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The traditional perception of groundwater food webs is that of short and poorly structured food chains extending to only few trophic levels. This is mainly attributed to the lack of light and the shortfall of substantial primary production as well as the shortage of dissolved organic carbon and nutrients. It is frequently argued that microbes form the basis of groundwater food webs. In reality, the obvious lack of comprehensive biofilms may point to detritus as the main common food source and an opportunistic and omnivorous feeding strategy with most groups and species. Also, the relative stability of groundwater environments may have led to low trait variability. However, recent research also contrasts the paradigm of poorly structured food chains against emerging convoluted mechanisms sustaining a great range of biotic diversity and functional complexity. In summary, understanding of the ecological and functional linkages among stygofaunal groups, and their interaction with the microbial community, is still in its infancy. Stygofauna sampled from three spots of a shallow alluvial aquifer in the vicinity of the Danube River in the city of Vienna, Austria, has been analyzed by means of bulk stable carbon and nitrogen isotope and gut microbiome analysis to disentangle food web interactions. Our preliminary data revealed the following pattern: plant material, terrestrial insects, and detritus show a narrow range in carbon (-32 to -25δ13C) and a wide range in nitrogen (-1.5 to 6.5 δ15N) isotope signatures. In terms of nitrogen isotope values, the next tropic level is represented by isopods and gastropods, followed by ostracods, oligochaetes and amphipods. The broader range of stygofauna in carbon isotope signatures indicate an additional food source; most probably microbes; gut microbiome analyses indicate differences with oligochaetes, gastropods, ostracodes, isopods and amphipodes, however, with a few individuals from the later three groups sharing a very similar gut microbiome; within the amphipods, there is obviously a difference in gut microbiome composition with species and sampling sites; carbon and nitrogen isotope signatures underline amphipod-species differences; microbiome diversity in all cases was less divers in the animal guts when compared to environmental water, sediment and detritus samples. plant material, terrestrial insects, and detritus show a narrow range in carbon (-32 to -25δ13C) and a wide range in nitrogen (-1.5 to 6.5 δ15N) isotope signatures. In terms of nitrogen isotope values, the next tropic level is represented by isopods and gastropods, followed by ostracods, oligochaetes and amphipods. The broader range of stygofauna in carbon isotope signatures indicate an additional food source; most probably microbes; gut microbiome analyses indicate differences with oligochaetes, gastropods, ostracodes, isopods and amphipodes, however, with a few individuals from the later three groups sharing a very similar gut microbiome; within the amphipods, there is obviously a difference in gut microbiome composition with species and sampling sites; carbon and nitrogen isotope signatures underline amphipod-species differences; microbiome diversity in all cases was less divers in the animal guts when compared to environmental water, sediment and detritus samples. Finally, we first collected evidence for anaerobic processes taking place in animal guts living in an oxic groundwater environment. In light of our data, putative groundwater food web interactions in the shallow alluvial aquifer are discussed.

Climate change has a massive impact on the global water cycle. Subsurface ecosystems, the earth largest reservoir of liquid freshwater, currently experience a significant increase in temperature and serious consequences from extreme hydrological events. Extended droughts as well as heavy rains and floods have measurable impacts on groundwater quality and availability. In addition, the growing water demand puts increasing pressure on the already vulnerable groundwater ecosystems. Global change induces undesired dynamics in the typically nutrient and energy poor aquifers that are home to a diverse and specialized microbiome and fauna. Current and future changes in subsurface environmental conditions, without doubt, alter the composition of communities, as well as important ecosystem functions, for instance the cycling of elements such as carbon and nitrogen. A key role is played by the microbes. Understanding the interplay of biotic and abiotic drivers in subterranean ecosystems is required to anticipate future effects of climate change on groundwater resources and habitats. This review summarizes potential threats to groundwater ecosystems with emphasis on climate change and the microbial world down below our feet in the water saturated subsurface.

Compound-specific isotope analysis (CSIA) can reveal mass-transfer limitations during biodegradation of organic pollutants by enabling the detection of masked isotope fractionation. Here, we applied CSIA to monitor the adaptive response of bacterial degradation in inoculated sediment to low contaminant concentrations over time. We characterized Aminobacter sp. MSH1 activity in a flow-through sediment tank in response to a transient supply of elevated 2,6-dichlorobenzamide (BAM) concentrations as a priming strategy and took advantage of an inadvertent intermittence to investigate the effect of short-term flow fluctuations. Priming and flow fluctuations yielded improved biodegradation performance and increased biodegradation capacity, as evaluated from bacterial activity and residual concentration time series. However, changes in isotope ratios in space and over time evidenced that mass transfer became increasingly limiting for degradation of BAM at low concentrations under such stimulated conditions, and that activity decreased further due to bacterial adaptation at low BAM (μg/L) levels. Isotope ratios, in conjunction with residual substrate concentrations, therefore helped identifying underlying limitations of biodegradation in such a stimulated system, offering important insight for future optimization of remediation schemes.

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”).

Groundwater fauna (stygofauna) comprises organisms that have adapted to the dark subterranean environment over a course of thousands and millions of years, typically having slow metabolisms and long life cycles. They are crucial players in the groundwater of oxygenic aquifers, and contribute to various ecosystem services. Today's knowledge of their sensitivity to anthropogenic impacts is incomplete and a critical analysis of the general relevance of local findings is lacking. In this review, we focus on those areas with the highest interference between humans and stygofauna: cities. Here is where local pollution by various contaminants and heat strongly stresses the unique groundwater ecosystems. It is demonstrated that it is difficult to discern the influence of individual factors from the findings reported in field studies, and to extrapolate laboratory results to field conditions. The effects of temperature increase and chemical pollution vary strongly between tested species and test conditions. In general, previous findings indicate that heating, especially in the long-term, will increase mortality, and less adapted species are at risk of vanishing from their habitats. The same may be true for salinity caused by road de-icing in cold urban areas. Furthermore, high sensitivities were shown for ammonium, which will probably be even more pronounced with rising temperatures resulting in altered biodiversity patterns. Toxicity of heavy metals, for a variety of invertebrates, increases with time and chronic exposure. Our current knowledge reveals diverse potential impacts on groundwater fauna by urban pollution, but our insights gained so far can only be validated by standardized and long-term test concepts.

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.

An integrative interdisciplinary approach is currently developed to investigate groundwater systems in alpine and prealpine environments and how they respond to hydrological extremes such as droughts, heavy rain and floods in terms of water quantity, hydrochemical quality, and ecological status. The new approach is aimed at improving the understanding of the interaction between physical, chemical, and biological processes in groundwater responses to extreme events as well as developing indicators suitable for an integrative monitoring and management of the aquifers. For this purpose, observation wells of the existing state hydrographic monitoring net have been selected within the Austrian part of the Mur river basin, stretching from the alpine origin to the national border in the foreland. The investigation area thus comprises diverse hydrogeological settings and land-use types. The selected observation wells have long-term records of groundwater levels and are used for sampling campaigns under different hydrological conditions. Groundwater level fluctuations are evaluated using drought indices and statistical approaches, such as auto-correlation and cross-correlation with precipitation and stream stages. Our hydrochemical analyses of groundwater and surface waters also consider compounds indicative of agricultural sources (e.g., nitrate), wastewater-borne micro-pollutants, and stable isotopes of water. These indicators are used to identify different drivers controlling water origin and quality. The ecological status is characterized using microbiological measures, such as total number of bacteria and microbial activity, groundwater fauna, and the qualitative composition of dissolved organic matter (DOM). First results demonstrate a deterioration of water quality from groundwater to surface water and from the alpine region towards the foreland, corresponding to the more intense agricultural and urban land use in the foreland. Linkages between water quality and hydrological conditions are currently being evaluated and will be further examined using UV-Vis spectrometry for high-resolution in-situ monitoring of water quality changes (DOM and nitrate) at selected observation wells.

In this presentation we provide a brief overview on the strategic selection of representative groundwater wells and lessons learned.The inter-disciplinary project “Integrative Groundwater Assessment”, looks into the effects of extreme hydro-meteorological events on the quantity and the chemical and biological quality of groundwater. Focus is on the Austrian Mur catchment, an area reaching from its alpine spring (~2000 m asl) down to the Slovenian border (~200 m asl). More than 500 state operated groundwater observation wells are available over the 400 km of the river’s course, taking private wells not into account. For state operated wells, time series for water levels are publicly available which allows for simply using all the data i.e. using big data approaches [1, 2, 3] – albeit with some issues [4].However, for water quality, such time series rarely exist and if so, they often do not cover all specific parameters one needs, asking for targeted sampling campaigns. The availability of hundreds of wells seems like a benefit. However, the identification of wells that are representative and suitable for sampling regarding both chemical and biological parameters is a challenging taskIn consequence, we went through a multi-step process of planning a sampling campaign that should fulfill the following requirements: Coverage of the entire stream section from alpine to lowland regions Coverage of different land uses in the river valley Realization of well transects from the river through the complete local aquifer Wells allow sampling of groundwater for the analysis of physical-chemical and biological parameters Historical data of groundwater quantity and quality aspects are available Assessing the available metadata and taking into account the very helpful advice of stakeholders, already reduced the number of representative wells considerably. In order to obtain a consistent data set, another set of wells had to be dismissed, to allow for the same sampling and monitoring procedures at every location. Finally, out in the field, wells that were found damaged or out of order, led to a further reduction. Thus we ended up with only 45 wells suitable for our specific purposes, However, using specific strategies for data analysis as outlined in [3] and [4] and application of a novel groundwater ecological assessment scheme (D-A-C Index [5]) showed that even the substantially reduced number of wells provides a very good coverage of the various regions in the Mur catchment. In a further step, the results from two sampling campaigns and subsequent data analysis will be used to select an even smaller subset of wells where novel multi-parameter spectral dataloggers are going to be installed, enabling us to monitor various quality data in an very high temporal resolution.References:[1] https://doi.org/10.5194/egusphere-egu2020-8148[2] https://doi.org/10.1016/j.ejrh.2019.100597[3] https://doi.org/10.1007/s12665-018-7469-4[4] Haas et al (2020)Tiny steps towards Big Data - Freud und Leid der Arbeit mit großen Grundwasserdatensätzen.Tagungsband 2020. Grundwasser und Flusseinzugsgebiete - Prozesse, Daten und Modelle.[5] https://doi.org/10.1016/j.watres.2019.114902

Organic contaminant degradation by suspended bacteria in chemostats has shown that isotope fractionation decreases dramatically when pollutant concentrations fall below the (half-saturation) Monod constant. This masked isotope fractionation implies that membrane transfer is slow relative to the enzyme turnover at μg L–1 substrate levels. Analogous evidence of mass transfer as a bottleneck for biodegradation in aquifer settings, where microbes are attached to the sediment, is lacking. A quasi-two-dimensional flow-through sediment microcosm/tank system enabled us to study the aerobic degradation of 2,6-dichlorobenzamide (BAM), while collecting sufficient samples at the outlet for compound-specific isotope analysis. By feeding an anoxic BAM solution through the center inlet port and dissolved oxygen (DO) above and below, strong transverse concentration cross-gradients of BAM and DO yielded zones of low (μg L–1) steady-state concentrations. We were able to simulate the profiles of concentrations and isotope ratios of the contaminant plume using a reactive transport model that accounted for a mass-transfer limitation into bacterial cells, where apparent isotope enrichment factors *ε decreased strongly below concentrations around 600 μg/L BAM. For the biodegradation of organic micropollutants, mass transfer into the cell emerges as a bottleneck, specifically at low (μg L–1) concentrations. Neglecting this effect when interpreting isotope ratios at field sites may lead to a significant underestimation of biodegradation., Please provide a synopsis

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.

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

Dissolved organic matter (DOM) in fresh groundwater is generally low in concentration compared to other fresh waters. However, the overall amount of groundwater DOM is huge, as there is 100 times more fresh groundwater than fresh surface water. To date, research on groundwater DOM has merely focused on specific threats to humans such as e.g. DOM and heavy metal complexations and DOM from hydrocarbon contamination. Only few studies targeted to understand DOM as energy source of groundwater food webs and the role of groundwater DOM in the global carbon cycle. While research on these two subjects in surface waters flourish, a comprehensive, large-scale study of groundwater is still missing. Since a major fraction of Earth’s microbial biomass is found in the subsurface, mainly in aquifers, this represents a major knowledge gap. Moreover, researchers found that groundwater DOM concentrations worldwide increase alarmingly. Here, for the first time, we examine DOM properties and heterogeneity in a large-scale approach with regards to aquifer characteristics and physical-chemical as well as microbial features. We hypothesize that groundwater DOM quality shows high diversity and plays an important, yet complex role in these ecosystems, where bioavailability is influenced by intrinsic molecular properties, as well as environmental conditions.We analyzed 1000 water samples from 100 groundwater bodies all over Austria with regards to their DOM quantity, quality and bacterial abundance (BA). From fluorescence excitation-emission-matrices (EEMs) we derived indices and components to describe DOM quality. Next, we explored this data with principal component analysis, where we used convex-hull areas to estimate the heterogeneity of DOM composition within the groundwater bodies. In parallel, the similarity of DOM quality was evaluated with self-organizing maps on EEMs to test if we captured the heterogeneity of the data set sufficiently with the previous analysis. DOM quantity and quality was then related to BA and physical-chemical parameters.Our results show that water from fractured and karstic aquifers exhibit significantly higher terrestrial DOM origin and less degraded DOM than porous aquifers. This result can be explained by abiotic factors such as adsorption of large, aromatic compounds, as well as biological factors, specifically, larger surface areas for biofilm development in porous aquifers. The latter is supported by our observation that porous aquifers showed higher BA values. Remarkably, we found that BA was related to different DOM quality in each aquifer type: In porous aquifers BA was related to large, aromatic DOM molecules indicating that these are important for bacterial growth, while in fractured and karstic aquifers BA was related to fulvics and highly degraded humic compounds. Bacterial growth and degradation of complex DOM might be limited by low retention times in some of these aquifers. Also, we found that groundwater bodies located in river valleys display high heterogeneity in DOM quality spanning across the whole DOM compositional diversity found in this study. This finding could either be explained by surface water infiltration in some parts and younger groundwater or the fact that river valleys are main settlement areas.

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

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.

Aromatic hydrocarbons belong to the most abundant contaminants in groundwater systems. They can serve as carbon and energy source for a multitude of indigenous microorganisms. Predictions of contaminant biodegradation and microbial growth in contaminated aquifers are often vague because the parameters of microbial activity in the mathematical models used for predictions are typically derived from batch experiments, which don’t represent conditions in the field. In order to improve our understanding of key drivers of natural attenuation and the accuracy of predictive models, we conducted comparative experiments in batch and sediment flow-through systems with varying concentrations of contaminant in the inflow and flow velocities applying the aerobic Pseudomonas putida strain F1 and the denitrifying Aromatoleum aromaticum strain EbN1. We followed toluene degradation and bacterial growth by measuring toluene and oxygen concentrations and by direct cell counts. In the sediment columns, the total amount of toluene degraded by P. putida F1 increased with increasing source concentration and flow velocity, while toluene removal efficiency gradually decreased. Results point at mass transfer limitation being an important process controlling toluene biodegradation that cannot be assessed with batch experiments. We also observed a decrease in the maximum specific growth rate with increasing source concentration and flow velocity. At low toluene concentrations, the efficiencies in carbon assimilation within the flow-through systems exceeded those in the batch systems. In all column experiments the number of attached cells plateaued after an initial growth phase indicating a specific “carrying capacity” depending on contaminant concentration and flow velocity. Moreover, in all cases, cells attached to the sediment dominated over those in suspension, and toluene degradation was performed practically by attached cells only. The observed effects of varying contaminant inflow concentration and flow velocity on biodegradation could be captured by a reactive-transport model. By monitoring both attached and suspended cells we could quantify the release of new-grown cells from the sediments to the mobile aqueous phase. Studying flow velocity and contaminant concentrations as key drivers of contaminant transformation in sediment flow-through microcosms improves our system understanding and eventually the prediction of microbial biodegradation at contaminated sites. Electronic supplementary material The online version of this article (10.1007/s10532-018-9824-2) contains supplementary material, which is available to authorized users.

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.

Earth’s ecosystems provide a multitude of goods and functions, recently conceptualized under the term ecosystem services (ES; [1]). Many of these services and their associated ecosystems have received considerable attention, but groundwater ecosystems, soils, and sediments that are hidden below our feet are often overlooked. In fact, subsurface ecosystems deliver services of immense societal and economic value, most prominently the purification of water through (1) nutrient cycling; (2) biodegradation of contaminants; (3) inactivation and elimination of pathogens; and (4) storage and transmission of water that can mitigate floods and provide a stable water supply during droughts. Several of these services are directly connected to the presence and activity of the microorganisms and metazoans living in groundwater. We argue that, due to global and climate change, many of the groundwater ecosystem services are at serious risk. The pressures on groundwater ecosystems include aspects of global change such as local (point) and diffuse (non-point) sources of contamination, and overexploitation of groundwater resources. Moreover, even though groundwater ecosystems are located below ground, their organisms and the services they provide are affected by climate change—inter alia through changes in temperature regime as well as changes in recharge patterns and hydrological conditions due to floods and droughts.

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.

At numerous groundwater sites worldwide, natural dissolved organic matter (DOM) is quantitatively complemented with petroleum hydrocarbons. To date, research has been focused almost exclusively on the contaminants, but detailed insights of the interaction of contaminant biodegradation, dominant redox processes, and interactions with natural DOM are missing. This study linked on-site high resolution spatial sampling of groundwater with high resolution molecular characterization of DOM and its relation to groundwater geochemistry across a petroleum hydrocarbon plume cross-section. Electrospray- and atmospheric pressure photoionization (ESI, APPI) ultrahigh resolution mass spectrometry (FT-ICR-MS) revealed a strong interaction between DOM and reactive sulfur species linked to microbial sulfate reduction, i.e., the key redox process involved in contaminant biodegradation. Excitation emission matrix (EEM) fluorescence spectroscopy in combination with Parallel Factor Analysis (PARAFAC) modeling attributed DOM samples to specific contamination traits. Nuclear magnetic resonance (NMR) spectroscopy evaluated the aromatic compounds and their degradation products in samples influenced by the petroleum contamination and its biodegradation. Our orthogonal high resolution analytical approach enabled a comprehensive molecular level understanding of the DOM with respect to in situ petroleum hydrocarbon biodegradation and microbial sulfate reduction. The role of natural DOM as potential cosubstrate and detoxification reactant may improve future bioremediation strategies.

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.

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.

Groundwater ecosystems are naturally low in productivity (oligotrophic) reflected by a comparably low standing stock of biomass and slow growth and reproduction of organisms. On the other hand, aquifers are, due to their dimensions, enormous (interim) reservoirs of carbon. However, since we typically lack detailed information on carbon and energy flow through groundwater food webs, it is difficult to predict effects of climate and global change on groundwater ecosystems performance with regard to ‘sink’ or ‘source’ of carbon. Applying stable isotope analysis, high end mass spectrometry, and flow cytometry in addition to a suite of established methods in microbiology and geochemistry, we started to dissect, at selected sites, the various pools and fluxes of organic carbon in aquifers, including dissolved organic matter, particulate organic matter, and organisms. Moreover we investigated the link between carbon (organic and inorganic) and microbial growth and productivity. Last but not least, we tried to uncover food sources for groundwater fauna and the connection to microbes. The work was done in collaboration with Roland Hofmann, Katrin Hug, Marion Gallus, Lucas Fillinger, Marina Spona-Friedl, Alexander Braun, Ramona Brejcha (Institute of Groundwater Ecology at the Helmholtz Zentrum München, Germany) and Jenny Uhl, Norbert Hertkorn, and Philip Schmitt-Kopplin (Research Unit Analytical Biogeochemistry at the Helmholtz Zentrum München). Groundwater rarely contained less than 1 mg/L DOC, however, with only a small fraction being readily biodegradable (BDOC, AOC). Unrecognized, 10-100 times the amount of organic carbon dissolved in groundwater was found to be adsorbed to the sediment matrix. Cell numbers and activities of microbes in aquifers typically ranged 1-3 orders below values from surface waters. In fact, carbon use efficiency (CUE) of groundwater bacterial communities was considerably lower than values from other oligotrophic aquatic environments, with a mean value of 5% (200 samples from 100 sites). There is ample evidence that biogeochemical processes are primarily linked to the sediment associated microbial communities that outnumber their suspended counterparts in terms of biomass and activity by 1-4 orders of magnitude. While groundwater fauna at many sites seem to live on plant derived organic matter and cannibalism, we also collected evidence for chemolithoautotrophic primary production fueling groundwater food webs.

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.

Hydrochemical solute concentrations in the shallow subsurface can be spatially highly variable within small scales, particularly at interfaces. However, most monitoring systems fail to capture these small scale variations. Within this study, we developed a high resolution multilevel well (HR-MLW) with which we monitored water across the interface of the unsaturated and saturated zone with a vertical resolution of 0.05-0.5 m. We installed three of these 4 m deep HR-MLWs in the riparian zone of a third-order river and analyzed for hydrochemical parameters and stable water isotopes. The results showed three distinct vertical zones (unsaturated zone, upper saturated zone, lower saturated zone) within the alluvial aquifer. A 2 m thick layer influenced by river water (upper saturated zone) was not captured by existing monitoring wells with higher screen length. Hydrochemical data (isotopes, total ions) were consistent in all HR-MLWs and showed similar variation over time emphasizing the reliability of the installed monitoring system. Further, the depths zones were also reflected in the NO

Microbial communities are the driving force behind the degradation of contaminants like aromatic hydrocarbons in groundwater ecosystems. However, little is known about the response of native microbial communities to contamination in pristine environments as well as their potential to recover from a contamination event. Here, we used an indoor aquifer mesocosm filled with sandy quaternary calciferous sediment that was continuously fed with pristine groundwater to study the response, resistance and resilience of microbial communities to toluene contamination over a period of almost two years, comprising 132 days of toluene exposure followed by nearly 600 days of recovery. We observed an unexpectedly high intrinsic potential for toluene degradation, starting within the first two weeks after the first exposure. The contamination led to a shift from oxic to anoxic, primarily nitrate-reducing conditions as well as marked cell growth inside the contaminant plume. Depth-resolved community fingerprinting revealed a low resistance of the native microbial community to the perturbation induced by the exposure to toluene. Distinct populations that were dominated by a small number of operational taxonomic units (OTUs) rapidly emerged inside the plume and at the plume fringes, partially replacing the original community. During the recovery period physico-chemical conditions were restored to the pristine state within about 35 days, whereas the recovery of the biological parameters was much slower and the community composition inside the former plume area had not recovered to the original state by the end of the experiment. These results demonstrate the low resilience of sediment-associated groundwater microbial communities to organic pollution and underline that recovery of groundwater ecosystems cannot be assessed solely by physico-chemical parameters.

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.

A protocol was developed for testing the ecotoxicological effects of volatile organic compounds (VOCs) on groundwater invertebrates. Test substance volatility was addressed in a “closed from start to analysis”-design. Since manifestation of toxic effects may be delayed in ‘slower metabolizing’ organisms such as groundwater fauna, a time-independent (TI-) approach was adopted. Toluene was used as a model substance and its toxicity to the groundwater amphipod Niphargus inopinatus was assessed as an example. The method evaluation process considered various methodological issues such as partitioning of the toxicant between the water and the gas phase (Henry equilibrium), the possible depletion of oxygen in closed test vials, as well as microbial biodegradation of the test substance. For N. inopinatus , an LC 50 , 14 days of 46.6 mg L −1 toluene was obtained. The ultimate LC 50 value was estimated at 23.3 mg L −1 toluene. No oxygen depletion occurred in the test vials and Henry equilibrium was found to be established after 6 h. The new test system proposed now awaits broad practical application.

Geothermal energy plays an increasingly important role as a renewable energy source. However, it induces temperature changes in natural thermally static groundwater ecosystems. Temperature impacts can considerably alter the groundwater chemical composition and quality, the metabolism of organisms, and, consequently, biogeochemical processes and ecosystem functions. Combining original data from current studies with a compact review of recent findings, we show that a moderate increase in groundwater/aquifer temperature [+5 to 10 Kelvin (K)] generally causes only minor changes in water chemistry, microbial biodiversity, and ecosystem function in non-contaminated and energy-poor (oligotrophic) groundwater systems. In aquifers that are contaminated with organics, nutrients, and heavy metals—typical in urban areas and at sites with intensive land use (e.g., agriculture)—and particularly at temperatures ≥30 °C as regularly reached when heat is actively stored in aquifers, significant changes in water quality and ecological patterns can result. Here most critical are the heat-related mobilization of organic matter and contaminants (e.g., arsenic), the reduction and depletion of dissolved oxygen, and consequently the consecutive shift to anaerobic redox processes that may produce toxic and corrosive products (e.g., hydrogen sulfide) and greenhouse gases (e.g., methane and carbon dioxide). Severe temperature alterations lead to a reduced biodiversity of the aquifer’s microbial community with the establishment of atypical thermophilic assemblages. Groundwater fauna, which is specifically adapted to the cold groundwater habitat, may be sensitive to thermal changes at temperature increases of only 5 K with long-term emigration or direct lethal effects. From an ecological point of view, long-lasting or reoccurring temperature alterations need to be carefully evaluated and regulated in the future. We suggest developing local and regional vulnerability concepts for the sustainable and ecologically sound use of subterranean heat and cold.

Multiple factors have been shown to influence the assembly of sediment microbial communities. We hypo thesized that in an organically polluted aquifer, the degree of contamination controls bacterial distribution patterns, superimposing other selective forces such as sediment and mineral properties. Groundwater and sediment samples were analyzed from distinct zones of a petroleum hydrocarbon contaminated sandy aquifer that correspond to different degrees of contamination: Zone 1, with a high concentration of dissolved contaminants (benzene, toluene, ethylbenzene, and xylenes); Zone 2, with high concentrations of sediment-bound polycyclic aromatic hydrocarbons (PAHs); and Zone 3, with only minor PAH contamination. Sediment analysis concentrated on 2 mineral fractions differing in many sediment properties, i.e. translucent quartz (TQ) and mica. Sediment bacterial communities were analyzed by DNA fingerprinting (terminal restriction fragment length polymorphism) and total cell counts. While Zone 1 exhibited highly similar communities on TQ and mica, the selective sorption of PAHs to mica revealed sediment bacterial communities with hardly any taxonomic units shared in Zone 2. Typical selective forces active in sediments of oligotrophic habitats, such as sediment mineral content and surface roughness, only gained influence in Zone 3. Similarly, the least contamination revealed the most pronounced differences in Shannon diversity, evenness, and total cell counts between the mineral fractions tested, with mica characterized by highest biomass and bacterial diversity. The role of contamination as a selective force is also underlined by the zone-specific dominance of key microbes involved in petroleum hydrocarbon degradation. Our re sults demonstrate that typical selective forces shaping aquifer sediment microbial communities are outcompeted by organic contamination.

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.

Environmental policy and in particular the European water legislation, in the framework of the EU Groundwater Directive, has started to consider groundwater not only as a resource but as a living ecosystem. A precondition for comprehensive groundwater protection is thus the assessment of the biological and ecological state. The assessment of ecosystems requires consideration of ecological criteria, which so far are not available for groundwater systems. In the framework of a national project, the German Federal Environment Agency (UBA) supports a consortium of scientists and stakeholders from water boards and regional environmental authorities to develop a first concept for an ecological assessment scheme for groundwater ecosystems. The attempts towards an integrative concept include the following steps: (i) selection of appropriate biological and ecological parameters, (ii) typology of groundwater ecosystems, (iii) derivation of a reference status (Leitbild) and natural background values for biological variables, (iv) identification of potential bioindicators and definition of threshold values, and (v) development of an assessment model. These proposed steps are discussed on the basis of a data set from two groundwater landscapes in Southern Germany. Investigations considered three different spatial units, i.e. the habitat unit at the local scale, and the aquifer type unit as well as the landscape unit at the regional scale. Fauna as well as bacterial communities could provide valuable ecological information on the ecosystems status. The paper reviews ‘state of the art’ knowledge and evaluates the near future perspectives for the development and implementation of groundwater ecosystems assessment programmes.

High-resolution depth-resolved monitoring was applied to groundwater and sediments samples in a tar oil contaminated aquifer. Today, it is not fully clear, whether groundwater-based lines of evidence are always sufficient to adequately assess natural attenuation (NA) potentials and processes going on in situ. Our data unveiled small-scale heterogeneities, steep physical-chemical and microbial gradients, as well as hot spots of contaminants and biodegradation in the supposedly homogeneous sandy aquifer. The comparison of basic geochemical data revealed a fairly good agreement between sediment and groundwater samples. Nevertheless, a comprehensive understanding of both BTEX and PAH distribution, as well as redox processes involving insoluble electron acceptors, i.e., iron reduction, clearly asks for consideration of both, sediment and groundwater analysis. A thin BTEX plume right below the groundwater table was visible only in groundwater, while significant amounts of PAHs were present in sediments from deeper zones of the aquifer. Indications for sulfate reduction as a dominant process involved in BTEX degradation were largely obtained from groundwater, while the role of iron reduction in degradation and possible sulfide oxidation at the capillary fringe and the upper BTEX plume fringe, as well as in deeper PAH-contaminated zones was evident from sediments. Moreover, sediment analyses were essential to meaningfully recover cell abundances, distribution, activity and composition of the bacterial community. Sediments harbored 97.7% of bacterial cells and displayed enzyme activities 5 to 6 orders of magnitude higher than groundwater samples. Bacterial community T-RFLP fingerprints revealed important distinctions, but also similarities in depth-resolved microbial community distribution in sediments and water. An apparently highly specialized degrader population was found to dominate the lower BTEX plume fringe. However, even though sediment data seemed to comprise most community information found also in groundwater, this relation did not apply vice versa. In summary, our results show that groundwater sampled at an appropriate scale may contain sufficient information to identify and localize dominant redox reactions, but clearly fails to unravel natural attenuation potentials. This clearly emphasizes the importance of both groundwater and sediment samples for truly assessing natural attenuation potentials and activities at organically contaminated aquifers.

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 A fundamental prerequisite of any remedial activity is a sound knowledge of both the biotic and abiotic processes involved in transport and degradation of contaminants. Investigations of these aspects in situ often seem infeasible due to the complexity of interacting processes. A simplified portrayal of nature can be facilitated in laboratory-based two-dimensional (2D) sediment flow-through microcosms. This paper describes the versatility of such simple aquifer model systems with respect to biodegradation of aromatic hydrocarbons, i.e. toluene and ethylbenzene, under various environmental conditions. Initially constructed to study non-reactive and bioreactive transport of organic contaminants in homogeneous porous media under steady state hydraulic conditions, experimental setups developed towards more realistic heterogeneous sediment packing and transient hydraulic conditions. High-resolution spatial and temporal sampling allowed to obtain new insights on the distribution of bioactivities in contaminant plumes and associated controlling and limiting factors. Major biodegradation activities in saturated porous sediments are located at the fringes of contaminant plumes and are driven by dispersive mixing. These hot-spots of contaminant biotransformation are characterized by steep physical–chemical gradients in the millimeter to centimeter range. Sediment heterogeneity, i.e. high-conductivity zones, was shown to significantly enhance transverse mixing and subsequently biodegradation. On the contrary, transient hydraulic conditions may generate intermediate disturbances to biodegrader populations and thus may interfere with optimized contaminant conversion. However, a bacterial strain aerobically degrading toluene, i.e. Pseudomonas putida F1, was shown to adapt to vertically moving contaminant plumes, in the way that it regained full biodegradation potential two-times faster in areas with a mid-term (days to weeks) contamination history than in areas not contaminated before. The 2D flow-through microcosms facilitated to combine a number of physicochemical and microbiological methods, such as high-resolution non-invasive oxygen measurements, conservative tracer tests, compound-specific isotope analysis (CSIA), fluorescence in situ hybridization (FISH), and numerical transport modelling, to name a few. Moreover, due to the defined and well-controlled operating conditions, these bench-scale flow-through systems allow to investigate theoretical concepts and to develop and test predictive models. They represent a valuable tool in helping to bridge the current knowledge gap concerning transport and degradation of contaminants in groundwater from the small-scale (i.e. oversimplified batch systems, disregarding transport processes) to the highly complex field conditions. The promising potential of applications is by far not exhausted. Further possibilities include testing ecological theories such as the resource-ratio theory, island biogeography, area-species richness relationships and relations between community structure, microbial abundance and process rates as well as the importance and effects of bacterial chemotaxis.

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.

Infiltration of surface water constitutes an important pillar in artificial groundwater recharge. However, insufficient transformation of organic carbon and nutrients, as well as clogging of sediments often cause major problems. The attenuation efficiency of dissolved organic carbon (DOC), nutrients and pathogens versus the risk of bioclogging for intermittent recharge were studied in an infiltration basin covered with different kinds of macrovegetation. The quality and concentration of organic carbon, major nutrients, as well as bacterial biomass, activity and diversity in the surface water, the porewater, and the sediment matrix were monitored over one recharge period. Additionally, the numbers of viral particles and Escherichia coli were assessed. Our study showed a fast establishment of high microbial activity. DOC and nutrients have sustainably been reduced within 1.2 m of sediment passage. Numbers of E. coli, which were high in the topmost centimetres of sediment porewater, dropped below the detection limit. Reed cover was found to be advantageous over bushes and trees, since it supported higher microbial activities along with a good infiltration and purification performance. Short-term infiltration periods of several days followed by a break of similar time were found suitable for providing high recharge rates, and good water purification without the risk of bioclogging.

Compound-specific stable isotope analysis (CSIA) is one of the most important methods for assessing biodegradation activities in contaminated aquifers. Although the concept is straightforward, the proof that the method cannot be only used for a qualitative analysis but also to quantify biodegradation in the subsurface was missing. We therefore performed a multitracer test in the field with ring-deuterated (d5) and completely (d8) deuterium-labeled toluene isotopologues (400 g) as reactive tracers as well as bromide as a conservative tracer. The compounds were injected into the anoxic zone of a BTEX plume located down-gradient of the contaminant source. Over a period of 4.5 months the tracer concentrations were analyzed at two control planes located 24 and 35 m downgradient of the injection well. Deuterium-labeled benzylsuccinate was found in the aquifer, indicating the anaerobic biodegradation of deuterated toluene via the benzylsuccinate synthase pathway. Three independent methods were applied to quantify biodegradation of deuterated toluene. First, fractionation of toluene-d8 and toluene-d5 using the Rayleigh equation and an appropriate laboratory-derived isotope fractionation factor was used for the calculation of the microbial decomposition of deuterated toluene isotopologues (CSIA-method). Second, the biodegradation was quantified by the changes of the concentrations of deuterated toluene relative to bromide. Both methods gave similar results, implying that the CSIA-method is a reliable tool to quantify biodegradation in contaminated aquifers. The results of both methods yielded a biodegradation of deuterated toluene isotopologues of approximately 23-29% for the first and 44-51% for the second control plane. Third, the mineralization of deuterated toluene isotopologues was verified by determination of the enrichment of deuterium in the groundwater. This method indicated that parts of deuterium were assimilated into the biomass of toluene degrading microorganisms.

Ecological and socioeconomic aspects of subterranean hydrosystems have changed during the past 40–50 years. The major environmental pressures (mainly anthropogenic ones) impact the quantity and quality of groundwater resources and the state of subsurface ecosystems, and it is expected that the environmental pressures on groundwater will continue, at least until 2025, unless new environmental policies change this state of affairs. The world demographic increase and the general rise of water demand constitute one of the major environmental pressures on groundwater ecosystems especially in less developed countries in Africa, Asia and South America. Specific human activities leading to the depletion of groundwater reserves include agricultural practices, landscape alteration, urbanization demand for domestic and public drinking water, various industrial activities such as thermoelectric production and mining, and the rise of tourism in coastal areas. Climate change is contributing to the water crisis too, especially in areas with arid climate and/or in some humid monsoonal countries. The overload of aquifers with pollutants derived from agriculture (fertilizers and pesticides), from industry (release of hydrocarbon chemicals, especially spills), from waste and industrial waters, from domestic and industrial landfills, from the infiltration of pollutants from surface and from the intrusion of saline water affect groundwater quality. The dangerous increase in contaminated subsurface sites with chemicals and microbial pathogens brings with it health risks to humans. Changes of redox condition in groundwater zones, changes of biological diversity, vegetation changes with modification of agriculture practices and impacts at the biosphere scale, such as the increase in the concentration of nitrous oxides in the atmosphere, all impact groundwater ecosystems. Groundwater ecosystems must be better investigated and understood. Economic, social and ecological lines of thinking have to be combined in order to achieve meaningful policies for the sustainable development of groundwater reserves and for the protection of subsurface ecosystems. Practical measures and ideas for the development of policies up to the 2025 time-horizon should improve the sustainable usage of the world's groundwater resources.

Biodegradation in contaminated aquifers has been shown to be most pronounced at the fringe of contaminant plumes, where mixing of contaminated water and ambient groundwater, containing dissolved electron acceptors, stimulates microbial activity. While physical mixing of contaminant and electron acceptor by transverse dispersion has been shown to be the major bottleneck for biodegradation in steady-state plumes, so far little is known on the effect of flow and transport dynamics (caused, e.g., by a seasonally fluctuating groundwater table) on biodegradation in these systems. Towards this end we performed experiments in quasi-two-dimensional flow-through microcosms on aerobic toluene degradation by Pseudomonas putida F1. Plume dynamics were simulated by vertical alteration of the toluene plume position and experimental results were analyzed by reactive-transport modeling. We found that, even after disappearance of the toluene plume for two weeks, the majority of microorganisms stayed attached to the sediment and regained their full biodegradation potential within two days after reappearance of the toluene plume. Our results underline that besides microbial growth, also maintenance and dormancy are important processes that affect biodegradation performance under transient environmental conditions and therefore deserve increased consideration in future reactive-transport modeling.

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.

Pristine and energy-limited aquifers are considered to have a low resistance and resilience towards organic pollution. An experiment in an indoor aquifer system revealed an unexpected high intrinsic potential for the attenuation of a short-term toluene contamination. A 30h pulse of 486mg of toluene, used as a model contaminant, and deuterated water (D2O) through an initially pristine, oxic, and organic carbon poor sandy aquifer revealed an immediate aerobic toluene degradation potential. Based on contaminant and tracer break-through curves, as well as mass balance analyses and reactive transport modelling, a contaminant removal of 40% over a transport distance of only 4.2m in less than one week of travel time was obtained. The mean first-order degradation rate constant was λ=0.178day-1, corresponding to a half-life time constant T1/2 of 3.87days. Toluene-specific stable carbon isotope analysis independently proved that the contaminant mass removal can be attributed to microbial biodegradation. Since average doubling times of indigenous bacterial communities were in the range of months to years, the aerobic biodegradation potential observed is assumed to be present and active in the pristine, energy-limited groundwater ecosystems at any time. Follow-up experiments and field studies will help to quantify the immediate natural attenuation potential of aquifers for selected priority contaminants and will try to identify the key-degraders within the autochthonous microbial communities.