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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.

A key question in microbial ecology is what the driving forces behind the persistence of large biodiversity in natural environments are. We studied a microbial community with more than 100 different types of species which evolved in a 15-years old bioreactor with benzene as the main carbon and energy source and nitrate as the electron acceptor. Using genome-centric metagenomics plus metatranscriptomics, we demonstrate that most of the community members likely feed on metabolic left-overs or on necromass while only a few of them, from families Rhodocyclaceae and Peptococcaceae, are candidates to degrade benzene. We verify with an additional succession experiment using metabolomics and metabarcoding that these few community members are the actual drivers of benzene degradation. As such, we hypothesize that high species richness is maintained and the complexity of a natural community is stabilized in a controlled environment by the interdependencies between the few benzene degraders and the rest of the community members, ultimately resulting in a food web with different trophic levels., Chrats Melkonian and colleagues use metagenomics and transcriptomics to study microbial dynamics in a 15-year old bioreactor. In contrast to what is expected in a system where benzene is the primary carbon and energy source, relatively few members of the microbial community can degrade this compound. The results have implications for understanding interdependencies within such a community.

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

The 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

A key question in microbial ecology is what the driving forces behind the persistence of large biodiversity in natural environments are. We studied a microbial community with more than 100 different types of species which evolved in a 15-years old bioreactor with benzene as the main carbon and free energy source and nitrate as the electron acceptor. We demonstrate that only a few community members are able to degrade benzene, and that most of the others feed on the metabolic left-overs or on the contents of dead cells making up a food web with different trophic levels. As a result of niche partitioning, a high species richness is maintained and the complexity of a natural community is stabilized in a relatively simple environment. This view highlights the importance of species interactions and interdependencies, which drive microbial community structure and function. These mechanisms may well be conserved across ecosystems. ### Competing Interest Statement The authors have declared no competing interest.

A key question in microbial ecology is what the driving forces behind the persistence of large biodiversity in natural environments are. We studied a microbial community with more than 100 different types of species which evolved in a 15-years old bioreactor with benzene as the main carbon and energy source and nitrate as the electron acceptor. Using genome-centric metagenomics plus metatranscriptomics, we demonstrate that most of the community members likely feed on metabolic left-overs or on necromass while only a few of them, from families Rhodocyclaceae and Peptococcaceae, are candidates to degrade benzene. We verify with an additional succession experiment using metabolomics and metabarcoding that these few community members are the actual drivers of benzene degradation. As such, we hypothesize that high species richness is maintained and the complexity of a natural community is stabilized in a controlled environment by the interdependencies between the few benzene degraders and the rest of the community members, ultimately resulting in a food web with different trophic levels.

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.

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.

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.

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.

The North Sea coast of the Dutch barrier island of Schiermonnikoog is covered by microbial mats that initiate a succession of plant communities that eventually results in the development of a densely vegetated salt marsh. The North Sea beach has a natural elevation running from the low water mark to the dunes resulting in gradients of environmental factors perpendicular to the beach. These gradients are due to the input of seawater at the low water mark and of freshwater from upwelling groundwater at the dunes and rainfall. The result is a natural and dynamic salinity gradient depending on the tide, rainfall and wind. We studied the microbial community composition in thirty three samples taken every ten meters along this natural salinity gradient by using denaturing gradient gel electrophoresis (DGGE) of rRNA gene fragments. We looked at representatives from each Domain of life (Bacteria, Archaea and Eukarya) and with a particular emphasis on Cyanobacteria. Analysis of the DGGE fingerprints together with pigment composition revealed three distinct microbial mat communities, a marine community dominated by diatoms as primary producers, an intermediate brackish community dominated by Cyanobacteria as primary producers and a freshwater community with Cyanobacteria and freshwater green algae.