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The input of nitrate and other agricultural pollutants in higher-order streams largely derives from first-order streams. The streambed as the transition zone between groundwater and stream water has a decisive impact on the attenuation of such pollutants. This reactivity is not yet well understood for lower-order agricultural streams, which are often anthropogenically altered and lack the streambed complexity allowing for extensive hyporheic exchange. Reactive hot spots in such streambeds have been hypothesized as a function of hydrology, which controls the local gaining (groundwater exfiltration) or losing (infiltration) of stream water. However, streambed microbial communities and activities associated with such reactive zones remain mostly uncharted. In this study, sediments of a first-order agriculturally impacted stream in southern Germany were investigated. Along with a hydraulic dissection of distinct gaining and losing reaches of the stream, community composition and the abundance of bacterial communities in the streambed were investigated using PacBio long-read sequencing of bacterial 16S rRNA gene amplicons, and qPCR of bacterial 16S rRNA and denitrification genes (nirK and nirS). We show that bidirectional water exchange between groundwater and the stream represents an important control for sediment microbiota, especially for nitrate-reducing populations. Typical heterotrophic denitrifiers were most abundant in a midstream net losing section, while up- and downstream net gaining sections were associated with an enrichment of sulfur-oxidizing potential nitrate reducers affiliated with Sulfuricurvum and Thiobacillus spp. Dispersal-based community assembly was found to dominate such spots of groundwater exfiltration. Our results indicate a coupling of N- and S-cycling processes in the streambed of an agricultural first-order stream, and a prominent control of microbiology by hydrology and hydrochemistry in situ. Such detailed local heterogeneities in exchange fluxes and streambed microbiomes have not been reported to date, but seem relevant for understanding the reactivity of lower-order streams., (Copyright © 2022. Published by Elsevier Ltd.)

Nitrate removal in oligotrophic environments is often limited by the availability of suitable organic electron donors. Chemolithoautotrophic bacteria may play a key role in denitrification in aquifers depleted in organic carbon. Under anoxic and circumneutral pH conditions, iron(II) was hypothesized to serve as an electron donor for microbially mediated nitrate reduction by Fe(II)-oxidizing (NRFeOx) microorganisms. However, lithoautotrophic NRFeOx cultures have never been enriched from any aquifer, and as such, there are no model cultures available to study the physiology and geochemistry of this potentially environmentally relevant process. Using iron(II) as an electron donor, we enriched a lithoautotrophic NRFeOx culture from nitrate-containing groundwater of a pyrite-rich limestone aquifer. In the enriched NRFeOx culture that does not require additional organic cosubstrates for growth, within 7 to 11 days, 0.3 to 0.5 mM nitrate was reduced and 1.3 to 2 mM iron(II) was oxidized, leading to a stoichiometric NO 3 - /Fe(II) ratio of 0.2, with N 2 and N 2 O identified as the main nitrate reduction products. Short-range ordered Fe(III) (oxyhydr)oxides were the product of iron(II) oxidation. Microorganisms were observed to be closely associated with formed minerals, but only few cells were encrusted, suggesting that most of the bacteria were able to avoid mineral precipitation at their surface. Analysis of the microbial community by long-read 16S rRNA gene sequencing revealed that the culture is dominated by members of the Gallionellaceae Removal of nitrate by microbial denitrification in groundwater is often limited by low concentrations of organic carbon. In these carbon-poor ecosystems, nitrate-reducing bacteria that can use inorganic compounds such as Fe(II) (NRFeOx) as electron donors could play a major role in nitrate removal. However, no lithoautotrophic NRFeOx culture has been successfully isolated or enriched from this type of environment, and as such, there are no model cultures available to study the rate-limiting factors of this potentially important process. Here, we present the physiology and microbial community composition of a novel lithoautotrophic NRFeOx culture enriched from a fractured aquifer in southern Germany. The culture is dominated by a putative Fe(II) oxidizer affiliated with the IMPORTANCE Removal of nitrate by microbial denitrification in groundwater is often limited by low concentrations of organic carbon. In these carbon-poor ecosystems, nitrate-reducing bacteria that can use inorganic compounds such as Fe(II) (NRFeOx) as electron donors could play a major role in nitrate removal. However, no lithoautotrophic NRFeOx culture has been successfully isolated or enriched from this type of environment, and as such, there are no model cultures available to study the rate-limiting factors of this potentially important process. Here, we present the physiology and microbial community composition of a novel lithoautotrophic NRFeOx culture enriched from a fractured aquifer in southern Germany. The culture is dominated by a putative Fe(II) oxidizer affiliated with the Gallionellaceae family and performs nitrate reduction coupled to Fe(II) oxidation leading to N 2 O and N 2 formation without the addition of organic substrates. Our analyses demonstrate that lithoautotrophic NRFeOx can potentially lead to nitrate removal in nitrate-contaminated aquifers.

The availability of oxygen is often a limiting factor for the degradation of aromatic hydrocarbons in subsurface environments. However, while both aerobic and anaerobic degraders have been intensively studied, degradation betwixt, under micro- or hypoxic conditions has rarely been addressed. It is speculated that in environments with limited, but sustained oxygen supply, such as in the vicinity of groundwater monitoring wells, hypoxic degradation may take place. A large diversity of subfamily I.2.C extradiol dioxygenase genes has been previously detected in a BTEX-contaminated aquifer in Hungary. Older literature suggests that such catabolic potentials could be associated to hypoxic degradation. Bacterial communities dominated by members of the Rhodocyclaceae were found, but the majority of the detected C23O genotypes could not be affiliated to any known bacterial degrader lineages. To address this, a stable isotope probing (SIP) incubation of site sediments with 13C7-toluene was performed under microoxic conditions. A combination of 16S rRNA gene amplicon sequencing and T-RFLP fingerprinting of C23O genes from SIP gradient fractions revealed the central role of degraders within the Rhodocyclaceae in hypoxic toluene degradation. The main assimilators of 13C were identified as members of the genera Quatrionicoccus and Zoogloea, and a yet uncultured group of the Rhodocyclaceae.

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 132days of toluene exposure followed by nearly 600days 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 35days, 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The degradation of benzene, toluene, ethylbenzene and xylene (BTEX) contaminants in groundwater relies largely on anaerobic processes. While the physiology and biochemistry of selected relevant microbes have been intensively studied, research has now started to take the generated knowledge back to the field, in order to trace the populations truly responsible for the anaerobic degradation of BTEX hydrocarbons in situ and to unravel their ecology in contaminated aquifers. Here, recent advances in our knowledge of the identity, diversity and ecology of microbes involved in these important ecosystem services are discussed. At several sites, distinct lineages within the Desulfobulbaceae, the Rhodocyclaceae and the Gram-positive Peptococcaceae have been shown to dominate the degradation of different BTEX hydrocarbons. Especially for the functional guild of anaerobic toluene degraders, specific molecular detection systems have been developed, allowing researchers to trace their diversity and distribution in contaminated aquifers. Their populations appear enriched in hot spots of biodegradation in situ 13 C-labelling experiments have revealed unexpected pathways of carbon sharing and obligate syntrophic interactions to be relevant in degradation. Together with feedback mechanisms between abiotic and biotic habitat components, this promotes an enhanced ecological perspective of the anaerobic degradation of BTEX hydrocarbons, as well as its incorporation into updated concepts for site monitoring and bioremediation., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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.

Hydrocarbon contaminants in groundwater can be degraded by microbes under different redox settings, forming hot spots of degradation especially at the fringes of contaminant plumes. At a tar-oil-contaminated aquifer in Germany, it was previously shown that the distribution of anaerobic toluene degraders as traced via catabolic and ribosomal marker genes is highly correlated to zones of increased anaerobic degradation at the lower fringe of the plume. Here, we trace the respective distribution of aerobic toluene degraders over a fine-scale depth transect of sediments taken at the upper fringe of the plume and below, based on the analysis of 16S rRNA genes as well as catabolic markers in intervals of 3-10 cm. Well-defined small-scale distribution maxima of typical aerobic degrader lineages within the Pseudomonadaceae, Comamonadaceae and Burkholderiaceae are revealed over the redox gradient. An unexpected maximal abundance of 9.2 × 10⁶ toluene monooxygenase (tmoA) genes per g of sediment was detected in the strongly reduced plume core, and gene counts did not increase towards the more oxidized upper plume fringe. This may point towards unusual ecological controls of these yet unidentified aerobic degraders, and indicates that competitive niche partitioning between aerobic and anaerobic hydrocarbon degraders in the field is not yet fully understood. These findings demonstrate the potential of catabolic marker gene assays in elaborating the ecology of contaminant plumes, which is a prerequisite for developing integrated monitoring strategies for natural attenuation., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The characterization of microbial community structure via 16S rRNA gene profiling has been greatly advanced in recent years by the introduction of amplicon pyrosequencing. The possibility of barcoding gives the opportunity to massively screen multiple samples from environmental or clinical sources for community details. However, an on-going debate questions the reproducibility and semi-quantitative rigour of pyrotag sequencing, similar to the early days of community fingerprinting. In this study we demonstrate the reproducibility of bacterial 454 pyrotag sequencing over biological and technical replicates of aquifer sediment bacterial communities. Moreover, we explore the potential of recovering specific template ratios via quantitatively defined template spiking to environmental DNA. We sequenced pyrotag libraries of triplicate sediment samples taken in annual sampling campaigns at a tar oil contaminated aquifer in Düsseldorf, Germany. The abundance of dominating lineages was highly reproducible with a maximal standard deviation of ~4% read abundance across biological, and ~2% across technical replicates. Our workflow also allows for the linking of read abundances within defined assembled pyrotag contigs to that of specific 'in vivo' fingerprinting signatures. Thus we demonstrate that both terminal restriction fragment length polymorphism (T-RFLP) analysis and pyrotag sequencing are capable of recovering highly comparable community structure. Overall diversity was roughly double in amplicon sequencing. Pyrotag libraries were also capable of linearly recovering increasing ratios (up to 20%) of 16S rRNA gene amendments from a pure culture of Aliivibrio fisheri spiked to sediment DNA. Our study demonstrates that 454 pyrotag sequencing is a robust and reproducible method, capable of reliably recovering template abundances and overall community structure within natural microbial communities.

Bioavailability of electron acceptors is probably the most limiting factor in the restoration of anoxic, contaminated environments. The oxidation of contaminants such as aromatic hydrocarbons, particularly in aquifers, often depends on the reduction of ferric iron or sulphate. We have previously detected a highly active fringe zone beneath a toluene plume at a tar-oil-contaminated aquifer in Germany, where a specialized community of contaminant degraders codominated by Desulfobulbaceae and Geobacteraceae had established. Although on-site geochemistry links degradation to sulphidogenic processes, dominating catabolic (benzylsuccinate synthase α-subunit, bssA) genes detected in situ appeared to be more related to those of Geobacter spp. Therefore, a stable isotope probing (SIP) incubation of sediment samples with (13)C(7)-toluene and comparative electron acceptor amendment was performed. We introduce pyrosequencing of templates from SIP microcosms as a powerful new strategy in SIP gradient interpretation (Pyro-SIP). Our results reveal the central role of Desulfobulbaceae in sulphidogenic toluene degradation in situ, and affiliate the detected bssA genes to this lineage. This and the absence of (13)C-labelled DNA of Geobacter spp. in SIP gradients preclude their relevance as toluene degraders in situ. In contrast, Betaproteobacteria related to Georgfuchsia spp. became labelled under iron-reducing conditions. Furthermore, secondary toluene degraders belonging to the Peptococcaceae detected in both treatments suggest the possibility of functional redundancy among anaerobic toluene degraders on site., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

While most studies using RNA-stable isotope probing (SIP) to date have focused on ribosomal RNA, the detection of 13C-labeled mRNA has rarely been demonstrated. This approach could alleviate some of the major caveats of current non-target environmental "omics." Here, we demonstrate the feasibility of total RNA-SIP in an experiment where hydrocarbon-degrading microbes from a BTEX-contaminated aquifer were studied in microcosms with 13C-labeled toluene under microoxic conditions. From the total sequencing reads (∼30 mio. reads per density-resolved RNA fraction), an average of 1.2% of reads per sample were identified as non-rRNA, including mRNA. Members of the Rhodocyclaceae (including those related to Quatrionicoccus spp.) were most abundant and enriched in 13C-rRNA, while well-known aerobic degraders such as Pseudomonas spp. remained unlabeled. Transcripts related to cell motility, secondary metabolite formation and xenobiotics degradation were highly labeled with 13C. mRNA of phenol hydroxylase genes were highly labeled and abundant, while other transcripts of toluene-activation were not detected. Clear labeling of catechol 2,3-dioxygenase transcripts supported previous findings that some of these extradiol dioxygenases were adapted to low oxygen concentrations. We introduce a novel combination of total RNA-SIP with calculation of transcript-specific enrichment factors (EFs) in 13C-RNA, enabling a targeted approach to process-relevant gene expression in complex microbiomes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]