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Light driven primary production by plants is the main source of biomass in terrestrial ecosystems. But also in subsurface habitats like aquifers, life is fueled largely by this plant-derived biomass. Here, we investigate the degradation of plant-derived polysaccharides in a groundwater microbiome to identify the microbial key players involved, and compare them to those from soil of the groundwater recharge area. We quantified the activities of enzymes degrading the abundant plant polymers starch, cellulose and hemicellulose in oligotrophic groundwater samples, despite the low cell numbers present. Normalized to 16S rRNA gene copy numbers, these activities were only one order of magnitude lower than in soil. Stimulation of the groundwater microbiome with either starch or cellulose and hemicellulose led to changes of the enzymatic activity ratios, indicating autochthonous production of enzymes in response to the plant polymers. Furthermore, DNA stable isotope probing with 13C labelled plant polymers allowed us to identify microbes involved in the degradation of these compounds. In (hemi)cellulose microcosms, Bacteroidia and Candidatus Parcubacteria were active, while the active community in starch microcosms mostly comprised Candidatus Saccharibacteria, Cytophagia, and Actinobacteria. Not a single one of the active OTUs was also found to be labelled in soil microcosms. This indicates that the degradation of plant-derived polysaccharides in groundwater is driven by organisms completely distinct from those active in soil. The involvement of members of the candidate phyla Cand. Parcubacteria and Cand. Saccharibacteria, organisms known to be abundant in groundwater, in plant-derived organic matter degradation might strongly impact subsurface carbon cycling.

Microbial life in soil is fueled by dissolved organic matter (DOM) that leaches from the litter layer. It is well known that decomposer communities adapt to the available litter source, but it remains unclear if they functionally compete or synergistically address different litter types. Therefore, we decomposed beech, oak, pine and grass litter from two geologically distinct sites in a lab-scale decomposition experiment. We performed a correlative network analysis on the results of direct infusion HR-MS DOM analysis and cross-validated functional predictions from 16S rRNA gene amplicon sequencing and with DOM and metaproteomic analyses. Here we show that many functions are redundantly distributed within decomposer communities and that their relative expression is rapidly optimized to address litter-specific properties. However, community changes are likely forced by antagonistic mechanisms as we identified several natural antibiotics in DOM. As a consequence, the decomposer community is specializing towards the litter source and the state of decomposition (community divergence) but showing similar litter metabolomes (metabolome convergence). Our multi-omics-based results highlight that DOM not only fuels microbial life, but it additionally holds meta-metabolomic information on the functioning of ecosystems.

Background The highly diverse Cand . Patescibacteria are predicted to have minimal biosynthetic and metabolic pathways, which hinders understanding of how their populations differentiate in response to environmental drivers or host organisms. Their mechanisms employed to cope with oxidative stress are largely unknown. Here, we utilized genome-resolved metagenomics to investigate the adaptive genome repertoire of Patescibacteria in oxic and anoxic groundwaters, and to infer putative host ranges. Results Within six groundwater wells, Cand . Patescibacteria was the most dominant (up to 79%) super-phylum across 32 metagenomes sequenced from DNA retained on 0.2 and 0.1 µm filters after sequential filtration. Of the reconstructed 1275 metagenome-assembled genomes (MAGs), 291 high-quality MAGs were classified as Cand . Patescibacteria. Cand . Paceibacteria and Cand . Microgenomates were enriched exclusively in the 0.1 µm fractions, whereas candidate division ABY1 and Cand . Gracilibacteria were enriched in the 0.2 µm fractions. On average, Patescibacteria enriched in the smaller 0.1 µm filter fractions had 22% smaller genomes, 13.4% lower replication measures, higher proportion of rod-shape determining proteins, and of genomic features suggesting type IV pili mediated cell–cell attachments. Near-surface wells harbored Patescibacteria with higher replication rates than anoxic downstream wells characterized by longer water residence time. Except prevalence of superoxide dismutase genes in Patescibacteria MAGs enriched in oxic groundwaters (83%), no major metabolic or phylogenetic differences were observed. The most abundant Patescibacteria MAG in oxic groundwater encoded a nitrate transporter, nitrite reductase, and F-type ATPase, suggesting an alternative energy conservation mechanism. Patescibacteria consistently co-occurred with one another or with members of phyla Nanoarchaeota, Bacteroidota, Nitrospirota, and Omnitrophota. Among the MAGs enriched in 0.2 µm fractions,, only 8% Patescibacteria showed highly significant one-to-one correlation, mostly with Omnitrophota. Motility and transport related genes in certain Patescibacteria were highly similar to genes from other phyla (Omnitrophota, Proteobacteria and Nanoarchaeota). Conclusion Other than genes to cope with oxidative stress, we found little genomic evidence for niche adaptation of Patescibacteria to oxic or anoxic groundwaters. Given that we could detect specific host preference only for a few MAGs, we speculate that the majority of Patescibacteria is able to attach multiple hosts just long enough to loot or exchange supplies.

Background The candidate phyla radiation (CPR) comprises a widespread but poorly understood group of bacteria with limited cultured representatives, largely due to their metabolic dependencies on microbial hosts. In laboratory incubations, CPR often decline sharply in relative abundance, even when samples originate from natural environments where they dominate, such as groundwater, where they can represent over 50% of the microbiome. Suitable enrichment conditions and host interactions remain poorly defined. Results Here, we analyzed 16S rRNA gene amplicon data from 397 groundwater incubation samples across 31 treatments, including 22 under oxic conditions, to identify factors that promote CPR survival and growth. Despite an initial decline, CPR abundances recovered over longer incubation times, reaching up to 11–30% of the microbial community. In total, we detected 1410 CPR amplicon sequence variants (ASVs), spanning six major CPR classes commonly found in groundwater. Enrichment success was treatment-specific: Cand. Saccharimonadia dominated in incubations with polysaccharides (up to 31.4%), while Cand. Parcubacteria were enriched (> 23%) in treatments stimulating methylotrophs and autotrophs. ASV-specific growth rates based on quantitative PCR showed that some CPR doubled within 1–2 days, comparable to faster-growing non-CPR groundwater bacteria, while most CPR had doubling times around 15 days. Strikingly, although the relative abundance of many CPR ASVs showed positive correlation with anoxic conditions, overall CPR reached higher absolute abundances under oxic conditions than under anoxic conditions. Metabolic network analysis based on metagenome-assembled genomes revealed that up to 62% of annotated genes were associated with functions linked to oxic conditions. In fact, 25 CPR genomes encoded enzymes that directly utilize oxygen, challenging the long-standing view of CPR as strictly anaerobic, fermentative organisms. Conclusions Our findings demonstrate that diverse CPR lineages not only survive but actively grow in groundwater incubations, even under oxic conditions. The discovery of genes for oxygen-dependent reactions and substantial CPR enrichment in oxic treatments reveals unexpected metabolic flexibility, helping to explain their persistence and ecological success across a wide range of environments. 

Due to its high spatial resolution, Raman microspectroscopy allows for the analysis of single microbial cells. Since Raman spectroscopy analyzes the whole cell content, this method is phenotypic and can therefore be used to evaluate cellular changes. In particular, labeling with stable isotopes (SIPs) enables the versatile use and observation of different metabolic states in microbes. Nevertheless, static measurements can only analyze the present situation and do not allow for further downstream evaluations. Therefore, a combination of Raman analysis and cell sorting is necessary to provide the possibility for further research on selected bacteria in a sample. Here, a new microfluidic approach for Raman-activated continuous-flow sorting of bacteria using an optical setup for image-based particle sorting with synchronous acquisition and analysis of Raman spectra for making the sorting decision is demonstrated, showing that active cells can be successfully sorted by means of this microfluidic chip.

Background Microorganisms in groundwater ecosystems exist either as planktonic cells or as attached communities on aquifer rock surfaces. Attached cells outnumber planktonic ones by at least three orders of magnitude, suggesting a critical role in aquifer ecosystem function. However, particularly in consolidated carbonate aquifers, where research has predominantly focused on planktonic microbes, the metabolic potential and ecological roles of attached communities remain poorly understood. Results To investigate the differences between attached and planktonic communities, we sampled the attached microbiome from passive samplers filled with crushed carbonate rock exposed to oxic and anoxic groundwater in the Hainich Critical Zone Exploratory and compared it to a previously published, extensive dataset of planktonic communities from the same aquifer ecosystem. Microbial lifestyle (attached vs. planktonic) explained more variance in community composition than redox conditions, prompting us to further investigate its role in shaping functional and activity profiles. Metagenomic analysis revealed a striking taxonomic and functional segregation: the 605 metagenome-assembled genomes (MAGs) from attached communities were dominated by Proteobacteria (358 MAGs) and were enriched in genes for biofilm formation, chemolithoautotrophy, and redox cycling (e.g., iron and sulfur metabolism). In contrast, the 891 MAGs from planktonic communities were dominated by Cand. Patescibacteria (464 MAGs) and Nitrospirota (60 MAGs) and showed lower functional versatility. Only a few genera were shared, and even closely related MAGs (> 90% average nucleotide identity) differed in assembly size and metabolic traits, demonstrating lifestyle-specific functional adaptation. Analysis of active replication indicated that the active fraction of the attached community was primarily represented by the most abundant MAGs. Planktonic communities featured a higher fraction of active MAGs compared to attached communities, but overall with lower relative abundances. Conclusions The high abundance, metabolic specialization, and carbon fixation potential of attached microbes suggest that they are key drivers of subsurface biogeochemical processes. Carbonate aquifers may act as much larger inorganic carbon sinks than previously estimated based on CO 2 fixation rates of the planktonic communities alone. Our findings underscore the need to incorporate attached microbial communities into models of subsurface ecosystem function. 2o8uywypvETBQzovpcAATJ Video Abstract

Benzene is a major contaminant in various environments, but the mechanisms behind its biodegradation under strictly anoxic conditions are not yet entirely clear. Here we analyzed a benzene-degrading, sulfate-reducing enrichment culture originating from a benzene-contaminated aquifer by a metagenome-based functional metaproteomic approach, using protein-based stable isotope probing (protein-SIP). The time-resolved, quantitative analysis of carbon fluxes within the community supplied with either 13 C-labeled benzene or 13 C-labeled carbonate yielded different functional groups of organisms, with their peptides showing specific time dependencies of 13 C relative isotope abundance indicating different carbon utilization. Through a detailed analysis of the mass spectrometric (MS) data, it was possible to quantify the utilization of the initial carbon source and the metabolic intermediates. The functional groups were affiliated to Clostridiales, Deltaproteobacteria and Bacteroidetes/Chlorobi. The Clostridiales-related organisms were involved in benzene degradation, putatively by fermentation, and additionally used significant amounts of carbonate as a carbon source. The other groups of organisms were found to perform diverse functions, with Deltaproteobacteria degrading fermentation products and Bacteroidetes/Chlorobi being putative scavengers feeding on dead cells. A functional classification of identified proteins supported this allocation and gave further insights into the metabolic pathways and the interactions between the community members. This example shows how protein-SIP can be applied to obtain temporal and phylogenetic information about functional interdependencies within microbial communities.

Population genetics is a field of research that predates the current generations of sequencing technology. Those approaches, that were established before massively parallel sequencing methods, have been adapted to these new marker systems (in some cases involving the development of new methods) that allow genome‐wide estimates of the four major micro‐evolutionary forces—mutation, gene flow, genetic drift, and selection. Nevertheless, classic population genetic markers are still commonly used and a plethora of analysis methods and programs is available for these and high‐throughput sequencing (HTS) data. These methods employ various and diverse theoretical and statistical frameworks, to varying degrees of success, to estimate similar evolutionary parameters making it difficult to get a concise overview across the available approaches. Presently, reviews on this topic generally focus on a particular class of methods to estimate one or two evolutionary parameters. Here, we provide a brief history of methods and a comprehensive list of available programs for estimating micro‐evolutionary forces. We furthermore analyzed their usage within the research community based on popularity (citation bias) and discuss the implications of this bias for the software community. We found that a few programs received the majority of citations, with program success being independent of both the parameters estimated and the computing platform. The only deviation from a model of exponential growth in the number of citations was found for the presence of a graphical user interface (GUI). Interestingly, no relationship was found for the impact factor of the journals, when the tools were published, suggesting accessibility might be more important than visibility. Multiple booms in software analyzing population genetics have occurred with changing technology/techniques. Both established and novel techniques have been developed providing a plethora of different analysis methods and programs for the researcher. We aim to provide an overview for the major micro‐evolutionary forces over these methods, providing a resource for researchers to assess the diversity of software in the field.

Pelagic aggregates function as biological carbon pumps for transporting fixed organic carbon to sediments. In iron-rich (ferruginous) lakes, photoferrotrophic and chemolithoautotrophic bacteria contribute to CO2 fixation by oxidizing reduced iron, leading to the formation of iron-rich pelagic aggregates (iron snow). The significance of iron oxidizers in carbon fixation, their general role in iron snow functioning and the flow of carbon within iron snow is still unclear. Here, we combined a two-year metatranscriptome analysis of iron snow collected from an acidic lake with protein-based stable isotope probing to determine general metabolic activities and to trace 13CO2 incorporation in iron snow over time under oxic and anoxic conditions. mRNA-derived metatranscriptome of iron snow identified four key players (Leptospirillum, Ferrovum, Acidithrix, Acidiphilium) with relative abundances (59.6–85.7%) encoding ecologically relevant pathways, including carbon fixation and polysaccharide biosynthesis. No transcriptional activity for carbon fixation from archaea or eukaryotes was detected. 13CO2 incorporation studies identified active chemolithoautotroph Ferrovum under both conditions. Only 1.0–5.3% relative 13C abundances were found in heterotrophic Acidiphilium and Acidocella under oxic conditions. These data show that iron oxidizers play an important role in CO2 fixation, but the majority of fixed C will be directly transported to the sediment without feeding heterotrophs in the water column in acidic ferruginous lakes.

The terrestrial subsurface contains nearly all of Earth’s freshwater reserves and harbours the majority of our planet’s total prokaryotic biomass. Although genetic surveys suggest these organisms rely on in situ carbon fixation, rather than the photosynthetically derived organic carbon transported from surface environments, direct measurements of carbon fixation in the subsurface are absent. Using an ultra-low level 14 C-labelling technique, we estimate in situ carbon fixation rates in a carbonate aquifer. We find these rates are similar to those measured in oligotrophic marine surface waters and up to six-fold greater than those observed in the lower euphotic zone. Our empirical carbon fixation rates agree with nitrification rate data. Metagenomic analyses reveal abundant putative chemolithoautotrophic members of an uncharacterized order of Nitrospiria that may be behind the carbon fixation. On the basis of our determined carbon fixation rates, we conservatively extrapolate global primary production in carbonate groundwaters (10% of global reserves) to be 0.11 Pg carbon per year. These rates fall within the range found for oligotrophic marine surface waters, indicating a substantial contribution of in situ primary production to subsurface ecosystem processes. We further suggest that, just as phototrophs are for marine biogeochemical cycling, such subsurface carbon fixation is potentially foundational to subsurface trophic webs. Direct measurements of carbon fixation rates in groundwater suggest a substantial contribution of in situ primary production to subsurface ecosystem processes.