Iron coatings on carbonate rocks shape the attached bacterial aquifer community.

Most studies of groundwater ecosystems target planktonic microbes, which are easily obtained via water samples. In contrast, little is known about the diversity and function of microbes adhering to rock surfaces, particularly to consolidated rocks. To investigate microbial attachment to rock surfaces, we incubated rock chips from fractured aquifers in limestone-mudstone alternations in bioreactors fed with groundwater from two wells representing oxic and anoxic conditions. Half of the chips were coated with iron oxides, representing common secondary mineralization in fractured rock. Our time-series analysis showed bacteria colonizing the chips within two days, reaching cell numbers up to 4.16 × 105 cells/mm2 after 44 days. Scanning electron microscopy analyses revealed extensive colonization but no multi-layered biofilms, with chips from oxic bioreactors more densely colonized than from anoxic ones. Estimated attached-to-planktonic cell ratios yielded values of up to 106: 1 and 103: 1, for oxic and anoxic aquifers, respectively. We identified distinct attached and planktonic communities with an overlap between 17 % and 42 %. Oxic bioreactors were dominated by proteobacterial genera Aquabacterium and Rhodoferax, while Rheinheimera and Simplicispira were the key players of anoxic bioreactors. Motility, attachment, and biofilm formation traits were predicted in major genera based on groundwater metagenome-assembled genomes and reference genomes. Early rock colonizers appeared to be facultative autotrophs, capable of fixing CO2 to synthesize biomass and a biofilm matrix. Late colonizers were predicted to possess biofilm degrading enzymes such as beta-glucosidase, beta-galactosidase, amylases. Fe-coated chips of both bioreactors featured more potential iron reducers and oxidizers than bare rock chips. As secondary minerals can also serve as energy source, they might favor primary production and thus contribute to subsurface ecosystem services like carbon fixation. Since most subsurface microbes seem to be attached, their contribution to ecosystem services should be considered in future studies.

Metabolic versatility enables sulfur-oxidizers to dominate primary production in groundwater.

High rates of CO2 fixation and the genetic potential of various groundwater microbes for autotrophic activity have shown that primary production is an important source of organic C in groundwater ecosystems. However, the contribution of specific chemolithoautotrophic groups such as S-oxidizing bacteria (SOB) to groundwater primary production and their adaptation strategies remain largely unknown. Here, we stimulated anoxic groundwater microcosms with reduced S and sampled the microbial community after 1, 3 and 6 weeks. Genome-resolved metaproteomics was combined with 50at-% 13CO2 stable isotope probing to follow the C flux through the microbial food web and infer traits expressed by active SOB in the groundwater microcosms. Already after 7 days, 90% of the total microbial biomass C in the microcosms was replaced by CO2-derived C, increasing to 97% at the end of incubation. Stable Isotope Cluster Analysis revealed active autotrophs, characterized by a uniform 13C-incorporation of 45% in their peptides, to dominate the microbial community throughout incubation. Mixo- and heterotrophs, characterized by 10 to 40% 13C-incorporation, utilized the primarily produced organic C. Interestingly, obligate autotrophs affiliated with Sulfuricella and Sulfuritalea contained traits enabling the storage of elemental S in globules to maintain primary production under energy limitation. Others related to Sulfurimonas seemed to rapidly utilize substrates for fast proliferation, and most autotrophs further maximized their energy yield via efficient denitrification and the potential for H2 oxidation. Mixotrophic SOB, belonging to Curvibacter or Polaromonas, enhanced metabolic flexibility by using organic compounds to satisfy their C requirements. Time series data spanning eight years further revealed that key taxa of our microcosms composed up to 15% of the microbial groundwater community, demonstrating their in-situ importance. This showed that SOB, by using different metabolic strategies, are able to account for high rates of primary production in groundwater, especially at sites limited to geogenic nutrient sources. The widespread presence of SOB with traits such as S storage, H2 oxidation, and organic C utilization in many aquatic habitats further suggested that metabolic versatility governs S-fueled primary production in the environment.

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.

DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganisms.

Stable-isotope probing (SIP) enables researchers to target active populations within complex microbial communities, which is achieved by providing growth substrates enriched in heavy isotopes, usually in the form of 13C, 18O, or 15N. After growth on the substrate and subsequent extraction of microbial biomarkers, typically nucleic acids or proteins, the SIP technique is used for the recovery and analysis of isotope-labelled biomarkers from active microbial populations. In the years following the initial development of DNA- and RNA-based SIP, it was common practice to characterize labelled populations by targeted gene analysis. Such approaches usually involved fingerprint-based analyses or sequencing clone libraries containing 16S rRNA genes or functional marker gene amplicons. Although molecular fingerprinting remains a valuable approach for rapid confirmation of isotope labelling, recent advances in sequencing technology mean that it is possible to obtain affordable and comprehensive amplicon profiles, or even metagenomes and metatranscriptomes from SIP experiments. Not only can the abundance of microbial groups be inferred from metagenomes, but researchers can bin, assemble, and explore individual genomes to build hypotheses about the metabolic capabilities of labelled microorganisms. Analysis of labelled mRNA is a more recent advance that can provide independent metatranscriptome-based analysis of active microorganisms. The power of metatranscriptomics is that mRNA abundance often correlates closely with the corresponding activity of encoded enzymes, thus providing insight into microbial metabolism at the time of sampling. Together, these advances have improved the sensitivity of SIP methods and allowed using labelled substrates at environmentally relevant concentrations. Particularly as methods improve and costs continue to drop, we expect that the integration of SIP with multiple omics-based methods will become prevalent components of microbial ecology studies, leading to further breakthroughs in our understanding of novel microbial populations and elucidation of the metabolic function of complex microbial communities. In this chapter, we provide protocols for obtaining labelled DNA, RNA, and proteins that can be used for downstream omics-based analyses.

Bacterial Necromass Is Rapidly Metabolized by Heterotrophic Bacteria and Supports Multiple Trophic Levels of the Groundwater Microbiome.

Pristine groundwater is a highly stable environment with microbes adapted to dark, oligotrophic conditions. Input events like heavy rainfalls can introduce the excess particulate organic matter, including surface-derived microorganisms, thereby disturbing the groundwater microbiome. Some surface-derived bacteria will not survive this translocation, leading to an input of necromass to the groundwater. Here, we investigated the effects of necromass addition to the microbial community in fractured bedrock groundwater, using groundwater mesocosms as model systems. We followed the uptake of 13C-labeled necromass by the bacterial and eukaryotic groundwater community quantitatively and over time using a complementary protein-stable and DNA-stable isotope probing approach. Necromass was rapidly depleted in the mesocosms within 4 days, accompanied by a strong decrease in Shannon diversity and a 10-fold increase in bacterial 16S rRNA gene copy numbers. Species of Flavobacterium, Massilia, Rheinheimera, Rhodoferax, and Undibacterium dominated the microbial community within 2 days and were identified as key players in necromass degradation, based on a 13C incorporation of >90% in their peptides. Their proteomes comprised various proteins for uptake and transport functions and amino acid metabolization. After 4 and 8 days, the autotrophic and mixotrophic taxa Nitrosomonas, Limnohabitans, Paucibacter, and Acidovorax increased in abundance with a 13C incorporation between 0.5% and 23%. Likewise, eukaryotes assimilated necromass-derived carbon either directly or indirectly. Our data point toward a fast and exclusive uptake of labeled necromass by a few specialists followed by a concerted action of groundwater microorganisms, including autotrophs presumably fueled by released, reduced nitrogen and sulfur compounds generated during necromass degradation. IMPORTANCE Subsurface microbiomes provide essential ecosystem services, like the generation of drinking water. These ecosystems are devoid of light-driven primary production, and microbial life is adapted to the resulting oligotrophic conditions. Modern groundwater is most vulnerable to anthropogenic and climatic impacts. Heavy rainfalls, which will increase with climate change, can result in high surface inputs into shallow aquifers by percolation or lateral flow. These inputs include terrestrial organic matter and surface-derived microbes that are not all capable to flourish in aquatic subsurface habitats. Here, we investigated the response of groundwater mesocosms to the addition of bacterial necromass, simulating event-driven surface input. We found that the groundwater microbiome responds with a rapid bloom of only a few primary degraders, followed by the activation of typical groundwater autotrophs and mixotrophs, as well as eukaryotes. Our results suggest that this multiphase strategy is essential to maintain the balance of the groundwater microbiome to provide ecosystem services.

Microbial community functioning during plant litter decomposition.

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.

Phenotypic Differentiation of Autotrophic and Heterotrophic Bacterial Cells Using Raman-D2O Labeling.

Carbon cycling is one of the major biogeochemical processes driven by bacteria. Autotrophic bacteria convert carbon dioxide (CO2) into organic compounds that are used by heterotrophs. Mixotrophic bacteria can employ both autotrophy and heterotrophy for growth. The characterization of the lifestyle of individual cells is essential to understand the microbial activity and thus reveal the implication of bacteria in the carbon flux. In this study, we used groundwater bacteria to investigate the potential of Raman-D2O labeling in combination with chemometrics to identify the carbon assimilation strategies of bacteria. Classification models were built using principal component analysis (PCA) followed by linear discriminant analysis (LDA). Autotrophs assimilated a significantly higher amount (mean C-D ratio between 16.63 and 21.69%) of deuterium than heterotrophs. The C-D signal only provides information about the activity since it appears in the Raman-silent region, where no interference with the taxonomic information is expected. The classification between autotrophs and heterotrophs achieved an overall accuracy of 96.3%. In the validation step with an independent dataset containing species not included in the model, the PCA-LDA model achieved 100% accuracy. This demonstrated that the C-D signal contributed to the identification of autotrophic and heterotrophic bacterial cells. This work reports a robust, rapid, and nondestructive approach for the identification of single cells based on their carbon acquisition strategies. The present study foresees the potential of Raman-D2O labeling as a promising method for automated discrimination of in situ functional activities of bacteria in environmental systems.

Bolstering fitness via CO2 fixation and organic carbon uptake: mixotrophs in modern groundwater.

Current understanding of organic carbon inputs into ecosystems lacking photosynthetic primary production is predicated on data and inferences derived almost entirely from metagenomic analyses. The elevated abundances of putative chemolithoautotrophs in groundwaters suggest that dark CO2 fixation is an integral component of subsurface trophic webs. To understand the impact of autotrophically fixed carbon, the flux of CO2-derived carbon through various populations of subsurface microbiota must first be resolved, both quantitatively and temporally. Here we implement novel Stable Isotope Cluster Analysis to render a time-resolved and quantitative evaluation of 13CO2-derived carbon flow through a groundwater community in microcosms stimulated with reduced sulfur compounds. We demonstrate that mixotrophs, not strict autotrophs, were the most abundant active organisms in groundwater microcosms. Species of Hydrogenophaga, Polaromonas, Dechloromonas, and other metabolically versatile mixotrophs drove the production and remineralization of organic carbon. Their activity facilitated the replacement of 43% and 80% of total microbial carbon stores in the groundwater microcosms with 13C in just 21 and 70 days, respectively. The mixotrophs employed different strategies for satisfying their carbon requirements by balancing CO2 fixation and uptake of available organic compounds. These different strategies might provide fitness under nutrient-limited conditions, explaining the great abundances of mixotrophs in other oligotrophic habitats, such as the upper ocean and boreal lakes.

The economical lifestyle of CPR bacteria in groundwater allows little preference for environmental drivers.

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.

A brief history and popularity of methods and tools used to estimate micro-evolutionary forces.

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.

Insights into Autotrophic Activities and Carbon Flow in Iron-Rich Pelagic Aggregates (Iron Snow).

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.

Monitoring Deuterium Uptake in Single Bacterial Cells via Two-Dimensional Raman Correlation Spectroscopy.

Raman-stable isotope labeling using heavy water (Raman-D2O) is attracting great interest as a fast technique with various applications ranging from the identification of pathogens in medical samples to the determination of microbial activity in the environment. Despite its widespread applications, little is known about the fundamental processes of hydrogen-deuterium (H/D) exchange, which are crucial for understanding molecular interactions in microorganisms. By combining two-dimensional (2D) correlation spectroscopy and Raman deuterium labeling, we have investigated H/D exchange in bacterial cells under time dependence. Most C-H stretching signals decreased in intensity over time, prior to the formation of the C-D stretching vibration signals. The intensity of the C-D signal gradually increased over time, and the shape of the C-D signal was more uniform after longer incubation times. Deuterium uptake showed high variability between the bacterial genera and mainly led to an observable labeling of methylene and methyl groups. Thus, the C-D signal encompassed a combination of symmetric and antisymmetric CD2 and CD3 stretching vibrations, depending on the bacterial genera. The present study allowed for the determination of the sequential order of deuterium incorporation into the functional groups of proteins, lipids, and nucleic acids and hence understanding the process of biomolecule synthesis and the growth strategies of different bacterial taxa. We present the combination of Raman-D2O labeling and 2D correlation spectroscopy as a promising approach to gain a fundamental understanding of molecular interactions in biological systems.

Phylogenetic and metabolic diversity have contrasting effects on the ecological functioning of bacterial communities.

Quantifying the relative contributions of microbial species to ecosystem functioning is challenging, because of the distinct mechanisms associated with microbial phylogenetic and metabolic diversity. We constructed bacterial communities with different diversity traits and employed exoenzyme activities (EEAs) and carbon acquisition potential (CAP) from substrates as proxies of bacterial functioning to test the independent effects of these two aspects of biodiversity. We expected that metabolic diversity, but not phylogenetic diversity would be associated with greater ecological function. Phylogenetically relatedness should intensify species interactions and coexistence, therefore amplifying the influence of metabolic diversity. We examined the effects of each diversity treatment using linear models, while controlling for the other, and found that phylogenetic diversity strongly influenced community functioning, positively and negatively. Metabolic diversity, however, exhibited negative or non-significant relationships with community functioning. When controlling for different substrates, EEAs increased along with phylogenetic diversity but decreased with metabolic diversity. The strength of diversity effects was related to substrate chemistry and the molecular mechanisms associated with each substrate's degradation. EEAs of phylogenetically similar groups were strongly affected by within-genus interactions. These results highlight the unique flexibility of microbial metabolic functions that must be considered in further ecological theory development.

Influence of Carbon Sources on Quantification of Deuterium Incorporation in Heterotrophic Bacteria: A Raman-Stable Isotope Labeling Approach.

A rapid and reliable method for the differentiation between active and inactive bacteria at single cell level is urgently needed in many fields including clinical diagnosis and environmental microbiology, to understand the contribution of metabolically active bacteria in fundamental processes triggering environmental and public health risks. Here, using heavy water (D2O) with Raman-stable isotope labeling (Raman-D2O), we evaluated the reliability of the quantification of deuterium uptake, a well-known indicator for the general metabolic activity of bacteria. For this purpose, we based our study on the quantification of deuterium assimilation from heavy water into single bacterial cells to check the influence of carbon source and bacterial identity on the deuterium uptake. We show that compared to complex carbon substrates, the deuterium assimilation is higher in the presence of simpler substrates such as sugars but differs significantly among bacterial isolates. Despite this variability, the developed classification models could differentiate deuterium labeled and nonlabeled single cells with high sensitivity and specificity. Highlighting the variability between single bacterial cells, the study emphasizes the challenges in establishing a threshold in terms of deuterium uptake to distinguish deuterium labeled and nonlabeled cells. Overall, we show that the Raman-D2O approach, when coupled with chemometrics, constitutes a powerful approach for monitoring single bacterial cells.

SIP-Metaproteomics: Linking Microbial Taxonomy, Function, and Activity.

Stable isotope probing combined with metaproteomics enables the detection and characterization of active key species in microbial populations under near-natural conditions, which greatly helps to understand the metabolic functions of complex microbial communities. This is achieved by providing growth substrates labeled with heavy isotopes such as 13C, which will be assimilated into microbial biomass. After subsequent extraction of proteins and proteolytic cleavage into peptides, the heavy isotope enrichment can be detected by high-resolution mass spectrometric analysis, and linked to the functional and taxonomic characterization of these biomarkers. Here we provide protocols for obtaining isotopically labeled proteins and for downstream SIP-metaproteomics analysis.

Communal metabolism by Methylococcaceae and Methylophilaceae is driving rapid aerobic methane oxidation in sediments of a shallow seep near Elba, Italy.

The release of abiotic methane from marine seeps into the atmosphere is a major source of this potent greenhouse gas. Methanotrophic microorganisms in methane seeps use methane as carbon and energy source, thus significantly mitigating global methane emissions. Here, we investigated microbial methane oxidation at the sediment-water interface of a shallow marine methane seep. Metagenomics and metaproteomics, combined with 13 C-methane stable isotope probing, demonstrated that various members of the gammaproteobacterial family Methylococcaceae were the key players for methane oxidation, catalysing the first reaction step to methanol. We observed a transfer of carbon to methanol-oxidizing methylotrophs of the betaproteobacterial family Methylophilaceae, suggesting an interaction between methanotrophic and methylotrophic microorganisms that allowed for rapid methane oxidation. From our microcosms, we estimated methane oxidation rates of up to 871 nmol of methane per gram sediment per day. This implies that more than 50% of methane at the seep is removed by microbial oxidation at the sediment-water interface, based on previously reported in situ methane fluxes. The organic carbon produced was further assimilated by different heterotrophic microbes, demonstrating that the methane-oxidizing community supported a complex trophic network. Our results provide valuable eco-physiological insights into this specialized microbial community performing an ecosystem function of global relevance.

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.

Divergent microbial communities in groundwater and overlying soils exhibit functional redundancy for plant-polysaccharide degradation.

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.

Growth promotion and inhibition induced by interactions of groundwater bacteria.

Microorganisms can produce a plethora of secondary metabolites, some acting as signaling compounds and others as suppressing agents. As yet, the potential of groundwater microbes to produce antimicrobial compounds to increase their competitiveness against other bacteria has not been examined. In this study, we developed an AlamarBlue® based high-throughput screening method that allowed for a fast and highly standardized evaluation of both growth-inhibiting and -promoting metabolites. With this technique, 149 screened bacterial isolates were grown in monocultures and in 1402 co-cultures. Co-cultivation did not increase the frequency of growth inhibition against the two tested model organisms (Staphylococcus aureus 533R4 and Escherichia coli WA321) compared to monocultures. Mainly co-cultivation of Proteobacteria induced growth inhibition of both model organisms. Only slightly increased growth promotion of S. aureus 533R4 was observed. Growth-promoting effects on E. coli WA321 were observed by supernatants from co-cultures between Bacteroidetes and Firmicutes. With the standardized screening for both growth-inhibiting and -promoting effects, this method will enable further studies to elaborate and better understand complex inter-specific interactions and networks in aquatic communities as well as in other environments.

Comparative Genomics and Mutational Analysis Reveals a Novel XoxF-Utilizing Methylotroph in the Roseobacter Group Isolated From the Marine Environment.

The Roseobacter group comprises a significant group of marine bacteria which are involved in global carbon and sulfur cycles. Some members are methylotrophs, using one-carbon compounds as a carbon and energy source. It has recently been shown that methylotrophs generally require a rare earth element when using the methanol dehydrogenase enzyme XoxF for growth on methanol. Addition of lanthanum to methanol enrichments of coastal seawater facilitated the isolation of a novel methylotroph in the Roseobacter group: Marinibacterium anthonyi strain La 6. Mutation of xoxF5 revealed the essential nature of this gene during growth on methanol and ethanol. Physiological characterization demonstrated the metabolic versatility of this strain. Genome sequencing revealed that strain La 6 has the largest genome of all Roseobacter group members sequenced to date, at 7.18 Mbp. Multilocus sequence analysis (MLSA) showed that whilst it displays the highest core gene sequence similarity with subgroup 1 of the Roseobacter group, it shares very little of its pangenome, suggesting unique genetic adaptations. This research revealed that the addition of lanthanides to isolation procedures was key to cultivating novel XoxF-utilizing methylotrophs from the marine environment, whilst genome sequencing and MLSA provided insights into their potential genetic adaptations and relationship to the wider community.