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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.

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.

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

Aquifers are important reservoirs for organic carbon. A fundamental understanding of the role of groundwater ecosystems in carbon cycling, however, is still missing. Using sediment flow-through microcosms, long-term (171d) experiments were conducted to test two scenarios. First, aquifer sediment microbial communities received dissolved organic matter (DOM) at low concentration and typical to groundwater in terms of composition (DOM-1x). Second, sediments received an elevated concentration of DOM originating from soil (DOM-5x). Changes in DOM composition were analyzed via NMR and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Carbon production, physiological adaptations and biodiversity of groundwater, and sediment prokaryotic communities were monitored by total cell counts, substrate use arrays, and deep amplicon sequencing. The experiments showed that groundwater microbial communities do not react very fast to the sudden availability of labile organic carbon from soil in terms of carbon degradation and biomass production. It took days to weeks for incoming DOM being efficiently degraded and pronounced cell production occurred. Once conditioned, the DOM-1x supplied sediments mineralized 294(±230) μgC L-1 sed d-1, 10-times less than the DOM-5x fed sediment communities [2.9(±1.1) mgC L-1 sed d-1]. However, the overall biomass carbon production was hardly different in the two treatments with 13.7(±4.8) μgC L-1 sed d-1 and 14.3(±3.5) μgC L-1 sed d-1, respectively, hinting at a significantly lower carbon use efficiency with higher DOM availability. However, the molecularly more diverse DOM from soil fostered a higher bacterial diversity. Taking the irregular inputs of labile DOM into account, shallow aquifers are assumed to have a low resilience. Lacking a highly active and responsive microbial community, oligotrophic aquifers are at high risk of contamination with organic chemicals.

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.

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.

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.

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.

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.

The total number of prokaryotic cells in aquatic sediments is a crucial measure tightly linked to energetic constraints, microbial productivity, and ecosystem status. While counting cells in water samples is routine, reliable procedures for the determination of prokaryotic cells associated to aquatic sediments are especially needed. A protocol for the direct quantification of prokaryotic cell abundance in aquatic sediments was developed and tested with sandy material from pristine and contaminated aquifers. Individual steps required in sample preparation, including preservation, cell dislodgement, density gradient centrifugation providing sample purification, and staining, were critically evaluated and optimized. Quantification of cells is conducted by flow cytometry promoting a high-throughput sample processing. Although tested for only two different types of aquifer sediments, researchers may easily adapt the protocol for their individual samples and purposes, thus conducting a brief efficiency re-evaluation of individual steps highlighted in this study.

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.

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.

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.

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

The success of in situ bioremediation is often limited by the inability to bring bacteria in contact with the pollutant, which they will degrade. A bench-scale model aquifer was used to evaluate the impact of chemotaxis on the migration of bacteria toward the source of a chemical pollutant. The model was packed with sand and aqueous media was pumped across horizontally, simulating groundwater flow in a homogenous aquifer. A vertical gradient in chemoattractant was created by either a continuous injection of sodium benzoate or a pulse injection of sodium acetate. A pulse of chemotactic Pseudomonas putida F1 or a non-chemotactic mutant of the same species was injected below the attractant. The eluent was sampled at the microcosm outlet to generate vertical concentration profiles of the bacteria and chemoattractant. Moment analysis was used to determine the center and variance of the bacterial profiles. The center of the chemotactic bacterial population was located at an average of 0.74 ± 0.07 cm closer to the level at which the chemoattractant was injected than its non-chemotactic mutant in benzoate experiments (P < 0.015) and 0.4 ± 0.2 cm closer in acetate experiments (P < 0.05). The transverse dispersivity of the chemotactic bacteria was 4 ± 1 × 10(-3) cm higher in benzoate experiments than the transverse dispersivity of the non-chemotactic mutant and 1 ± 2 × 10(-3) cm higher in acetate experiments. These results underscore the contribution of chemotaxis to improve transport of bacteria to contaminant sources, potentially enhancing the effectiveness of in situ bioremediation.

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

The detailed understanding of in situ biodegradation of petroleum hydrocarbons in porous aquifers requires knowledge on biogeochemical gradients, the distribution of individual redox species and microorganisms. The generally limited spatial resolution of conventional monitoring wells, however, hampers appropriate characterization of small-scale gradients and thus localization of the relevant processes. Groundwater sampling across a BTEX plume in a sandy aquifer by means of a novel high-resolution multi-level well (HR-MLW) is presented here. The presence of distinct and steep biogeochemical gradients is demonstrated in the centimeter and decimeter scale, which could not be resolved with a conventional multi-level well. The thin BTEX plume with a vertical extension of only 80 cm exhibited a decline of contaminant concentrations by two orders of magnitude within a few centimeters in the upper and lower fringe zone. The small-scale distribution of sulfate, sulfide and Fe(II) in relation to the contaminants and elevated δ 34 S and δ 18 O values of groundwater sulfate strongly indicated sulfate and iron reduction to be the dominant redox processes involved in biodegradation. High microbial activities and biomass especially at the plume fringes and the slope of chemical gradients supported the concept that the latter are regulated by microbial processes and transverse dispersion, i.e. vertical mixing of electron donors and acceptors. Transverse dispersion therefore was suggested to be a driving factor controlling biodegradation in porous aquifers, but not exclusively limiting natural attenuation processes at this site. Broad overlapping zones of electron donors and electron acceptors point towards additional factors limiting anaerobic biodegradation in situ . The identification of small-scale gradients substantially contributed to a better understanding of biodegradation processes and hence is a prerequisite for the development of reliable predictive mathematical models and future remediation strategies.

Biodegradation is one of the most favored and sustainable means of removing organic pollutants from contaminated aquifers but the major steering factors are still surprisingly poorly understood. Growing evidence questions some of the established concepts for control of biodegradation. Here, we critically discuss classical concepts such as the thermodynamic redox zonation, or the use of steady state transport scenarios for assessing biodegradation rates. Furthermore, we discuss if the absence of specific degrader populations can explain poor biodegradation. We propose updated perspectives on the controls of biodegradation in contaminant plumes. These include the plume fringe concept, transport limitations, and transient conditions as currently underestimated processes affecting biodegradation.

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

Groundwater is a hidden part of the water cycle due to its subterranean nature but is highly connected to rivers, lakes, and wetlands. In this chapter, we review the physical basis of aquifers (groundwater reservoirs), their hydrodynamics, and hydrogeological parameters (porosity and permeability) that collectively define different types of aquifers. We explore the relationships between groundwater and surface water and define how aquifers function in terms of (1) groundwater flow and transport of solutes and particulate matter; (2) groundwater age, which affects ecosystem processes, physical and biological reactions, and groundwater resources and (3) modeling of the above processes. We also consider the chemical composition of groundwater and the origin of compounds and water-rock interactions that influence water quality. Finally, we discuss chemical and nutrient fluxes in aquifers and biogeochemical reactions, with a focus on oxygen and nitrogen.