Activity and electron donor preference of two denitrifying bacterial strains identified by Raman gas spectroscopy.

Human activities have greatly increased the input of reactive nitrogen species into the environment and disturbed the balance of the global N cycle. This imbalance may be offset by bacterial denitrification, an important process in maintaining the ecological balance of nitrogen. However, our understanding of the activity of mixotrophic denitrifying bacteria is not complete, as most research has focused on heterotrophic denitrification. The aim of this study was to investigate substrate preferences for two mixotrophic denitrifying bacterial strains, Acidovorax delafieldii and Hydrogenophaga taeniospiralis, under heterotrophic, autotrophic or mixotrophic conditions. This complex analysis was achieved by simultaneous identification and quantification of H2, O2, CO2, 14N2, 15N2 and 15N2O in course of the denitrification process with help of cavity-enhanced Raman spectroscopic (CERS) multi-gas analysis. To disentangle electron donor preferences for both bacterial strains, microcosm-based incubation experiments under varying substrate conditions were conducted. We found that Acidovorax delafieldii preferentially performed heterotrophic denitrification in the mixotrophic sub-experiments, while Hydrogenophaga taeniospiralis preferred autotrophic denitrification in the mixotrophic incubation. These observations were supported by stoichiometric calculations. The results demonstrate the prowess of advanced Raman multi-gas analysis to study substrate use and electron donor preferences in denitrification, based on the comprehensive quantification of complex microbial gas exchange processes.

Canopy Position Has a Stronger Effect than Tree Species Identity on Phyllosphere Bacterial Diversity in a Floodplain Hardwood Forest.

The phyllosphere is a challenging microbial habitat in which microorganisms can flourish on organic carbon released by plant leaves but are also exposed to harsh environmental conditions. Here, we assessed the relative importance of canopy position-top, mid, and bottom at a height between 31 and 20 m-and tree species identity for shaping the phyllosphere microbiome in a floodplain hardwood forest. Leaf material was sampled from three tree species-maple (Acer pseudoplatanus L.), oak (Quercus robur L.), and linden (Tilia cordata MILL.)-at the Leipzig canopy crane facility (Germany). Estimated bacterial species richness (Chao1) and bacterial abundances approximated by quantitative PCR of 16S rRNA genes exhibited clear vertical trends with a strong increase from the top to the mid and bottom position of the canopy. Thirty operational taxonomic units (OTUs) formed the core microbiome, which accounted for 77% of all sequence reads. These core OTUs showed contrasting trends in their vertical distribution within the canopy, pointing to different ecological preferences and tolerance to presumably more extreme conditions at the top position of the canopy. Co-occurrence analysis revealed distinct tree species-specific OTU networks, and 55-57% of the OTUs were unique to each tree species. Overall, the phyllosphere microbiome harbored surprisingly high fractions of Actinobacteria of up to 66%. Our results clearly demonstrate strong effects of the position in the canopy on phyllosphere bacterial communities in a floodplain hardwood forest and-in contrast to other temperate or tropical forests-a strong predominance of Actinobacteria.

Genome-inferred spatio-temporal resolution of an uncultivated Roizmanbacterium reveals its ecological preferences in groundwater.

Subsurface ecosystems like groundwater harbour diverse microbial communities, including small-sized, putatively symbiotic organisms of the Candidate Phyla Radiation, yet little is known about their ecological preferences and potential microbial partners. Here, we investigated a member of the superphylum Microgenomates (Cand. Roizmanbacterium ADI133) from oligotrophic groundwater using mini-metagenomics and monitored its spatio-temporal distribution using 16S rRNA gene analyses. A Roizmanbacteria-specific quantitative PCR assay allowed us to track its abundance over the course of 1 year within eight groundwater wells along a 5.4 km hillslope transect, where Roizmanbacteria reached maximum relative abundances of 2.3%. In-depth genomic analyses suggested that Cand. Roizmanbacterium ADI133 is a lactic acid fermenter, potentially able to utilize a range of complex carbon substrates, including cellulose. We hypothesize that it attaches to host cells using a trimeric autotransporter adhesin and inhibits their cell wall biosynthesis using a toxin-antitoxin system. Network analyses based on correlating Cand. Roizmanbacterium ADI133 abundances with amplicon sequencing-derived microbial community profiles suggested one potential host organism, classified as a member of the class Thermodesulfovibrionia (Nitrospirae). By providing lactate as an electron donor Cand. Roizmanbacterium ADI133 potentially mediates the transfer of carbon to other microorganisms and thereby is an important connector in the microbial community.

Biogeochemical Regimes in Shallow Aquifers Reflect the Metabolic Coupling of the Elements Nitrogen, Sulfur, and Carbon.

Near-surface groundwaters are prone to receive (in)organic matter input from their recharge areas and are known to harbor autotrophic microbial communities linked to nitrogen and sulfur metabolism. Here, we use multi-omic profiling to gain holistic insights into the turnover of inorganic nitrogen compounds, carbon fixation processes, and organic matter processing in groundwater. We sampled microbial biomass from two superimposed aquifers via monitoring wells that follow groundwater flow from its recharge area through differences in hydrogeochemical settings and land use. Functional profiling revealed that groundwater microbiomes are mainly driven by nitrogen (nitrification, denitrification, and ammonium oxidation [anammox]) and to a lesser extent sulfur cycling (sulfur oxidation and sulfate reduction), depending on local hydrochemical differences. Surprisingly, the differentiation potential of the groundwater microbiome surpasses that of hydrochemistry for individual monitoring wells. Being dominated by a few phyla (Bacteroidetes, Proteobacteria, Planctomycetes, and Thaumarchaeota), the taxonomic profiling of groundwater metagenomes and metatranscriptomes revealed pronounced differences between merely present microbiome members and those actively participating in community gene expression and biogeochemical cycling. Unexpectedly, we observed a constitutive expression of carbohydrate-active enzymes encoded by different microbiome members, along with the groundwater flow path. The turnover of organic carbon apparently complements for lithoautotrophic carbon assimilation pathways mainly used by the groundwater microbiome depending on the availability of oxygen and inorganic electron donors, like ammonium.IMPORTANCE Groundwater is a key resource for drinking water production and irrigation. The interplay between geological setting, hydrochemistry, carbon storage, and groundwater microbiome ecosystem functioning is crucial for our understanding of these important ecosystem services. We targeted the encoded and expressed metabolic potential of groundwater microbiomes along an aquifer transect that diversifies in terms of hydrochemistry and land use. Our results showed that the groundwater microbiome has a higher spatial differentiation potential than does hydrochemistry.

Archaeal Diversity and CO2 Fixers in Carbonate-/Siliciclastic-Rock Groundwater Ecosystems.

Groundwater environments provide habitats for diverse microbial communities, and although Archaea usually represent a minor fraction of communities, they are involved in key biogeochemical cycles. We analysed the archaeal diversity within a mixed carbonate-rock/siliciclastic-rock aquifer system, vertically from surface soils to subsurface groundwater including aquifer and aquitard rocks. Archaeal diversity was also characterized along a monitoring well transect that spanned surface land uses from forest/woodland to grassland and cropland. Sequencing of 16S rRNA genes showed that only a few surface soil-inhabiting Archaea were present in the groundwater suggesting a restricted input from the surface. Dominant groups in the groundwater belonged to the marine group I (MG-I) Thaumarchaeota and the Woesearchaeota. Most of the groups detected in the aquitard and aquifer rock samples belonged to either cultured or predicted lithoautotrophs (e.g., Thaumarchaeota or Hadesarchaea). Furthermore, to target autotrophs, a series of 13CO2 stable isotope-probing experiments were conducted using filter pieces obtained after filtration of 10,000 L of groundwater to concentrate cells. These incubations identified the SAGMCG Thaumarchaeota and Bathyarchaeota as groundwater autotrophs. Overall, the results suggest that the majority of Archaea on rocks are fixing CO2, while archaeal autotrophy seems to be limited in the groundwater.

Large fractions of CO2-fixing microorganisms in pristine limestone aquifers appear to be involved in the oxidation of reduced sulfur and nitrogen compounds.

The traditional view of the dependency of subsurface environments on surface-derived allochthonous carbon inputs is challenged by increasing evidence for the role of lithoautotrophy in aquifer carbon flow. We linked information on autotrophy (Calvin-Benson-Bassham cycle) with that from total microbial community analysis in groundwater at two superimposed-upper and lower-limestone groundwater reservoirs (aquifers). Quantitative PCR revealed that up to 17% of the microbial population had the genetic potential to fix CO2 via the Calvin cycle, with abundances of cbbM and cbbL genes, encoding RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) forms I and II, ranging from 1.14 × 10(3) to 6 × 10(6) genes liter(-1) over a 2-year period. The structure of the active microbial communities based on 16S rRNA transcripts differed between the two aquifers, with a larger fraction of heterotrophic, facultative anaerobic, soil-related groups in the oxygen-deficient upper aquifer. Most identified CO2-assimilating phylogenetic groups appeared to be involved in the oxidation of sulfur or nitrogen compounds and harbored both RubisCO forms I and II, allowing efficient CO2 fixation in environments with strong oxygen and CO2 fluctuations. The genera Sulfuricella and Nitrosomonas were represented by read fractions of up to 78 and 33%, respectively, within the cbbM and cbbL transcript pool and accounted for 5.6 and 3.8% of 16S rRNA sequence reads, respectively, in the lower aquifer. Our results indicate that a large fraction of bacteria in pristine limestone aquifers has the genetic potential for autotrophic CO2 fixation, with energy most likely provided by the oxidation of reduced sulfur and nitrogen compounds.

Phagotrophic protist diversity in the groundwater of a karstified aquifer - morphological and molecular analysis.

To clarify the structure of microbial food webs in groundwater, knowledge about the protist diversity and feeding strategies is essential. We applied cultivation-dependent approaches and molecular methods for further understanding of protist diversity in groundwater. Groundwater was sampled from a karstified aquifer located in the Thuringian Basin (Thuringia, Germany). Cultivable protist abundance estimated up to 8,000 cells/L. Eleven flagellates, 10 naked amoebae, and one ciliate morpho-species were detected in groundwater enrichment cultures. Most of the flagellates morpho-species, typically < 10 µm, were sessile or free swimming suspension feeders, e.g., Spumella spp., Monosiga spp., and mobile, surface-associated forms that grasp biofilms, e.g., Bodo spp. Naked amoebae, typically < 35 µm, that grasp biofilms were represented by, e.g., Vahlkampfia spp., Vannella spp., and Hartmanella spp. The largest fraction of the 18S rRNA gene sequences was affiliated with Spumella-like Stramenopiles. Besides, also sequences affiliated with fungi and metazoan grazers were detected in clone libraries of the groundwater. We hypothesize that small sized protist species take refuge in the structured surface of the fractures and fissures of the karstified aquifer and mainly feed on biofilm-associated or suspended bacteria.

Differential contribution of nitrifying prokaryotes to groundwater nitrification.

The ecophysiology of complete ammonia-oxidizing bacteria (CMX) of the genus Nitrospira and their widespread occurrence in groundwater suggests that CMX bacteria have a competitive advantage over ammonia-oxidizing bacteria (AOB) and archaea (AOA) in these environments. However, the specific contribution of their activity to nitrification processes has remained unclear. We aimed to disentangle the contribution of CMX, AOA and AOB to nitrification and to identify the environmental drivers of their niche differentiation at different levels of ammonium and oxygen in oligotrophic carbonate rock aquifers. CMX ammonia monooxygenase sub-unit A (amoA) genes accounted on average for 16 to 75% of the total groundwater amoA genes detected. Nitrification rates were positively correlated to CMX clade A associated phylotypes and AOB affiliated with Nitrosomonas ureae. Short-term incubations amended with the nitrification inhibitors allylthiourea and chlorate suggested that AOB contributed a large fraction to overall ammonia oxidation, while metaproteomics analysis confirmed an active role of CMX in both ammonia and nitrite oxidation. Ecophysiological niche differentiation of CMX clades A and B, AOB and AOA was linked to their requirements for ammonium, oxygen tolerance, and metabolic versatility. Our results demonstrate that despite numerical predominance of CMX, the first step of nitrification in oligotrophic groundwater appears to be primarily governed by AOB. Higher growth yields at lower ammonia turnover rates and energy derived from nitrite oxidation most likely enable CMX to maintain consistently high populations.

Predominance of thaumarchaeal ammonia oxidizer abundance and transcriptional activity in an acidic fen.

We investigated the abundance, community composition and transcriptional activity of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in the acidic fen Schlöppnerbrunnen (Germany) that was subjected to water table manipulations. Quantitative PCR targeting amoA gene copies and transcripts showed that AOA dominated the ammonia-oxidizing community in the upper 20 cm of the peat soil. Numbers of archaeal amoA gene copies and transcripts as well as the relative fraction of AOA of the total archaea decreased with depth. AOA-AmoA sequences were 96.2-98.9% identical to that of Candidatus Nitrosotalea devanaterra while bacterial AmoA sequences affiliated with Nitrosospira clusters 2 and 4. Archaeal but not bacterial amoA transcripts were detected in short-term laboratory incubations of peat that showed nitrifying activity. Nitrate accumulated in the peat pore water after 6 weeks of induced drought during a field experiment. Subsequent rewetting resulted in a significant decrease of AOA transcriptional activity, indicating that AOA responded to water table fluctuations on the transcriptional level. Our results suggest that nitrification in this fen is primarily linked to archaeal ammonia oxidation. pH and anoxia appear to be key factors regulating AOA community composition, vertical distribution and activity in acidic fens.

Sulfangolids, macrolide sulfate esters from Sorangium cellulosum.

Sulfangolids are the first sulfate ester containing secondary metabolites from myxobacteria. The metabolites 1-4 and the structurally related kulkenon (5) were isolated from different strains of the species Sorangium cellulosum. In the course of isolation all metabolites proved to be rather sensitive due to their conjugated double bond systems and the strong acidic nature of the sulfate ester in sulfangolids. The relative configuration of sulfangolid C (3) was assigned by extensive 1D and 2D NMR analysis and molecular modelling. In addition, the biosynthesis of 3 was studied by feeding experiments.

Ammonium availability affects the ratio of ammonia-oxidizing bacteria to ammonia-oxidizing archaea in simulated creek ecosystems.

The ammonia-oxidizing microbial community colonizing clay tiles in flow channels changed in favor of ammonia-oxidizing bacteria during a 12-week incubation period even at originally high ratios of ammonia-oxidizing archaea to ammonia-oxidizing bacteria (AOB). AOB predominance was established more rapidly in flow channels incubated at 350 µM NH(4)(+) than in those incubated at 50 or 20 µM NH(4)(+). Biofilm-associated potential nitrification activity was first detected after 28 days and was positively correlated with bacterial but not archaeal amoA gene copy numbers.

Groundwater bacterial communities evolve over time in response to recharge.

Time series analyses are a crucial tool for uncovering the patterns and processes shaping microbial communities and their functions, especially in aquatic ecosystems. Subsurface aquatic environments are perceived to be more stable than surface oceans and lakes, due to the lack of sunlight, the absence of photosysnthetically-driven primary production, low temperature variations, and oligotrophic conditions. However, periodic groundwater recharge should affect the structure and succession of groundwater microbiomes. To disentangle the long-term temporal changes in bacterial communities of shallow fractured bedrock groundwater, and identify the drivers of the observed patterns, we analysed bacterial 16S rRNA gene sequencing data for samples collected monthly from three groundwater wells over a six-year period (n = 230) along a hillslope recharge area. We showed that the bacterial communities in the groundwater of limestone-mudstone alternations were not stable over time and exhibited non-linear dissimilarity patterns which corresponded to periods of groundwater recharge. Further, we observed an increase in dissimilarity over time (generalized additive model P < 0.001) indicating that the successive recharge events result in communities that are increasingly more dissimilar to the initial reference time point. The sampling period was able to explain up to 29.5% of the variability in bacterial community composition and the impact of recharge events on the groundwater microbiome was linked to the strength of the recharge and local environmental selection. Many groundwater bacteria originated from the recharge-related sources (mean = 66.5%, SD = 15.1%) and specific bacterial taxa were identified as being either enriched or repressed during recharge events. Overall, similar to surface aquatic environments, the microbiomes in shallow fractured-rock groundwater vary through time, though we revealed groundwater recharges as unique driving factors for these patterns. The high temporal resolution employed here highlights the dynamics of bacterial communities in groundwater, which is an essential resource for the provision of clean drinking water; understanding the biological complexities of these systems is therefore crucial.

Environmental selection shapes the formation of near-surface groundwater microbiomes.

Hydrodynamics drives both stochastic and deterministic community assembly in aquatic habitats, by translocating microbes across geographic barriers and generating changes in selective pressures. Thus, heterogeneity of hydrogeological settings and episodic surface inputs from recharge areas might play important roles in shaping and maintaining groundwater microbial communities. Here we took advantage of the Hainich Critical Zone Exploratory to disentangle mechanisms of groundwater microbiome differentiation via a three-year observation in a setting of mixed carbonate-siliciclastic alternations along a hillslope transect. Variation partitioning of all data elucidated significant roles of hydrochemistry (35.0%) and spatial distance (18.6%) but not of time in shaping groundwater microbiomes. Groundwater was dominated by rare species (99.6% of OTUs), accounting for 25.9% of total reads, whereas only 26 OTUs were identified as core species. The proximity to the recharge area gave prominence to high microbial diversity coinciding with high surface inputs. In downstream direction, the abundance of rare OTUs decreased whereas core OTUs abundance increased up to 47% suggesting increasing selection stress with a higher competition cost for colonization. In general, environmental selection was the key mechanism driving the spatial differentiation of groundwater microbiomes, with N-compounds and dissolved oxygen as the major determinants, but it was more prominent in the upper aquifer with low flow velocity. Across the lower aquifer with higher flow velocity, stochastic processes appeared to be additionally important for community assembly. Overall, this study highlights the impact of surface and subsurface conditions, as well as flow regime and related habitat accessibility, on groundwater microbiomes assembly.

Effect of lake trophic status and rooted macrophytes on community composition and abundance of ammonia-oxidizing prokaryotes in freshwater sediments.

Communities of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in freshwater sediments and those in association with the root system of the macrophyte species Littorella uniflora, Juncus bulbosus, and Myriophyllum alterniflorum were compared for seven oligotrophic to mesotrophic softwater lakes and acidic heathland pools. Archaeal and bacterial ammonia monooxygenase alpha-subunit (amoA) gene diversity increased from oligotrophic to mesotrophic sites; the number of detected operational taxonomic units was positively correlated to ammonia availability and pH and negatively correlated to sediment C/N ratios. AOA communities could be grouped according to lake trophic status and pH; plant species-specific communities were not detected, and no grouping was apparent for AOB communities. Relative abundance, determined by quantitative PCR targeting amoA, was always low for AOB (<0.05% of all prokaryotes) and slightly higher for AOA in unvegetated sediment and AOA in association with M. alterniflorum (0.01 to 2%), while AOA accounted for up to 5% in the rhizospheres of L. uniflora and J. bulbosus. These results indicate that (i) AOA are at least as numerous as AOB in freshwater sediments, (ii) aquatic macrophytes with substantial release of oxygen and organic carbon into their rhizospheres, like L. uniflora and J. bulbosus, increase AOA abundance; and (iii) AOA community composition is generally determined by lake trophy, not by plant species-specific interactions.

Distribution and rate of microbial processes in an ammonia-loaded air filter biofilm.

The in situ activity and distribution of heterotrophic and nitrifying bacteria and their potential interactions were investigated in a full-scale, two-section, trickling filter designed for biological degradation of volatile organics and NH(3) in ventilation air from pig farms. The filter biofilm was investigated by microsensor analysis, fluorescence in situ hybridization, quantitative PCR, and batch incubation activity measurements. In situ aerobic activity showed a significant decrease through the filter, while the distribution of ammonia-oxidizing bacteria (AOB) was highly skewed toward the filter outlet. Nitrite oxidation was not detected during most of the experimental period, and the AOB activity therefore resulted in NO(2)(-), accumulation, with concentrations often exceeding 100 mM at the filter inlet. The restriction of AOB to the outlet section of the filter was explained by both competition with heterotrophic bacteria for O(2) and inhibition by the protonated form of NO(2)(-), HNO(2). Product inhibition of AOB growth could explain why this type of filter tends to emit air with a rather constant NH(3) concentration irrespective of variations in inlet concentration and airflow.

DNA- and RNA- Derived Fungal Communities in Subsurface Aquifers Only Partly Overlap but React Similarly to Environmental Factors.

Recent advances in high-throughput sequencing (HTS) technologies have revolutionized our understanding of microbial diversity and composition in relation to their environment. HTS-based characterization of metabolically active (RNA-derived) and total (DNA-derived) fungal communities in different terrestrial habitats has revealed profound differences in both richness and community compositions. However, such DNA- and RNA-based HTS comparisons are widely missing for fungal communities of groundwater aquifers in the terrestrial biogeosphere. Therefore, in this study, we extracted DNA and RNA from groundwater samples of two pristine aquifers in the Hainich CZE and employed paired-end Illumina sequencing of the fungal nuclear ribosomal internal transcribed spacer 2 (ITS2) region to comprehensively test difference/similarities in the "total" and "active" fungal communities. We found no significant differences in the species richness between the DNA- and RNA-derived fungal communities, but the relative abundances of various fungal operational taxonomic units (OTUs) appeared to differ. We also found the same set of environmental parameters to shape the "total" and "active" fungal communities in the targeted aquifers. Furthermore, our comparison also underlined that about 30%-40% of the fungal OTUs were only detected in RNA-derived communities. This implies that the active fungal communities analyzed by HTS methods in the subsurface aquifers are actually not a subset of supposedly total fungal communities. In general, our study highlights the importance of differentiating the potential (DNA-derived) and expressed (RNA-derived) members of the fungal communities in aquatic ecosystems.

Predominance of Cand. Patescibacteria in Groundwater Is Caused by Their Preferential Mobilization From Soils and Flourishing Under Oligotrophic Conditions.

Despite the widely observed predominance of Cand. Patescibacteria in subsurface communities, their input source and ecophysiology are poorly understood. Here we study mechanisms of the formation of a groundwater microbiome and the subsequent differentiation of Cand. Patescibacteria. In the Hainich Critical Zone Exploratory, Germany, we trace the input of microorganisms from forested soils of preferential recharge areas through fractured aquifers along a 5.4 km hillslope well transect. Cand. Patescibacteria were preferentially mobilized from soils and constituted 66% of species-level OTUs shared between seepage and shallow groundwater. These OTUs, mostly related to Cand. Kaiserbacteraceae, Cand. Nomurabacteraceae, and unclassified UBA9983 at the family level, represented a relative abundance of 71.4% of the Cand. Patescibacteria community at the shallowest groundwater well, and still 44.4% at the end of the transect. Several Cand. Patescibacteria subclass-level groups exhibited preferences for different conditions in the two aquifer assemblages investigated: Cand. Kaiserbacteraceae surprisingly showed positive correlations with oxygen concentrations, while Cand. Nomurabacteraceae were negatively correlated. Co-occurrence network analysis revealed a central role of Cand. Patescibacteria in the groundwater microbial communities and pointed to potential associations with specific organisms, including abundant autotrophic taxa involved in nitrogen, sulfur and iron cycling. Strong associations among Cand. Patescibacteria themselves further suggested that for many groups within this phylum, distribution was mainly driven by conditions commonly supporting a fermentative life style without direct dependence on specific hosts. We propose that import from soil, and community differentiation driven by hydrochemical conditions, including the availability of organic resources and potential hosts, determine the success of Cand. Patescibacteria in groundwater environments.

Archaea dominate the ammonia-oxidizing community in the rhizosphere of the freshwater macrophyte Littorella uniflora.

Archaeal and bacterial ammonia monooxygenase genes (amoA) had similar low relative abundances in freshwater sediment. In the rhizosphere of the submersed macrophyte Littorella uniflora, archaeal amoA was 500- to >8,000-fold enriched compared to bacterial amoA, suggesting that the enhanced nitrification activity observed in the rhizosphere was due to ammonia-oxidizing Archaea.

First insights into the living groundwater mycobiome of the terrestrial biogeosphere.

Although fungi play important roles in biogeochemical cycling in aquatic ecosystems and have received a great deal of attention, much remains unknown about the living fractions of fungal communities in aquifers of the terrestrial subsurface in terms of diversity, community dynamics, functional roles, the impact of environmental factors and presence of fungal pathogens. Here we address this gap in knowledge by using RNA-based high throughput pair-end illumina sequencing analysis of fungal internal transcribed spacer (ITS) gene markers, to target the living fractions of groundwater fungal communities from fractured alternating carbonate-/siliciclastic-rock aquifers of the Hainich Critical Zone Exploratory. The probed levels of the hillslope multi-storey aquifer system differ primarily in their oxygen and nitrogen content due to their different connections to the surface. We discovered highly diverse living fungal communities (384 Operational Taxonomic Units, OTUs) with different taxonomic affiliations and ecological functions. The observed fungal communities primarily belonged to three phyla: Ascomycota, Basidiomycota and Chytridiomycota. Perceived dynamics in the composition of living fungal communities were significantly shaped by the concentration of ammonium in the moderately agriculturally impacted aquifer system. Apart from fungal saprotrophs, we also detected living plant and animal pathogens for the first time in this aquifer system. This work also demonstrates that the RNA-based high throughput pair-end illumina sequencing method can be used in future for water quality monitoring in terms of living fungal load and subsequent risk assessments. In general, this study contributes towards the growing knowledge of aquatic fungi in terrestrial subsurface biogeosphere.