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Despite the high relevance of anaerobic ammonium oxidation (anammox) for nitrogen loss from marine systems, its relative importance compared to denitrification has less been studied in freshwater ecosystems, and our knowledge is especially scarce for groundwater. Surprisingly, phospholipid fatty acids (PLFA)-based studies identified zones with potentially active anammox bacteria within two superimposed pristine limestone aquifer assemblages of the Hainich Critical Zone Exploratory (CZE; Germany). We found anammox to contribute an estimated 83% to total nitrogen loss in suboxic groundwaters of these aquifer assemblages at rates of 3.5-4.7 nmol L-1 d-1, presumably favored over denitrification by low organic carbon availability. Transcript abundances of hzsA genes encoding hydrazine synthase exceeded nirS and nirK transcript abundances encoding denitrifier nitrite reductase by up to two orders of magnitude, providing further support of a predominance of anammox. Anammox bacteria, dominated by groups closely related to Cand. Brocadia fulgida, constituted up to 10.6% of the groundwater microbial community and were ubiquitously present across the two aquifer assemblages with indication of active anammox bacteria even in the presence of 103 μmol L-1 oxygen. Co-occurrence of hzsA and amoA gene transcripts encoding ammonia mono-oxygenase suggested coupling between aerobic and anaerobic ammonium oxidation under suboxic conditions. These results clearly demonstrate the relevance of anammox as a key process driving nitrogen loss from oligotrophic groundwater environments, which might further be enhanced through coupling with incomplete nitrification.

Summary: 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. [ABSTRACT FROM AUTHOR]

Summary: Microbial activity is key in understanding the contribution of microbial communities to ecosystem functions. Metabolic labelling with heavy water (D2O) leads to the formation of carbon–deuterium bonds in active microorganisms. We illustrated how D2O labelling allows monitoring of metabolic activity combined with a functional characterization of active populations in complex microbial communities. First, we demonstrated by single cell Raman microspectroscopy that all measured bacterial cells from groundwater isolates growing in complex medium with D2O were labelled. Next, we conducted a labelling approach with the total groundwater microbiome in D2O amended microcosms. Deuterium was incorporated in most measured cells, indicating metabolic activity in the oligotrophic groundwater. Moreover, we spiked the groundwater microbiome with organic model compounds. We discovered that heterotrophs assimilating veratric acid, a lignin derivative, showed higher labelling than heterotrophs assimilating methylamine, a degradation product of biomass. This difference can be explained by dilution of the deuterium through hydrogen from the organic compounds. Metaproteomics identified Sphingomonadaceae and Microbacteriaceae as key players in veratric acid degradation, and the metabolic pathways employed. Methylamine, in contrast, stimulated various proteobacterial genera. We propose this combined approach of Raman microspectroscopy and metaproteomics for elucidating the complex metabolic response of microbial populations to different stimuli. [ABSTRACT FROM AUTHOR]

Despite the high relevance of anaerobic ammonium oxidation (anammox) for nitrogen loss from marine systems, its relative importance compared to denitrification has less been studied in freshwater ecosystems, and our knowledge is especially scarce for groundwater. Surprisingly, phospholipid fatty acids (PLFA)-based studies identified zones with potentially active anammox bacteria within two superimposed pristine limestone aquifer assemblages of the Hainich Critical Zone Exploratory (CZE; Germany). We found anammox to contribute an estimated 83% to total nitrogen loss in suboxic groundwaters of these aquifer assemblages at rates of 3.5-4.7 nmol L-1 d-1, presumably favored over denitrification by low organic carbon availability. Transcript abundances of hzsA genes encoding hydrazine synthase exceeded nirS and nirK transcript abundances encoding denitrifier nitrite reductase by up to two orders of magnitude, providing further support of a predominance of anammox. Anammox bacteria, dominated by groups closely related to Cand. Brocadia fulgida, constituted up to 10.6% of the groundwater microbial community and were ubiquitously present across the two aquifer assemblages with indication of active anammox bacteria even in the presence of 103 μmol L-1 oxygen. Co-occurrence of hzsA and amoA gene transcripts encoding ammonia mono-oxygenase suggested coupling between aerobic and anaerobic ammonium oxidation under suboxic conditions. These results clearly demonstrate the relevance of anammox as a key process driving nitrogen loss from oligotrophic groundwater environments, which might further be enhanced through coupling with incomplete nitrification. [ABSTRACT FROM AUTHOR]

Near-surface groundwaters are prone to receive (in)organic matter input from their recharge areas and are known to harbour 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, anammox) and to a lesser extent sulfur cycling (sulfur oxidation and sulfate reduction), dependent on local hydrochemical differences. Surprisingly, the differentiation potential of the groundwater microbiome surpasses that of hydrochemistry for individual monitoring wells. Dominated by few phyla (Bacteroidetes, Proteobacteria, Planctomycetes, 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 dependent on the availability of oxygen and inorganic electron donors like ammonium. [ABSTRACT FROM AUTHOR]

Microbial activity is key in understanding the contribution of microbial communities to ecosystem functions. Metabolic labelling with heavy water (D 2 O) leads to the formation of carbon-deuterium bonds in active microorganisms. We illustrated how D 2 O labelling allows monitoring of metabolic activity combined with a functional characterization of active populations in complex microbial communities. First, we demonstrated by single cell Raman microspectroscopy that all measured bacterial cells from groundwater isolates growing in complex medium with D 2 O were labelled. Next, we conducted a labelling approach with the total groundwater microbiome in D 2 O amended microcosms. Deuterium was incorporated in most measured cells, indicating metabolic activity in the oligotrophic groundwater. Moreover, we spiked the groundwater microbiome with organic model compounds. We discovered that heterotrophs assimilating veratric acid, a lignin derivative, showed higher labelling than heterotrophs assimilating methylamine, a degradation product of biomass. This difference can be explained by dilution of the deuterium through hydrogen from the organic compounds. Metaproteomics identified Sphingomonadaceae and Microbacteriaceae as key players in veratric acid degradation, and the metabolic pathways employed. Methylamine, in contrast, stimulated various proteobacterial genera. We propose this combined approach of Raman microspectroscopy and metaproteomics for elucidating the complex metabolic response of microbial populations to different stimuli., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)