The nitrogen cycle plays a major role in aquatic nitrogen transformations, including in the terrestrial subsurface. However, the variety of transformations remains understudied. To determine how nitrogen cycling microorganisms respond to different aquifer chemistries, we sampled groundwater with varying nutrient and oxygen contents. Genes and transcripts involved in major nitrogen-cycling pathways were quantified from 55 and 26 sites, respectively, and metagenomes and metatranscriptomes were analyzed from a subset of oxic and dysoxic sites (0.3-1.1 mg/L bulk dissolved oxygen). Nitrogen-cycling mechanisms (e.g. ammonia oxidation, denitrification, dissimilatory nitrate reduction to ammonium) were prevalent and highly redundant, regardless of site-specific physicochemistry or nitrate availability, and present in 40% of reconstructed genomes, suggesting that nitrogen cycling is a core function of aquifer communities. Transcriptional activity for nitrification, denitrification, nitrite-dependent anaerobic methane oxidation and anaerobic ammonia oxidation (anammox) occurred simultaneously in oxic and dysoxic groundwater, indicating the availability of oxic-anoxic interfaces. Concurrent activity by these microorganisms indicates potential synergisms through metabolite exchange across these interfaces (e.g. nitrite and oxygen). Fragmented denitrification pathway encoding and transcription was widespread among groundwater bacteria, although a considerable proportion of associated transcriptional activity was driven by complete denitrifiers, especially under dysoxic conditions. Despite large differences in transcription, the capacity for the final steps of denitrification was largely invariant to aquifer conditions, and most genes and transcripts encoding N2O reductases were the atypical Sec-dependant type, suggesting energy-efficiency prioritization. Results provide insights into the capacity for cooperative relationships in groundwater communities, and the richness and complexity of metabolic mechanisms leading to the loss of fixed nitrogen.

Anammox is increasingly shown to play a major role in the aquatic nitrogen cycle and can outcompete heterotrophic denitrification in environments low in organic carbon. Given that aquifers are characteristically oligotrophic, anammox may represent a major route for the removal of fixed nitrogen in these environments, including agricultural nitrogen, a common groundwater contaminant.

Aquifers are populated by highly diverse microbial communities, including unusually small bacteria and archaea. The recently described Patescibacteria (or Candidate Phyla Radiation) and DPANN radiation are characterized by ultra-small cell and genomes sizes, resulting in limited metabolic capacities and probable dependency on other organisms to survive. We applied a multi-omics approach to characterize the ultra-small microbial communities over a wide range of aquifer groundwater chemistries. Results expand the known global range of these unusual organisms, demonstrate the wide geographical range of over 11,000 subsurface-adapted Patescibacteria, Dependentiae and DPANN archaea, and indicate that prokaryotes with ultra-small genomes and minimalistic metabolism are a characteristic feature of the terrestrial subsurface. Community composition and metabolic activities were largely shaped by water oxygen content, while highly site-specific relative abundance profiles were driven by a combination of groundwater physicochemistries (pH, nitrate-N, dissolved organic carbon). We provide insights into the activity of ultra-small prokaryotes with evidence that they are major contributors to groundwater community transcriptional activity. Ultra-small prokaryotes exhibited genetic flexibility with respect to groundwater oxygen content, and transcriptionally distinct responses, including proportionally greater transcription invested into amino acid and lipid metabolism and signal transduction in oxic groundwater, along with differences in taxa transcriptionally active. Those associated with sediments differed from planktonic counterparts in species composition and transcriptional activity, and exhibited metabolic adaptations reflecting a surface-associated lifestyle. Finally, results showed that groups of phylogenetically diverse ultra-small organisms co-occurred strongly across sites, indicating shared preferences for groundwater conditions.

Background: Anaerobic ammonium oxidation (anammox) is important for converting bioavailable nitrogen into dinitrogen gas, particularly in carbon poor environments. Yet, the diversity and prevalence of anammox bacteria in the terrestrial subsurface – a typically oligotrophic environment – is little understood across different geochemical conditions. To determine the distribution and activity of anammox bacteria across a range of aquifer lithologies and physicochemistries, we analysed 16S rRNA genes, metagenomes and metatranscriptomes, and quantified hydrazine synthase genes and transcripts sampled from 59 groundwater wells distributed over 1 240 km2. Results: Data indicate that anammox-associated bacteria (class Brocadiae) and the anammox process are prevalent in aquifers (identified in aquifers with sandy-gravel, sandsilt and volcanic lithologies). While Brocadiae diversity decreased with increasing DO, Brocadiae 16S rRNA genes and hydrazine synthase genes and transcripts (hydrazine synthase, hzsB) were detected across a wide range of bulk groundwater dissolved oxygen (DO) concentrations (0 – 10 mg/L). Anammox genes and transcripts (hzsB) correlated significantly with those involved in bacterial and archaeal ammonia oxidation (ammonia monooxygenase, amoA), which could represent a major source of nitrite for anammox. Differences in anammox community composition were strongly associated with DO and bore depth (and to a lesser extent pH and phosphate), revealing niche differentiation among anammox bacteria in groundwater that was largely driven by water oxygen contents, and not ammonium/nitrite. Eight Brocadiae genomes (63-95% estimated completeness) reconstructed from a subset of groundwater sites belong to 2 uncharacterized families and 6 novel species (based on average nucleotide identity). Distinct groups of these genomes dominated the anammox-associated community at dysoxic and oxic sites, further reflecting the influence of DO on Brocadiae composition. Six of the genomes (dominating dysoxic or oxic sites) have genes characteristic of anammox (hydrazine synthase and/or dehydrogenase). These genes, in addition to aerotolerance genes, belonging to four Brocadiae genomes, were transcriptionally active, although transcript numbers clearly highest in dyoxic groundwater. Conclusions: Our findings indicate anammox bacteria contribute to loss of fixed N across diverse anoxic-to-oxic aquifer conditions, and that this is likely supported by nitrite from aerobic ammonia oxidation. Results provide an insight into the distribution and activity of anammox bacteria across distinct aquifer physicochemisties.

Bacterial genomes are highly dynamic entities, mostly due to the extent of horizontal gene transfer (HGT) occurring in these organisms. HGT is thought to be the main driver of genetic variation and adaptation to local environment in bacteria. However, little is known about the modalities of HGT within natural microbial communities, especially the implications of genetic exchange for streamlined microorganisms such as Patescibacteria (Candidate Phyla Radiation). We searched for evidence of genetic exchange in 125 Patescibacteria genomes recovered from aquifer environments and detected the presence of hundreds of genomic islands, individually transferred genes and prophage combined, with up to 29% of genome length attributed to HGT. Results show that most individual gene transfer events occurred between Patescibacteria, but donors were also phylogenetically diverse groundwater microorganisms. Using gene donor-recipient information, we identified one potential host (Omnitrophota) of the ultra-small bacteria, and confirmed this by matching relative abundance patterns across 16 groundwater samples. A wide variety of metabolic functions were introduced in Patescibacteria genomes by HGT including transcription, translation and DNA replication, recombination and repair. This study illustrates the evolutionarily dynamic nature of Patescibacteria genomes despite the constraints of streamlining, and that HGT in these organisms is also mediated via viral infection.

<p>Extensive peatland rewetting efforts have recently been proposed to restore these key terrestrial carbon storage systems in order to mitigate greenhouse gas (GHG) emissions. However, little is known about the effects of rewetting on peat microbial functions that are linked to GHG fluxes. A better understanding of which biotic and abiotic factors control these processes in rewetted peatlands is crucial to help guide restoration decisions with maximum climate benefits. Here, we present results exploring the effects of peat nutrient status (nutrient-rich vs. nutrient-poor) and N loading on microbial processes and GHG (carbon dioxide, methane, and nitrous oxide) production and consumption patterns in two rewetted fens. We used an automated incubation system coupled with a gas chromatograph to monitor microbial functions and GHG dynamics in rewetted peat samples under different treatments. Samples were collected at the start of a running year-long mesocosm experiment, where peat is incubated with controlled N concentrations and vegetation composition.</p><p>The start point incubation data show that N loading, but not the inherent peat nutrient status, promoted N related processes such as nitrification and denitrification. Both methane production and consumption were higher in nutrient-rich peat, and were inhibited by the presence of nitrate and ammonium respectively. Methane production kinetics displayed variable patterns between nutrient-rich and -poor peat (higher initial production rate in nutrient-rich peat), yet the total amount of methane produced was similar between fens. Results also suggest that the availability of other electron acceptors than oxygen tended to increase anoxic carbon dioxide production rates in rewetted peatlands. Overall, these findings indicate that differences in chemical composition between the two similar peatland types (fens) can lead to variable GHG dynamics after rewetting, and that controls of soil functions are site-specific.</p><p>We aim to use results from the endpoint of the mesocosm experiment (after 1 year of incubation) to investigate the impact of vegetation composition on soil functions, and whether N loading leads to acclimatization of GHG-related microbial functions in rewetted fens using transcriptomics combined with targeted incubations.</p>