ.

In light of recent alarming trends in human population growth, climate change, and other environmental modifications, a “Warning to humanity” manifesto was published in BioScience in 2017. This call reiterated most of the ideas originally expressed by the Union of Concerned Scientists in 1992, including the fear that we are “pushing Earth's ecosystems beyond their capacities to support the web of life.” As subterranean biologists, we take this opportunity to emphasize the global importance and the conservation challenges associated with subterranean ecosystems. They likely represent the most widespread nonmarine environments on Earth, but specialized subterranean organisms remain among the least documented and studied. Largely overlooked in conservation policies, subterranean habitats play a critical role in the function of the web of life and provide important ecosystem services. We highlight the main threats to subterranean ecosystems and propose a set of effective actions to protect this globally important natural heritage.

Several studies have analyzed biogeographic distribution patterns of microbial communities across broad spatial scales. However, it is often unclear to what extent differences in community composition across different regions are caused by dispersal limitation or selection, and if selection is caused by local environmental conditions alone or additional broad-scale region-specific factors. This is especially true for groundwater environments, which have been understudied in this context relative to other non-subsurface habitats. Here, we analyzed microbial community composition based on exact 16S rRNA amplicon sequence variants (ASVs) from four geographically separated aquifers located in different regions along a latitudinal transect of ∼700 km across Germany. Using a combination of variation partitioning and ecological null models revealed that differences in microbial community composition were mainly the product of selection imposed by local environmental conditions and to a smaller but still significant extent dispersal limitation and drift across regions. Only ∼23% of the total variation in microbial community composition remained unexplained, possibly due to underestimated effects of dispersal limitation among local communities within regions and temporal drift. No evidence was found for selection due to region-specific factors independent of local environmental conditions., Biogeography of microbial communities in groundwater ecosystems.

The biodegradation of organic pollutants in aquifers is often restricted to the fringes of contaminant plumes where steep countergradients of electron donors and acceptors are separated by limited dispersive mixing. However, long-distance electron transfer (LDET) by filamentous ‘cable bacteria’ has recently been discovered in marine sediments to couple spatially separated redox half reactions over centimeter scales. Here we provide primary evidence that such sulfur-oxidizing cable bacteria can also be found at oxic–anoxic interfaces in aquifer sediments, where they provide a means for the direct recycling of sulfate by electron transfer over 1–2-cm distance. Sediments were taken from a hydrocarbon-contaminated aquifer, amended with iron sulfide and saturated with water, leaving the sediment surface exposed to air. Steep geochemical gradients developed in the upper 3 cm, showing a spatial separation of oxygen and sulfide by 9 mm together with a pH profile characteristic for sulfur oxidation by LDET. Bacterial filaments, which were highly abundant in the suboxic zone, were identified by sequencing of 16S rRNA genes and fluorescence in situ hybridization (FISH) as cable bacteria belonging to the Desulfobulbaceae. The detection of similar Desulfobulbaceae at the oxic–anoxic interface of fresh sediment cores taken at a contaminated aquifer suggests that LDET may indeed be active at the capillary fringe in situ. The biodegradation of organic pollutants in aquifers is often restricted to the fringes of contaminant plumes where steep countergradients of electron donors and acceptors are separated by limited dispersive mixing. However, long-distance electron transfer (LDET) by filamentous ‘cable bacteria’ has recently been discovered in marine sediments to couple spatially separated redox half reactions over centimeter scales. Here we provide primary evidence that such sulfur-oxidizing cable bacteria can also be found at oxic–anoxic interfaces in aquifer sediments, where they provide a means for the direct recycling of sulfate by electron transfer over 1–2-cm distance. Sediments were taken from a hydrocarbon-contaminated aquifer, amended with iron sulfide and saturated with water, leaving the sediment surface exposed to air. Steep geochemical gradients developed in the upper 3 cm, showing a spatial separation of oxygen and sulfide by 9 mm together with a pH profile characteristic for sulfur oxidation by LDET. Bacterial filaments, which were highly abundant in the suboxic zone, were identified by sequencing of 16S rRNA genes and fluorescence in situ hybridization (FISH) as cable bacteria belonging to the Desulfobulbaceae. The detection of similar Desulfobulbaceae at the oxic–anoxic interface of fresh sediment cores taken at a contaminated aquifer suggests that LDET may indeed be active at the capillary fringe in situ.

Groundwaters provide the vast majority of unfrozen freshwater resources on the planet, but our knowledge of subsurface ecosystems is surprisingly limited. Stygofauna, or stygobionts -subterranean obligate aquatic animals - provide ecosystem services such as grazing biofilms and maintaining water quality, but we know little about how their ecosystems function. The cryptic nature of groundwaters, together with the high degree of local endemism and stygofaunal site-specific adaptations, represent major obstacles for the field. To overcome these challenges, and integrate biodiversity and ecosystem function, requires a holistic design drawing on classical ecology, taxonomy, molecular ecology and geochemistry. This study presents an approach based on the integration of existing concepts in groundwater ecology with three more novel scientific techniques: compound specific stable isotope analysis (CSIA) of amino acids, radiocarbon analysis (14C) and DNA analyses of environmental samples, stygofauna and gut contents. The combination of these techniques allows elucidation of aspects of ecosystem function that are often obscured in small invertebrates and cryptic systems. Carbon (δ13C) and nitrogen (δ15N) CSIA provides a linkage between biogeochemical patterns and ecological dynamics. It allows the identification of stygofaunal food web structures and energy flows based on the metabolic pathway of specific amino groups. Concurrently, 14C provides complementary data on the carbon recycling and incorporation within the stygobiotic trophic webs. Changes in groundwater environmental conditions (e.g. aquifer recharge), and subsequent community adaptations, can be pinpointed via the measurementof the radiocarbon fingerprint of water, sediment and specimens. DNA analyses are a rapidly expanding approach in ecology. eDNA is mainly employed as a biomonitoring tool, while metabarcoding of individuals and/or gut contents provides insight into diet regimes. In all cases, the application of the approaches in combination provides more powerful data than any one alone. By combining quantitative (CSIA and 14C) and qualitative (eDNA and DNA metabarcoding) approaches via Bayesian Mixing Models (BMM), linkages can be made between community composition, energy and nutrient sources in the system, and trophic function. This suggested multidisciplinary design will contribute to a more thorough comprehension of the biogeochemical and ecological patterns within these undervalued but essential ecosystems.

Our daily life depends on many services delivered by the planet’s ecosystems. Groundwater ecosystems deliver services that are of immense societal and economic value, such as: 1) purification of water and its storage in good quality for decades and centuries, 2) active biodegradation of anthropogenic contaminants and inactivation and elimination of pathogens, 3) nutrient recycling, and 4) mitigation of floods and droughts. Many of these services are directly connected to the presence and activity of specific organisms, microorganisms, or metazoa. Sustainable protection and management of important groundwater ecosystem services will require quantitative understanding of processes at different spatial and temporal scales and assessment of their resistance and resilience with regard to common anthropogenic impacts. Our review compiles known groundwater ecosystem services and, where appropriate, highlights important research gaps.

Despite the active and intense treatment of wastewater, pathogenic microorganisms and viruses are frequently introduced into the aquatic environment. For most human pathogens, however, this is a rather hostile place, where starvation, continuous inactivation, and decay generally occur, rather than successful reproduction. Nevertheless, a great diversity of the pathogenic microorganisms can be detected, in particular, in the surface waters receiving wastewater. Pathogen survival depends majorly on abiotic factors such as irradiation, changes in water ionic strength, temperature, and redox state. In addition, inactivation is enhanced by the biotic interactions in the environment. Although knowledge of the antagonistic biotic interactions has been available since a long time, certain underlying processes and mechanisms still remain unclear. Others are well-appreciated and increasingly are applied to the present research. Our review compiles and discusses the presently known biotic interactions between autochthonous microbes and pathogens introduced into the aquatic environment, including protozoan grazing, virus-induced bacterial cell lysis, antimicrobial substances, and predatory bacteria. An overview is provided on the present knowledge, as well as on the obvious research gaps. Individual processes that appear promising for future applications in the aquatic environment are presented and discussed.

International audience; Groundwater ecosystems constitute the largest terrestrial freshwater biome. They are dark, extremely low in energy and do not provide much space but they contain an unexpectedly high diversity of living forms showing characteristic adaptive features. The restricted accessibility along with the enormous 'invisible' heterogeneity challenged for a long time testing of scientific theories and unraveling of ecosystem functioning. Triggered by an improved interdisciplinarity, comprehensive sampling strategies and current developments in biotechnology and statistical analysis, groundwater ecology gains momentum entering a new era of research. We are only beginning to understand adaptive mechanisms, species distribution patterns and ecosystem functioning. Ninety-five percent of global liquid freshwater is stored in the terrestrial subsurface constituting a major source of water for drinking, irrigation and industrial purposes. There is an urgent need to integrate evolutionary and ecological research for developing a holistic perspective of the functional roles of biodiversity and ecosystem services and predicting global changes under alternative groundwater resource use scenarios.

Continuous research in the field of water and health is important to ensure high standards of hygiene and to improve microbial risk assessment and water management. Various waterborne outbreaks induced by pathogenic bacteria, viruses and protozoa have been reported in recent decades. Pathogens derived from human and animal feces have been played frequently a key role in contaminations of freshwater. Reasons for the presence of pathogens in drinking water systems are insufficient wastewater treatment, contaminated raw water or damaged water supply systems. Numerous routes of transmission and the distribution of individual pathogens by raw water and drinking water constitute potential hazards to public health. Recently, different research activities have been initiated in Germany, which focus on the examination of pathogens in water to establish a modern microbial risk assessment according to the high-tech strategy employed by the federal government. This review article provides a compilation of the pathogens that are important in German water bodies (wastewater, surface water, groundwater, and drinking water) and includes coverage of German research on pathogens and technologies for reducing these pathogens in water, challenges to research, and recent developments in concentration and detection methods for pathogens. Finally, current knowledge gaps and ongoing research questions are highlighted.

CO(2) fixation is one of the most important processes on the Earth's surface, but our current understanding of the occurrence and importance of chemolithoautotrophy in the terrestrial subsurface is poor. Groundwater ecosystems, especially at organically polluted sites, have all the requirements for autotrophic growth processes, and CO(2) fixation is thus suggested to contribute significantly to carbon flux in these environments. We explored the potential for autotrophic CO(2) fixation in microbial communities of a petroleum hydrocarbon-contaminated aquifer by detection of functional marker genes (cbbL, cbbM), encoding different forms of the key enzyme RubisCO of the Calvin-Benson-Bassham cycle. Quantification of (red-like) cbbL genes revealed highest numbers at the upper fringe of the contaminant plume and the capillary fringe where reduced sulphur and iron species are regularly oxidized in the course of groundwater table changes. Functional gene sequences retrieved from this area were most closely related to sequences of different thiobacilli. Moreover, several cultures could be enriched from fresh aquifer material, all of which are able to grow under chemolithoautotrophic conditions. A novel, nitrate-reducing, thiosulfate-oxidizing bacterial strain, recently described as Thiobacillus thiophilus D24TN(T) sp. nov., was shown to carry and transcribe RubisCO large-subunit genes of form I and II. Enzyme tests proved the actual activity of RubisCO in this strain.

The use of groundwater as a carrier of thermal energy is an important source of sustainable heating and cooling. However, the effects of thermal use on geochemical and biological aquifer characteristics are poorly understood. Here, we have assessed the impacts of heat discharge on an uncontaminated, shallow aquifer by monitoring the hydrogeochemical, bacterial and faunal parameters at an active thermal discharge facility. The observed variability between wells was considerable. Yet, no significant temperature impacts on bacterial or faunal abundance and on bacterial productivity were observed. Also, we did not observe an improved survival or growth of coliforms with temperature. In contrast, the diversity of bacterial terminal restriction fragment (T-RF) length polymorphism fingerprints and faunal populations was either positively or negatively affected by temperature, respectively, and the abundance of selected T-RFs was clearly temperature dependent. Canonical correspondence analysis indicated that both the impact of temperature and of surface water from a nearby river, were important drivers of aquifer biotic variability. These results demonstrate that aquifer thermal energy discharge can affect aquifer bacteria and fauna, while at the same time controlling only a minor part of the total seasonal and spatial variability and therefore posing no likely threat to ecosystem functioning and drinking water protection in uncontaminated, shallow aquifers.

Summary 1. Groundwater ecosystems offer vast and complex habitats for diverse microbial communities. Here we review the current status of groundwater microbial biodiversity research with a focus on Bacteria and Archaea and on the prospects of modern techniques for enhancing our understanding of microbial biodiversity patterns and their relation to environmental conditions. 2. The enormous volume of the saturated terrestrial underground forms the largest habitat for microorganisms on earth. Up to 40% of prokaryotic biomass on earth is hidden within this terrestrial subsurface. Besides representing a globally important pool of carbon and nutrients in organisms, these communities harbour a degree of microbial diversity only marginally explored to date. 3. Although first observations of groundwater microbiota date back to Antonie van Leeuwenhoek in 1677, the systematic investigation of groundwater microbial biodiversity has gained momentum only within the last few decades. These investigations were initiated by an increasing awareness of the importance of aquifer microbiota for ecosystem services and functioning, including the provision of drinking water and the degradation of contaminants. 4. The development of sampling techniques suitable for microbiological investigations as well as the application of both cultivation-based and molecular methods has yielded substantial insights into microbial communities in contaminated aquifers, whereas knowledge of microbial biodiversity in pristine habitats is still poor at present. 5. Several novel phylogenetic lineages have been described from groundwater habitats, but to date no clearly ‘endemic’ subsurface microbial phyla have been identified. The future will show if the rather low diversity generally found in pristine oligotrophic aquifers is a fact or just a result of low abundances and insufficient resolution of today’s methods. Refined approaches complemented by statistically rigorous applications of biodiversity estimates are urgently needed. 6. Factors identified to control microbial diversity in aquifers include spatial heterogeneity, temporal variability and disturbances such as pollution with chemical anthropogenic contaminants. Although first insights into the importance of individual biogeochemical processes may be obtained from surveys of microbial diversity within functional groups, direct links to groundwater ecosystem functioning have rarely been established so far.

Groundwater ecology merged during the second part of the 20 th century with modern ecological practice after having adopted the ‘ecosystem concept’. The latter was first applied to karstic systems and separately for alluvial non-consolidated aquifers along surface running waters. Today groundwater ecosystems are studied within a multi- and transdisciplinary framework at various spatial and temporal scales by experts dealing with microbiology, the ecology and systematics of meio- and macro-fauna, geochemistry, hydrogeology and mathematical modelling. A further paradigmatic change occured with the recognition that subterranean assemblages of organisms are formed by both hypogean and epigean taxa. The biological diversity in subterranean ecosystems can be much higher than earlier thought and may even exceed surface diversity in some taxa. This largely unrecognized biodiversity in many cases deserves environmental protection. A third phase in the development of groundwater ecology has occured over the last 15 years with the incorporation of socio-economic research topics within groundwater ecology (GIBERT et al., 1994a) and in this sense today we have the “New Groundwater Ecology”.

Healthy aquifers deliver important ecosystem services, e.g. the purification of infiltrating water and the storage of high-quality water over decades in significant quantities. The functioning of terrestrial and surface aquatic ecosystems directly depends on groundwater and vice versa. Nowadays, legislation has started to consider groundwater not only as a resource but as a living ecosystem. The assessment of ecosystems requires consideration of ecological criteria, which, so far, are not available for groundwater systems. In the framework of a project supported by the German Federal Environment Agency (UBA), a first concept for the ecological assessment of groundwater ecosystems is developed. Steps to be taken are introduced, with a strong focus on microbes as potential bioindicators. These include (1) the typology of groundwater ecosystems, (2) the derivation of natural background values, (3) the identification of potential bioindicators, and (4) the development of an assessment model. First successes and difficulties associated with these challenges, e.g. the lack of simple correlations between abiotic and biotic variables, are discussed on the basis of a data set from a local and regional aquifer in NE Germany. The need for collaboration between ecologists, hydrogeologists and geochemists, as well as the application of modern approaches such as multivariate statistics, is emphasized.

The fate of organic contaminants in groundwater environments is controlled by a multitude of abiotic and biotic processes, such as transport, dilution, dispersion, and chemical or microbial degradation. The last is considered the only sustainable component of natural attenuation (9, 46). Due to large carbon loads and the low rates of oxygen replenishment to groundwater systems, oxygen is rapidly depleted upon contaminant impact (2). Therefore, within hydrocarbon-contaminated aquifers, anoxic contaminant plumes with distinct redox compartments are formed, where microbial guilds capable of using locally available electron donors and acceptors are active (9). Biodegradation processes occur at different rates in these redox zones (46), but it is still poorly understood which plume compartments are most relevant for net contaminant removal. Especially, overlapping countergradients of electron donors and acceptors are assumed to be hot spots of biodegradation (3, 12, 53, 54), a hypothesis that has recently been summarized as the “plume fringe concept” (4). Such redox gradients may be extant on very fine spatial scales; thus, a detailed characterization of redox species and intrinsic microbiota at appropriate spatial resolutions is a prerequisite for a better understanding of biodegradation processes. It is especially relevant to ask how the spatial distribution of plume compartments and degradation processes is correlated to local microbial communities in general and whether the distribution of specific contaminant degraders can inform us about the occurrence and localization of the respective processes. Local microbial community composition has been shown to yield important insights into the microbes characteristic of different contaminated zones, as well as into their putative involvement in specific transformation processes in aquifers (1, 13, 27, 39). However, the monitoring of microbial capacities and their distribution at contaminated sites as a basis for assessing natural attenuation and for promoting biota-based site management options is still in its infancy. This may be attributed partially to the fact that conventional multilevel groundwater sampling is performed usually with a depth resolution of meters (24, 49). This spatial resolution has been suggested to be inadequate for truly assessing ongoing natural attenuation processes (57). In this study, to address these questions, we have characterized microbial community distribution across a high-resolution depth profile of a tar oil-impacted aquifer at a former gasworks location, where hydrocarbon degradation has been reported to depend mainly on sulfate reduction (14, 59). We have specifically traced as a model system intrinsic populations of anaerobic toluene degraders by using their benzylsuccinate synthase (bssA) genes as specific catabolic markers (58). Benzylsuccinate synthase (Bss) is the key enzyme of anaerobic toluene oxidation and has repeatedly been proven to be a valuable functional marker gene for unknown anaerobic toluene degraders (6, 55, 58). Thus, we have shown for the Flingern site in Dusseldorf, Germany, that local anaerobic toluene degraders are dominated by an as-yet unaffiliated lineage of environmental bssA genes, tentatively termed “F1-cluster” bssA, which has ∼90% amino acid similarity to known geobacterial bssA genes (58). However, the identities of the degraders carrying these bssA genes, as well as their quantitative distribution and relevance for net toluene degradation in the contaminant plume, remain to be addressed. At the site, we have collected sediment cores and installed a unique high-resolution multilevel monitoring well (3). Using fine-scale hydrogeochemical measurements as well as spatially resolved qualitative and quantitative molecular microbial community analyses, we show that a highly specialized degrader community, as well as a surprisingly high abundance of specific anaerobic toluene degraders, resides within the biogeochemical gradient zone underneath the plume core. These findings show that the distribution of specific aquifer microbiota and redox and degradation processes in contaminated aquifers are tightly coupled.

Polycyclic aromatic hydrocarbons (PAHs) are among the most important contaminants of groundwater. The 2- and 3-ring PAHs are of particular concern because they are water soluble in the 1-200 μg/l range and are transported with the groundwater over significant distances. Anaerobic degradation of PAH has been demonstrated in several microcosm studies with nitrate, ferric iron, or sulfate as electron acceptors and under methanogenic conditions. The biochemical degradation pathways were studied with naphthalene-degrading pure and enrichment cultures and revealed that 2-naphthoic acid is a central metabolite. Naphthalene is activated by addition of a C₁-unit to generate 2-naphthoic acid, whereas methylnaphthalene is activated by addition of fumarate to the methyl group and further degraded to 2-naphthoic acid. In the central 2-naphthoic acid degradation pathway the ring system is reduced prior to ring cleavage generating e.g. 5,6,7,8-tetrahydro-2-naphthoic acid. The ring cleavage produces metabolites such as 2-carboxycyclohexylacetic acid indicating that further degradation goes via cyclohexane derivatives and not via aromatic compounds. Anaerobic degradation of PAH has also been demonstrated in situ in contaminated aquifers by identification of compound specific metabolites and using stable isotope fraction studies. Detection of specific metabolites of anaerobic PAH degradation such as naphthyl-2-methylsuccinate indicated anaerobic degradation of 2-methylnaphthalene in situ whereas 2-naphthoic acid was indicative of naphthalene and 2-methylnaphthalene degradation. Other carboxylic acids that were detected in groundwater indicated anaerobic degradation of a wide range of PAH and heterocyclic compounds. Degradation of naphthalenes in contaminated aquifers could also be confirmed by carbon stable isotope shifts in the residual substrate fraction. © 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Since nearly one hundred years Traunsee experiences the import of tons of liquid and solid waste originating from salt and soda production. Today, the lake exhibits chloride concentrations of up to 170 mg L-1 and 19% of the lake floor are directly or indirectly influenced by industrial deposits (ID). Based on the comparison of several microbial parameters in unaffected, directly affected and intermediate lake bottom sediments, the ecological integrity of the lake was evaluated. The highly alkaline ID, which were exclusively colonized by microorganisms, harbored a bacterial community reduced by a factor of 10 in abundance and biomass compared to undisturbed sediment areas within the lake. The bacterial community of ID was furthermore characterized by a reduced content of actively respiring cells (INT-formazan reduction), a lower frequency of dividing cells (FDC) and a significantly reduced cell and biomass production. A 80 to 90% reduction in carbon recycling is estimated for the area exclusively covered by ID. Protists, although occasionally absent from the industrial sediments, were in general found to be less sensitive to the contaminant stress. Differences in alkalinity and dissolved organic carbon (DOC) concentrations of sediment porewaters as well as the total organic content and C/N ratios of sediments partly explain the microbial pattern observed at the various sampling sites. Possible consequences of the continuous industrial tailings for the whole lake ecosystem and the validation of the ecological integrity are discussed.

Traunsee is a deep oligotrophic lake in Austria characterised by an artificial enrichment of chloride in the hypolimnion (up to 170 mg L-1) caused by waste disposal of soda and salt industries. Protists were collected monthly over one year, observed alive and after Quantitative Protargol Staining (ciliates) or via epifluorescence microscopy (heterotrophic flagellates). Three sites within the lake (0–40 m depths) were compared to deeper water layers from 60–160 m depths where chloride concentrations and conductivity were increased. In addition, we observed the protozooplankton of two neighbouring lakes, i.e. reference systems, during one sampling occasion. In Traunsee the abundance of ciliates was low (200–36 600 cells L-1) in contrast to high species diversity (at least 60 different species; HS = 2.6) throughout the year. The main pelagic species in terms of abundance were small oligotrichs and prostomatids like Rimostrombidium brachykinetum/hyalinum, Balanion planctonicum and Urotricha spp. throughout the investigation period. Among free-living heterotrophic flagellates, which occurred at densities of 40–2800 cells mL-1, small morphotypes dominated in the pelagial. No differences at the community level between the three lakes could be observed and pelagic ciliates and flagellates seemed not to be affected by increased chloride concentrations or by enhanced conductivity.

An improved assessment for the enumeration of aquatic sediment bacteria is introduced. Sediment samples were stained with DAPI and counterstained with SYBR Green II. This, for the first time, allows the researcher to clearly distinguish between bacteria and nonspecifically stained bacteria-like particles. A strong linear correlation was observed between total number of attached bacteria and sediment grain size in sandy fractions of lotic, lentic and subsurface environments. This trend did not continue into the silt and clay fraction. Ratios of attached to free-living bacteria in our studies spanned three orders of magnitude and varied with sediment type. Sediment habitats which were exposed to anthropogenic disturbance and/or contamination always had lower biofilm-porewater ratios than obviously undisturbed sites. Biofilm-porewater ratios are therefore suggested as valuable indicators for the characterization of sediment systems.

Temperature stress in invertebrates is known to be reflected by changes in catecholamine levels. However, the mechanisms of stress response are not fully understood. Groundwater and surface water amphipods are expected to be differently adapted to temperature elevations due to the different temperature regimes in their habitats and consequently, show a different stress response. No data have been published so far regarding the effects of stress on catecholamine patterns in groundwater invertebrates and accordingly, comparisons with surface water fauna are also missing. In this study, we compared the average catecholamine levels in two taxonomically related amphipod species: Niphargus inopinatus , living in groundwater with constant water temperatures throughout the year, and Gammarus pulex , a surface water stream amphipod frequently exposed to diurnal and seasonal temperature fluctuations. Furthermore, we tracked the immediate changes in whole-animal catecholamine levels in response to heat stress in both species. Pronounced differences in the catecholamine levels of the two species became apparent, with the average dopamine (DA) level of N. inopinatus being almost 1000 times higher than that in G. pulex . The noradrenaline (NA) concentrations in N. inopinatus were on average two orders of magnitude higher than in G. pulex , and for adrenaline (A), the difference constituted one order of magnitude. When exposed to short-term heat stress, both species showed a response in terms of catecholamine levels, but the observed patterns were different. In N. inopinatus , temperature stress was reflected by the appearance of adrenaline, while in G. pulex a significant increase in noradrenaline levels occurred in the treatment with the highest temperature elevation.

Linked to diverse biological processes, groundwater ecosystems deliver essential services to mankind, the most important of which is the provision of drinking water. In contrast to surface waters, ecological aspects of groundwater systems are ignored by the current European Union and national legislation. Groundwater management and protection measures refer exclusively to its good physicochemical and quantitative status. Current initiatives in developing ecologically sound integrative assessment schemes by taking groundwater fauna into account depend on the initial classification of subsurface bioregions. In a large scale survey, the regional and biogeographical distribution patterns of groundwater dwelling invertebrates were examined for many parts of Germany. Following an exploratory approach, our results underline that the distribution patterns of invertebrates in groundwater are not in accordance with any existing bioregional classification system established for surface habitats. In consequence, we propose to develope a new classification scheme for groundwater ecosystems based on stygoregions.

Seasonal patterns of groundwater and sediment microbial communities were explored in a hydrologically dynamic alpine oligotrophic porous aquifer, characterized by pronounced groundwater table fluctuations. Rising of the groundwater level in consequence of snow melting water recharge was accompanied by a dramatic drop of bacterial Shannon diversity in groundwater from H '=3.22+/-0.28 in autumn and winter to H '=1.31+/-0.35 in spring and summer, evaluated based on T-RFLP community fingerprinting. Elevated numbers of bacteria in groundwater in autumn followed nutrient inputs via recharge from summer rains and correlated well with highest concentrations of assimilable organic carbon. Sterile sediments incubated to groundwater in monitoring wells were readily colonized reaching maximum cell densities within 2months, followed by a consecutive but delayed increase and leveling-off of bacterial diversity. After 1year of incubation, the initially sterile sediments exhibited a similar number of bacteria and Shannon diversity when compared to vital sediment from a nearby river incubated in parallel. The river bed sediment microbial communities hardly changed in composition, diversity, and cell numbers during 1year of exposure to groundwater. Summing up, the seasonal hydrological dynamics were found to induce considerable dynamics of microbial communities suspended in groundwater, while sediment communities seem unaffected and stable in terms of biomass and diversity.

The use of ecological criteria for the assessment of aquatic ecosystem status is routine for surface waters. So far no ecological parameters are considered for the assessment and monitoring of groundwater quality. It has been well known for decades that aquifers are ecosystems harbouring a vast diversity of invertebrates and microorganisms. The growing knowledge on groundwater microbial and faunal communities as well as the molecular and statistical tools available form a solid ground for the development of first ecologically sound assessment schemes. The sensitivity of groundwater communities towards impacts from land use and surface waters is exemplarily demonstrated by a data set of two geologically similar but hydrologically partially separated aquifer systems. Subgroups of the fauna in groundwater (stygobites vs. stygophiles and stygoxenes) successfully indicated elevated nitrate impacts linked to land use activities. Within the microbial communities, impacts from land use are mirrored by high bacterial biodiversity values atypical for pristine groundwater of comparable systems. The data show that there is legitimate hope for the application of ecological criteria for groundwater quality assessment in the future.

Implementation of the European Union (EU) Water Framework Directive (WFD 2000) and its planned supplement ‘Directive on the Protection of Groundwater against Pollution’ (EU GWD [European Union Groundwater Directive] 2003) demands an initial characterization of all groundwater bodies by national and regional authorities. The main criteria considered in the Directives to define the groundwater (GW) status are quality (chemical) and quantity, but there is the obvious omission of ecological perspective in these. A directive for a comprehensive policy dealing with GW protection at the level of the EU is a prerequisite for human welfare. Additionally, recognition of GW-dependent ecosystems (GDEs) in the EU is equally important for their sustainable management. GW management and policy should recognize the ecological functions of GW and their interactions with GDEs. As they can stretch across national and regional boundaries, political recognition of their importance and the necessity for ecological consistency of management plans have to be emphasized. During the last three decades, a number of researchers have focused their attention on the dynamics and functioning of GW ecosystems and this work could serve as a basis for identifying impacts of changes in key attributes of GDEs. Detailed aspects of GW ecosystems have been comprehensively dealt with (Gibert et al. 1994; Wilkens et al. 2000; Griebler et al. 2001; Danielopol et al. 2003). Changes in key attributes of GW ecosystems have had consequences for the environment in the EU and USA (Klijn & Witte 1999; Winter 1999; Sophocleous 2002; Winter et al. 2003).

Fractionation of stable carbon isotopes upon degradation of trichlorobenzenes was studied under aerobic and anaerobic conditions. Mineralization of 1,2,4-trichlorobenzene by the aerobic strain Pseudomonas sp. P51 which uses a dioxygenase for the initial enzymatic reaction was not accompanied by a significant isotope fractionation. In contrast, reductive dehalogenation by the anaerobic strain Dehalococcoides sp. strain CBDB1 revealed average isotope enrichment factors (ε) between -3.1 and -3.7 for 1,2,3- and 1,2,4-trichlorobenzene, respectively. The significant isotope fractionation during reductive dehalogenation would allow tracing the in situ biodegradation of halogenated benzenes in contaminated anoxic aquifers, whereas the lack of isotope fractionation during aerobic transformation limits the use of this approach in oxic environments. © 2004 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.

A new method to determine microbial (bacterial and fungal) activity in various freshwater habitats is described. Based on microbial reduction of dimethyl sulfoxide (DMSO) to dimethyl sulfide (DMS), our DMSO reduction method allows measurement of the respiratory activity in interstitial water, as well as in the water column. DMSO is added to water samples at a concentration (0.75% [vol/vol] or 106 mM) high enough to compete with other naturally occurring electron acceptors, as determined with oxygen and nitrate, without stimulating or inhibiting microbial activity. Addition of NaN3, KCN, and formaldehyde, as well as autoclaving, inhibited the production of DMS, which proves that the reduction of DMSO is a biotic process. DMSO reduction is readily detectable via the formation of DMS even at low microbial activities. All water samples showed significant DMSO reduction over several hours. Microbially reduced DMSO is recovered in the form of DMS from water samples by a purge and trap system and is quantified by gas chromatography and detection with a flame photometric detector. The DMSO reduction method was compared with other methods commonly used for assessment of microbial activity. DMSO reduction activity correlated well with bacterial production in predator-free batch cultures. Cell-production-specific DMSO reduction rates did not differ significantly in batch cultures with different nutrient regimes but were different in different growth phases. Overall, a cell-production-specific DMSO reduction rate of 1.26 × 10−17± 0.12 × 10−17mol of DMS per produced cell (mean ± standard error;R2= 0.78) was calculated. We suggest that the relationship of DMSO reduction rates to thymidine and leucine incorporation is linear (theR2values ranged from 0.783 to 0.944), whereas there is an exponential relationship between DMSO reduction rates and glucose uptake, as well as incorporation (theR2values ranged from 0.821 to 0.931). Based on our results, we conclude that the DMSO reduction method is a nonradioactive alternative to other methods commonly used to assess microbial activity.

We used specially designed microcosms filled with natural substrate to study microbial colonization in a shallow aquifer. Sterilized sediments were exposed to 3 types of groundwater varying in physical, chemical and biological characteristics: (1) pristine groundwater (site PI 92); (2) groundwater in an observation well at a pristine site (OMV 11); and (3) contaminated groundwater at a landfill site (OMV 5). The number of suspended bacteria was always highest at the landfill site (4.0 ± 4.2 [standard deviation, SD] x 10 6 cells cm -3 ), i.e. on average 16 times higher than in the well water (2.5 ± 3.0 x 10 5 cells cm -3 ) and 96 times higher than in the pristine groundwater (4.1 ± 1.3 x 10 4 cells cm -3 ). Sediments in the microcosms were rapidly colonized and the total number of attached bacteria after 10 mo of exposure was highest at the landfill site (1.8 ± 0.4 × 10 8 cells cm -3 ) followed by the sediment incubated in well water (1.5 ± 0.5 x 10 8 cells cm -3 ) and in pristine groundwater (5.0 ± 1.5 x 10 7 cells cm -3 ). As estimated from image analysis, attached cells from the landfill site were on average characterized by higher cell carbon contents (28 ± 36 fg C cell -1 ) than at the well water (24 ± 23 fgC cell -1 ) and the pristine groundwater site (21 ± 23 fgC cell -1 ). The ratio of attached to suspended bacteria after 10 mo of exposure was highest in the microcosm incubated in pristine groundwater (1657:1) and lowest at the contaminated site (59:1). On the basis of our results we emphasize the importance of attached microbial communities in porous subsurface systems and underline the need for groundwater as well as sediment samples for a serious microbiological characterization of the subsurface. Furthermore, the ratio of attached to suspended bacteria in shallow aquifer systems is suggested to be an indicator of prevailing nutrient concentrations.