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The discovery of the Archaea is a major scientific hallmark of the twentieth century. Since then, important features of their cell biology, physiology, ecology, and diversity have been revealed. Over the course of some 40 years, the diversity of known archaea has expanded from 2 to about 30 phyla comprising over 20,000 species. Most of this archaeal diversity has been revealed by environmental 16S rRNA gene amplicon sequencing surveys using a broad range of universal and targeted primers. Of the few primers that target a large fraction of known archaeal diversity, all display a bias against recently discovered lineages, which limits studies aiming to survey overall archaeal diversity. Induced by genomic exploration of archaeal diversity, and improved phylogenomics approaches, archaeal taxonomic classification has been frequently revised. Due to computational limitations and continued discovery of new lineages, a stable archaeal phylogeny is not yet within reach. Obtaining phylogenetic and taxonomic consensus of archaea should be a high priority for the archaeal research community.

Despite the surge of microbial genome data, experimental testing is important to confirm inferences about the cell biology, ecological roles and evolution of microorganisms. As the majority of archaeal and bacterial diversity remains uncultured and poorly characterized, culturing is a priority. The growing interest in and need for efficient cultivation strategies has led to many rapid methodological and technological advances. In this Review, we discuss common barriers that can hamper the isolation and culturing of novel microorganisms and review emerging, innovative methods for targeted or high-throughput cultivation. We also highlight recent examples of successful cultivation of novel archaea and bacteria, and suggest key microorganisms for future cultivation attempts.

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. Electronic supplementary material The online version of this article (10.1007/s00248-020-01565-y) contains supplementary material, which is available to authorized users.

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.

Effective mentoring implies a two-way relationship in which mentees obtain benefits from the knowledge and training provided by mentors, and mentors gain the possibility of contemplating and learning new perspectives, of self-evaluating their roles and, in consequence, growing as professionals. Mentorship relationships cannot be separated from cultural and societal backgrounds. Thus, they often reflect systemic biases requiring active effort to counteract institutional inequities. Such efforts, particularly when formalized as programs, expand training opportunities for both mentors and mentees. Mentorship networks, in which multiple mentor-mentee relationships are involved, therefore increase collective performance by magnifying resources. Importantly, mentorship exceeds the relationships of students and their direct supervisors (often reflected in co-authorship in publications), and in fact, mentors are often purposely picked outside the direct publication network. A recent large data analysis by AlShebli et al. (2020) showed results of presumed “mentor-protege” relationships after mining millions of coauthor pairs in publications over time, suggesting gender-insensitive changes in institutional mentorship policies based on value-skewed academic success. Because mentorship engages a broader sense of community in academia, mentorship outcomes cannot be quantified solely by the impact of publications.

Despite the surge of microbial genome data, experimental testing is important to confirm inferences about the cell biology, ecological roles and evolution of microorganisms. As the majority of archaeal and bacterial diversity remains uncultured and poorly characterized, culturing is a priority. The growing interest in and need for efficient cultivation strategies has led to many rapid methodological and technological advances. In this Review, we discuss common barriers that can hamper the isolation and culturing of novel microorganisms and review emerging, innovative methods for targeted or high-throughput cultivation. We also highlight recent examples of successful cultivation of novel archaea and bacteria, and suggest key microorganisms for future cultivation attempts.

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.

Assembling microbial and phage genomes from metagenomes is a powerful and appealing method to understand structure-function relationships in complex environments. In order to compare the recovery of genomes from microorganisms and their phages from groundwater, we generated shotgun metagenomes with Illumina sequencing accompanied by long reads derived from the Oxford Nanopore sequencing platform. Assembly and metagenome-assembled genome (MAG) metrics for both microbes and viruses were determined from Illumina-only assemblies and a hybrid assembly approach. Strikingly, the hybrid approach more than doubled the number of mid to high-quality MAGs (> 50% completion, < 10% redundancy), generated nearly four-fold more phage genomes, and improved all associated genome metrics relative to the Illumina only method. The hybrid assemblies yielded MAGs that were on average 7.8% more complete, with 133 fewer contigs and a 14 kbp greater N50. Furthermore, the longer contigs from the hybrid approach generated microbial MAGs that had a higher proportion of rRNA genes. We demonstrate this usefulness by linking microbial MAGs containing 16S rRNA genes with extensive amplicon dataset. This work provides quantitative data to inform a cost-benefit analysis on the decision to supplement shotgun metagenomic projects with long reads towards the goal of recovering genomes from environmentally abundant groups.

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.

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.

Groundwater ecosystems face the challenge of energy limitation due to the absence of light-driven primary production. Lack of space and low oxygen availability might further contribute to generally assumed low food web complexity. Chemolithoautotrophy provides additional input of carbon within the subsurface, however, we still do not understand how abundances of chemolithoautotrophs, differences in surface carbon input, and oxygen availability control subsurface food web complexity. Using a molecular approach, we aimed to disentangle the different levels of potential trophic interactions in oligotrophic groundwater along a hillslope setting of alternating mixed carbonate-/siliciclastic bedrock with contrasting hydrochemical conditions and hotspots of chemolithoautotrophy. Across all sites, groundwater harbored diverse protist communities including Ciliophora, Cercozoa, Centroheliozoa, and Amoebozoa but correlations with hydrochemical parameters were less pronounced for eukaryotes compared to bacteria. Ciliophora-affiliated reads dominated the eukaryotic data sets across all sites. DNA-based evidence for the presence of metazoan top predators such as Cyclopoida (Arthropoda) and Stenostomidae (Platyhelminthes) was only found at wells where abundances of functional genes associated with chemolithoautotrophy were 10–100 times higher compared to wells without indications of these top predators. At wells closer to recharge areas with presumably increased inputs of soil-derived substances and biota, fungi accounted for up to 85% of the metazoan-curated eukaryotic sequence data, together with a low potential for chemolithoautotrophy. Although we did not directly observe higher organisms, our results point to the existence of complex food webs with several trophic levels in oligotrophic groundwater. Chemolithoautotrophy appears to provide strong support to more complex trophic interactions, feeding in additional biomass produced by light-independent CO2-fixation.

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.

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.