Raman-Activated, Interactive Sorting of Isotope-Labeled Bacteria.

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.

Redefining the phylogenetic and metabolic diversity of phylum Omnitrophota.

The candidate phylum Omnitrophica-recently termed Omnitrophota, and originally known as OP3-is an understudied bacterial clade that has primarily been found in aquatic ecosystems. To characterize the diversity and ecology of this phylum, we reconstructed 55 Omnitrophota metagenome-assembled genomes (MAGs) from a well-characterized groundwater system within central Germany and placed them within the context of publicly available genomes. Seven clades were identified, four of which contained novel genomes obtained from our groundwater system. All clades exhibited the capacity for type IV pili, type II secretion systems, glycogen storage, and carbohydrate degradation. Only the characterized Cand. Omnitrophus magneticus genome exhibited functions associated with magnetosome construction. Clades were characterized by sets of traits rather than unique pathways, which were then used to infer ecological strategies. These lifestyles consisted of mixotrophs, obligate fermenters, and versatile respiratory heterotrophs. Patterns in 16S rRNA gene amplicons from a 6 years, monthly sampled groundwater time-series dataset reflected the persistent and widespread occurrence of Clade 7 Wood-Ljungdahl utilizing mixotrophs and highlight this group as a core member of the groundwater community. Overall, this study uncovered, characterized, and contextualized the metabolic and phylogenetic diversity within phylum Omnitrophota, and predicts that environmental populations may mediate both nitrogen and sulfur cycling, along with organic matter production and degradation within aquatic 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.

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.

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.

Extracellular and Intracellular Lanthanide Accumulation in the Methylotrophic Beijerinckiaceae Bacterium RH AL1.

Recent work with Methylorubrum extorquens AM1 identified intracellular, cytoplasmic lanthanide storage in an organism that harnesses these metals for its metabolism. Here, we describe the extracellular and intracellular accumulation of lanthanides in the Beijerinckiaceae bacterium RH AL1, a newly isolated and recently characterized methylotroph. Using ultrathin-section transmission electron microscopy (TEM), freeze fracture TEM (FFTEM), and energy-dispersive X-ray spectroscopy, we demonstrated that strain RH AL1 accumulates lanthanides extracellularly at outer membrane vesicles (OMVs) and stores them in the periplasm. High-resolution elemental analyses of biomass samples revealed that strain RH AL1 can accumulate ions of different lanthanide species, with a preference for heavier lanthanides. Its methanol oxidation machinery is supposedly adapted to light lanthanides, and their selective uptake is mediated by dedicated uptake mechanisms. Based on transcriptome sequencing (RNA-seq) analysis, these presumably include the previously characterized TonB-ABC transport system encoded by the lut cluster but potentially also a type VI secretion system. A high level of constitutive expression of genes coding for lanthanide-dependent enzymes suggested that strain RH AL1 maintains a stable transcript pool to flexibly respond to changing lanthanide availability. Genes coding for lanthanide-dependent enzymes are broadly distributed taxonomically. Our results support the hypothesis that central aspects of lanthanide-dependent metabolism partially differ between the various taxa. IMPORTANCE Although multiple pieces of evidence have been added to the puzzle of lanthanide-dependent metabolism, we are still far from understanding the physiological role of lanthanides. Given how widespread lanthanide-dependent enzymes are, only limited information is available with respect to how lanthanides are taken up and stored in an organism. Our research complements work with commonly studied model organisms and showed the localized storage of lanthanides in the periplasm. This storage occurred at comparably low concentrations. Strain RH AL1 is able to accumulate lanthanide ions extracellularly and to selectively utilize lighter lanthanides. The Beijerinckiaceae bacterium RH AL1 might be an attractive target for developing biorecovery strategies to obtain these economically highly demanded metals in environmentally friendly ways.

The multidimensionality of soil macroecology.

The recent past has seen a tremendous surge in soil macroecological studies and new insights into the global drivers of one-quarter of the biodiversity of the Earth. Building on these important developments, a recent paper in Global Ecology and Biogeography outlined promising methods and approaches to advance soil macroecology. Among other recommendations, White and colleagues introduced the concept of a spatial three-dimensionality in soil macroecology by considering the different spheres of influence and scales, as soil organism size ranges vary from bacteria to macro- and megafauna. Here, we extend this concept by discussing three additional dimensions (biological, physical, and societal) that are crucial to steer soil macroecology from pattern description towards better mechanistic understanding. In our view, these are the requirements to establish it as a predictive science that can inform policy about relevant nature and management conservation actions. We highlight the need to explore temporal dynamics of soil biodiversity and functions across multiple temporal scales, integrating different facets of biodiversity (i.e., variability in body size, life-history traits, species identities, and groups of taxa) and their relationships to multiple ecosystem functions, in addition to the feedback effects between humans and soil biodiversity. We also argue that future research needs to consider effective soil conservation policy and management in combination with higher awareness of the contributions of soil-based nature's contributions to people. To verify causal relationships, soil macroecology should be paired with local and globally distributed experiments. The present paper expands the multidimensional perspective on soil macroecology to guide future research contents and funding. We recommend considering these multiple dimensions in projected global soil biodiversity monitoring initiatives.

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.

Inclusion of Oxford Nanopore long reads improves all microbial and viral metagenome-assembled genomes from a complex aquifer system.

Assembling microbial and viral genomes from metagenomes is a powerful and appealing method to understand structure-function relationships in complex environments. To compare the recovery of genomes from microorganisms and their viruses from groundwater, we generated shotgun metagenomes with Illumina sequencing accompanied by long reads derived from the Oxford Nanopore Technologies (ONT) sequencing platform. Assembly and metagenome-assembled genome (MAG) metrics for both microbes and viruses were determined from an Illumina-only assembly, ONT-only assembly, and a hybrid assembly approach. The hybrid approach recovered 2× more mid to high-quality MAGs compared to the Illumina-only approach and 4× more than the ONT-only approach. A similar number of viral genomes were reconstructed using the hybrid and ONT methods, and both recovered nearly fourfold more viral genomes than the Illumina-only approach. While yielding fewer MAGs, the ONT-only approach generated MAGs with a high probability of containing rRNA genes, 3× higher than either of the other methods. Of the shared MAGs recovered from each method, the ONT-only approach generated the longest and least fragmented MAGs, while the hybrid approach yielded the most complete. This work provides quantitative data to inform a cost-benefit analysis of the decision to supplement shotgun metagenomic projects with long reads towards the goal of recovering genomes from environmentally abundant groups.

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.

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.

Mixotrophic Iron-Oxidizing Thiomonas Isolates from an Acid Mine Drainage-Affected Creek.

Natural attenuation of heavy metals occurs via coupled microbial iron cycling and metal precipitation in creeks impacted by acid mine drainage (AMD). Here, we describe the isolation, characterization, and genomic sequencing of two iron-oxidizing bacteria (FeOB) species: Thiomonas ferrovorans FB-6 and Thiomonas metallidurans FB-Cd, isolated from slightly acidic (pH 6.3), Fe-rich, AMD-impacted creek sediments. These strains precipitated amorphous iron oxides, lepidocrocite, goethite, and magnetite or maghemite and grew at a pH optimum of 5.5. While Thiomonas spp. are known as mixotrophic sulfur oxidizers and As oxidizers, the FB strains oxidized Fe, which suggests they can efficiently remove Fe and other metals via coprecipitation. Previous evidence for Thiomonas sp. Fe oxidation is largely ambiguous, possibly because of difficulty demonstrating Fe oxidation in heterotrophic/mixotrophic organisms. Therefore, we also conducted a genomic analysis to identify genetic mechanisms of Fe oxidation, other metal transformations, and additional adaptations, comparing the two FB strain genomes with 12 other Thiomonas genomes. The FB strains fall within a relatively novel group of Thiomonas strains that includes another strain (b6) with solid evidence of Fe oxidation. Most Thiomonas isolates, including the FB strains, have the putative iron oxidation gene cyc2, but only the two FB strains possess the putative Fe oxidase genes mtoAB The two FB strain genomes contain the highest numbers of strain-specific gene clusters, greatly increasing the known Thiomonas genetic potential. Our results revealed that the FB strains are two distinct novel species of Thiomonas with the genetic potential for bioremediation of AMD via iron oxidation.IMPORTANCE As AMD moves through the environment, it impacts aquatic ecosystems, but at the same time, these ecosystems can naturally attenuate contaminated waters via acid neutralization and catalyzing metal precipitation. This is the case in the former Ronneburg uranium-mining district, where AMD impacts creek sediments. We isolated and characterized two iron-oxidizing Thiomonas species that are mildly acidophilic to neutrophilic and that have two genetic pathways for iron oxidation. These Thiomonas species are well positioned to naturally attenuate AMD as it discharges across the landscape.

Rhizobactin B is the preferred siderophore by a novel Pseudomonas isolate to obtain iron from dissolved organic matter in peatlands.

Bacteria often release diverse iron-chelating compounds called siderophores to scavenge iron from the environment for many essential biological processes. In peatlands, where the biogeochemical cycle of iron and dissolved organic matter (DOM) are coupled, bacterial iron acquisition can be challenging even at high total iron concentrations. We found that the bacterium Pseudomonas sp. FEN, isolated from an Fe-rich peatland in the Northern Bavarian Fichtelgebirge (Germany), released an unprecedented siderophore for its genus. High-resolution mass spectrometry (HR-MS) using metal isotope-coded profiling (MICP), MS/MS experiments, and nuclear magnetic resonance spectroscopy (NMR) identified the amino polycarboxylic acid rhizobactin and a novel derivative at even higher amounts, which was named rhizobactin B. Interestingly, pyoverdine-like siderophores, typical for this genus, were not detected. With peat water extract (PWE), studies revealed that rhizobactin B could acquire Fe complexed by DOM, potentially through a TonB-dependent transporter, implying a higher Fe binding constant of rhizobactin B than DOM. The further uptake of Fe-rhizobactin B by Pseudomonas sp. FEN suggested its role as a siderophore. Rhizobactin B can complex several other metals, including Al, Cu, Mo, and Zn. The study demonstrates that the utilization of rhizobactin B can increase the Fe availability for Pseudomonas sp. FEN through ligand exchange with Fe-DOM, which has implications for the biogeochemical cycling of Fe in this peatland.

Lanthanide-Dependent Methylotrophs of the Family Beijerinckiaceae: Physiological and Genomic Insights.

Methylotrophic bacteria use methanol and related C1 compounds as carbon and energy sources. Methanol dehydrogenases are essential for methanol oxidation, while lanthanides are important cofactors of many pyrroloquinoline quinone-dependent methanol dehydrogenases and related alcohol dehydrogenases. We describe here the physiological and genomic characterization of newly isolated Beijerinckiaceae bacteria that rely on lanthanides for methanol oxidation. A broad physiological diversity was indicated by the ability to metabolize a wide range of multicarbon substrates, including various sugars, and organic acids, as well as diverse C1 substrates such as methylated amines and methylated sulfur compounds. Methanol oxidation was possible only in the presence of low-mass lanthanides (La, Ce, and Nd) at submicromolar concentrations (>100 nM). In a comparison with other Beijerinckiaceae, genomic and transcriptomic analyses revealed the usage of a glutathione- and tetrahydrofolate-dependent pathway for formaldehyde oxidation and channeling methyl groups into the serine cycle for carbon assimilation. Besides a single xoxF gene, we identified two additional genes for lanthanide-dependent alcohol dehydrogenases, including one coding for an ExaF-type alcohol dehydrogenase, which was so far not known in Beijerinckiaceae Homologs for most of the gene products of the recently postulated gene cluster linked to lanthanide utilization and transport could be detected, but for now it remains unanswered how lanthanides are sensed and taken up by our strains. Studying physiological responses to lanthanides under nonmethylotrophic conditions in these isolates as well as other organisms is necessary to gain a more complete understanding of lanthanide-dependent metabolism as a whole.IMPORTANCE We supplemented knowledge of the broad metabolic diversity of the Beijerinckiaceae by characterizing new members of this family that rely on lanthanides for methanol oxidation and that possess additional lanthanide-dependent enzymes. Considering that lanthanides are critical resources for many modern applications and that recovering them is expensive and puts a heavy burden on the environment, lanthanide-dependent metabolism in microorganisms is an exploding field of research. Further research into how isolated Beijerinckiaceae and other microbes utilize lanthanides is needed to increase our understanding of lanthanide-dependent metabolism. The diversity and widespread occurrence of lanthanide-dependent enzymes make it likely that lanthanide utilization varies in different taxonomic groups and is dependent on the habitat of the microbes.

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.

14C-Free Carbon Is a Major Contributor to Cellular Biomass in Geochemically Distinct Groundwater of Shallow Sedimentary Bedrock Aquifers.

Despite the global significance of the subsurface biosphere, the degree to which it depends on surface organic carbon (OC) is still poorly understood. Here, we compare stable and radiogenic carbon isotope compositions of microbial phospholipid fatty acids (PLFAs) with those of in situ potential microbial C sources to assess the major C sources for subsurface microorganisms in biogeochemical distinct shallow aquifers (Critical Zone Exploratory, Thuringia Germany). Despite the presence of younger OC, the microbes assimilated 14C-free OC to varying degrees; ~31% in groundwater within the oxic zone, ~47% in an iron reduction zone, and ~70% in a sulfate reduction/anammox zone. The persistence of trace amounts of mature and partially biodegraded hydrocarbons suggested that autochthonous petroleum-derived hydrocarbons were a potential 14C-free C source for heterotrophs in the oxic zone. In this zone, Δ14C values of dissolved inorganic carbon (-366 ± 18‰) and 11MeC16:0 (-283 ± 32‰), an important component in autotrophic nitrite oxidizers, were similar enough to indicate that autotrophy is an important additional C fixation pathway. In anoxic zones, methane as an important C source was unlikely since the 13C-fractionations between the PLFAs and CH4 were inconsistent with kinetic isotope effects associated with methanotrophy. In the sulfate reduction/anammox zone, the strong 14C-depletion of 10MeC16:0 (-942 ± 22‰), a PLFA common in sulfate reducers, indicated that those bacteria were likely to play a critical part in 14C-free sedimentary OC cycling. Results indicated that the 14C-content of microbial biomass in shallow sedimentary aquifers results from complex interactions between abundance and bioavailability of naturally occurring OC, hydrogeology, and specific microbial metabolisms.

Tracking active groundwater microbes with D2 O labelling to understand their ecosystem function.

Microbial activity is key in understanding the contribution of microbial communities to ecosystem functions. Metabolic labelling with heavy water (D2 O) leads to the formation of carbon-deuterium bonds in active microorganisms. We illustrated how D2 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 D2 O were labelled. Next, we conducted a labelling approach with the total groundwater microbiome in D2 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.

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.

Plants, microorganisms, and soil temperatures contribute to a decrease in methane fluxes on a drained Arctic floodplain.

As surface temperatures are expected to rise in the future, ice-rich permafrost may thaw, altering soil topography and hydrology and creating a mosaic of wet and dry soil surfaces in the Arctic. Arctic wetlands are large sources of CH4 , and investigating effects of soil hydrology on CH4 fluxes is of great importance for predicting ecosystem feedback in response to climate change. In this study, we investigate how a decade-long drying manipulation on an Arctic floodplain influences CH4 -associated microorganisms, soil thermal regimes, and plant communities. Moreover, we examine how these drainage-induced changes may then modify CH4 fluxes in the growing and nongrowing seasons. This study shows that drainage substantially lowered the abundance of methanogens along with methanotrophic bacteria, which may have reduced CH4 cycling. Soil temperatures of the drained areas were lower in deep, anoxic soil layers (below 30 cm), but higher in oxic topsoil layers (0-15 cm) compared to the control wet areas. This pattern of soil temperatures may have reduced the rates of methanogenesis while elevating those of CH4 oxidation, thereby decreasing net CH4 fluxes. The abundance of Eriophorum angustifolium, an aerenchymatous plant species, diminished significantly in the drained areas. Due to this decrease, a higher fraction of CH4 was alternatively emitted to the atmosphere by diffusion, possibly increasing the potential for CH4 oxidation and leading to a decrease in net CH4 fluxes compared to a control site. Drainage lowered CH4 fluxes by a factor of 20 during the growing season, with postdrainage changes in microbial communities, soil temperatures, and plant communities also contributing to this reduction. In contrast, we observed CH4 emissions increased by 10% in the drained areas during the nongrowing season, although this difference was insignificant given the small magnitudes of fluxes. This study showed that long-term drainage considerably reduced CH4 fluxes through modified ecosystem properties.

Schwertmannite formation at cell junctions by a new filament-forming Fe(II)-oxidizing isolate affiliated with the novel genus Acidithrix.

A new acidophilic iron-oxidizing strain (C25) belonging to the novel genus Acidithrix was isolated from pelagic iron-rich aggregates ('iron snow') collected below the redoxcline of an acidic lignite mine lake. Strain C25 catalysed the oxidation of ferrous iron [Fe(II)] under oxic conditions at 25 °C at a rate of 3.8 mM Fe(II) day(-1) in synthetic medium and 3.0 mM Fe(II) day(-1) in sterilized lake water in the presence of yeast extract, producing the rust-coloured, poorly crystalline mineral schwertmannite [Fe(III) oxyhydroxylsulfate]. During growth, rod-shaped cells of strain C25 formed long filaments, and then aggregated and degraded into shorter fragments, building large cell-mineral aggregates in the late stationary phase. Scanning electron microscopy analysis of cells during the early growth phase revealed that Fe(III)-minerals were formed as single needles on the cell surface, whereas the typical pincushion-like schwertmannite was observed during later growth phases at junctions between the cells, leaving major parts of the cell not encrusted. This directed mechanism of biomineralization at specific locations on the cell surface has not been reported from other acidophilic iron-oxidizing bacteria. Strain C25 was also capable of reducing Fe(III) under micro-oxic conditions which led to a dissolution of the Fe(III)-minerals. Thus, strain C25 appeared to have ecological relevance for both the formation and transformation of the pelagic iron-rich aggregates at oxic/anoxic transition zones in the acidic lignite mine lake.