Microbial Dark Matter Research Articles

High-throughput single-cell sequencing of activated sludge microbiome

Wastewater treatment plants (WWTPs) represent one of biotechnology's largest and most critical applications, playing a pivotal role in environmental protection and public health. In WWTPs, activated sludge (AS) plays a major role in removing contaminants and pathogens from wastewater. While metagenomics has advanced our understanding of microbial communities, it still faces challenges in revealing the genomic heterogeneity of cells, uncovering the microbial dark matter, and establishing precise links between genetic elements and their host cells as a bulk method. These issues could be largely resolved by single-cell sequencing, which can offer unprecedented resolution to show the unique genetic information. Here we show the high-throughput single-cell sequencing to the AS microbiome. The single-amplified genomes (SAGs) of 15,110 individual cells were clustered into 2,454 SAG bins. We find that 27.5% of the genomes in the AS microbial community represent potential novel species, highlighting the presence of microbial dark matter. Furthermore, we identified 1,137 antibiotic resistance genes (ARGs), 10,450 plasmid fragments, and 1,343 phage contigs, with shared plasmid and phage groups broadly distributed among hosts, indicating a high frequency of horizontal gene transfer (HGT) within the AS microbiome. Complementary analysis using 1,529 metagenome-assembled genomes from the AS samples allowed for the taxonomic classification of 98 SAG bins, which were previously unclassified. Our study establishes the feasibility of single-cell sequencing in characterizing the AS microbiome, providing novel insights into its ecological dynamics, and deepening our understanding of HGT processes, particularly those involving ARGs. Additionally, this valuable tool could monitor the distribution, spread, and pathogenic hosts of ARGs both within AS environments and between AS and other environments, which will ultimately contribute to developing a health risk evaluation system for diverse environments within a One Health framework.

Multi-omics insights into the function and evolution of sodium benzoate biodegradation pathway in Benzoatithermus flavus gen. nov., sp. nov. from hot spring

Biodegradation stands as an eco-friendly and effective approach for organic contaminant remediation. However, research on microorganisms degrading sodium benzoate contaminants in extreme environments remains limited. In this study, we report to display the isolation of a novel hot spring enriched cultures with sodium benzoate (400 mg/L) as the sole carbon source. The results revealed that the phylum Pseudomonadota was the potential sodium benzoate degrader and a novel genus within the family Geminicoccaceae of this phylum. The isolated strain was named Benzoatithermus flavus SYSU G07066T and was isolated from HNT-2 hot spring samples. Genomic analysis revealed that SYSU G07066T carried benABC genes and physiological experiments indicated the ability to utilize sodium benzoate as a sole carbon source for growth, which was further confirmed by transcriptomic data with expression of benABC. Phylogenetic analysis suggested that Horizontal Gene Transfer (HGT) plays a significant role in acquiring sodium benzoate degradation capability among prokaryotes, and SYSU G07066T might have acquired benABC genes through HGT from the family Acetobacteraceae. The discovery of the first microorganism with sodium benzoate degradation function from a hot spring enhances our understanding of the diverse functions within the family Geminicoccaceae. This study unearths the first novel genus capable of efficiently degrading sodium benzoate and its evolution history at high temperatures, holding promising industrial applications, and provides a new perspective for further exploring the application potential of hot spring “microbial dark matter”.

Decipher syntrophies within C2-C4 organic acids-degrading anaerobic microbiomes: A multi-omic exploration

In methanogenic ecosystems, carboxylic acid degradation is a crucial process facilitated by specialized bacteria working in syntrophy with methanogens. This study explores the transcriptomic responses in 178 metagenome-assembled genomes upon changes in feeding composition, specifically lactate, butyrate, propionate, and acetate, in four lab-scale bioreactors. Methanothrix soehngenii and Methanoculleus sp. emerged as key biomethanation contributors. Through metatranscriptomics, machine learning-based functional annotation, and flux balance analysis the underlying microbial interactions and dynamics were deciphered. Syntrophomonadaceae and Mesotoga species maintained mutualistic metabolite exchanges with hydrogenotrophic methanogens, degrading primarily butyrate and lactate, respectively. Acetate was mainly consumed by Smithellaceae sp., in competition with M. soehngenii in all reactors. Furthermore, the acetoclastic archaeon exhibited a previously undocumented capability to metabolize lactate, thereby confirming the prevalence of acetotrophic pathway. Transcriptomic profiles revealed an additional layer of complexity, where propionate dismutation and beta-oxidation pathways are interconnected by the exchanges of butyrate between putative syntrophs, including Syntrophomonadaceae and Smithellaceae species. Integrating multi-omics data with genome-scale metabolic modeling enabled the accurate reconstruction of dynamics within the controlled ecosystem. This composite novel approach was applied to the AD system to unveil the intricate relationships operating within the microbiome to promote thriving. Results elucidated the still poorly explored organization of the so-called microbial dark matter.

Exploring nature's filters: Peat-mineral mix for low and high-strength oilfield produced water reclamation

Nature-based solutions are encouraged for treating oilfield produced water from oil and gas extraction, a crucial undertaking that aligns with the Canadian oil sands industry's ambitious goal of zero waste, and the globally recognized Sustainable Development Goals (SDGs) pertaining to water conservation and ecosystem preservation. This study explored the use of peat-mineral mix (PMM), a leftover of inevitable oil sands mining, for treating low and high-strength wastewaters during biofiltration, which contained large molecular weight (44.3 kDa), which include alcohols, aliphatics, aromatics, and ketones, and can impart high toxicity to both fauna and flora (MicroTox: 99 %). The breakthrough curve indicated an effective initial adsorption phase driven by advection within the column dynamics. For complete organics removal and mechanistic insights, the wastewater was re-circulated in a continuous mode for up to 42 days. Here, we found that chemical oxygen demand was reduced from ∼85,000 mg/L to ∼965 mg/L). Kinetics investigations along with physicochemical characterization of PMM and wastewater suggested that chemisorption and anaerobic digestion contributed to the overall removal of contaminants. Chemisorption, led by hydrogen bonding and hydrophobic interactions, was the dominant mechanism, with a limited contribution from physical adsorption (surface area: 2.85 m2/g). The microbial community within the PMM bed was rich/diverse (Shannon > 6.0; Chao1 > 600), with ∼ 50 % unclassified phylotypes representing ‘microbial dark matter’. High electric conductivity (332.1 μS cm−1) of PMM and the presence of Geobacter, syntrophs, and Methanosaeta suggest that direct interspecies electron transfer was likely occurring during anaerobic digestion. Both low and high-strength wastewaters showed effective removal of dissolved organics (e.g., naphthenic acids, acid extractable fraction, oil and grease content), nutrients, and potentially toxic metals. The successful use of PMM in treating oilfield produced water offers promising avenues for embracing nature-based remediation solutions at oil refining sites.

Effects of operational parameters on bacterial communities in Hong Kong and global wastewater treatment plants.

Wastewater treatment plants (WWTPs) are an indispensable component of modern cities, as they can remove pollutants in wastewater to prevent anthropogenic activities. Activated sludge (AS) is a fundamental wastewater treatment process and it harbors a highly complex microbial community that forms the main components and contains functional groups. Unveiling "who is there" is a long-term goal of the research on AS microbiology. High-throughput sequencing provides insights into the inventory diversity of microbial communities to an unprecedented level of detail. At present, the analysis of communities in WWTPs usually comes from a specific WWTP and lacks comparisons and verification among different WWTPs. The wide-scale and long-term sampling project and research in this study could help us evaluate the AS community more accurately to find the similarities and different results for different WWTPs in Hong Kong and other regions of the world.

Towards high-throughput parallel imaging and single-cell transcriptomics of microbial eukaryotic plankton.

Single-cell transcriptomics has the potential to provide novel insights into poorly studied microbial eukaryotes. Although several such technologies are available and benchmarked on mammalian cells, few have been tested on protists. Here, we applied a microarray single-cell sequencing (MASC-seq) technology, that generates microscope images of cells in parallel with capturing their transcriptomes, on three species representing important plankton groups with different cell structures; the ciliate Tetrahymena thermophila, the diatom Phaeodactylum tricornutum, and the dinoflagellate Heterocapsa sp. Both the cell fixation and permeabilization steps were adjusted. For the ciliate and dinoflagellate, the number of transcripts of microarray spots with single cells were significantly higher than for background spots, and the overall expression patterns were correlated with that of bulk RNA, while for the much smaller diatom cells, it was not possible to separate single-cell transcripts from background. The MASC-seq method holds promise for investigating "microbial dark matter", although further optimizations are necessary to increase the signal-to-noise ratio.

Metabolic profiling of petroleum-degrading microbial communities incubated under high-pressure conditions.

While pressure is a significant characteristic of petroleum reservoirs, it is often overlooked in laboratory studies. To clarify the composition and metabolic properties of microbial communities under high-pressure conditions, we established methanogenic and sulfate-reducing enrichment cultures under high-pressure conditions using production water from the Jilin Oilfield in China. We utilized a metagenomics approach to analyze the microbial community after a 90-day incubation period. Under methanogenic conditions, Firmicutes, Deferribacteres, Ignavibacteriae, Thermotogae, and Nitrospirae, in association with the hydrogenotrophic methanogen Archaeoglobaceae and acetoclastic Methanosaeta, were highly represented. Genomes for Ca. Odinarchaeota and the hydrogen-dependent methylotrophic Ca. Methanosuratus were also recovered from the methanogenic culture. The sulfate-reducing community was dominated by Firmicutes, Thermotogae, Nitrospirae, Archaeoglobus, and several candidate taxa including Ca. Bipolaricaulota, Ca. Aminicenantes, and Candidate division WOR-3. These candidate taxa were key pantothenate producers for other community members. The study expands present knowledge of the metabolic roles of petroleum-degrading microbial communities under high-pressure conditions. Our results also indicate that microbial community interactions were shaped by syntrophic metabolism and the exchange of amino acids and cofactors among members. Furthermore, incubation under in situ pressure conditions has the potential to reveal the roles of microbial dark matter.

A sequential one-pot approach for rapid and convenient characterization of putative restriction-modification systems.

The elucidation of the molecular basis of virus-host coevolutionary interactions is boosted with state-of-the-art sequencing technologies. However, the sequence-only information is often insufficient to output a conclusive argument without biochemical characterizations. We proposed a 1-day and one-pot approach to confirm the exact function of putative restriction-modification (R-M) genes that presumably mediate microbial coevolution. The experiments mainly focused on a series of putative R-M enzymes from a deep-sea virus and its host bacterium. The results quickly unveiled unambiguous substrate specificities, superior catalytic performance, and unique sequence preferences for two new restriction enzymes (capable of cleaving DNA) and two new methyltransferases (capable of modifying DNA with methyl groups). The reality of the functional R-M system reinforced a model of mutually beneficial interactions with the virus in the deep-sea microbial ecosystem. The cell culture-independent approach also holds great potential for exploring novel and biotechnologically significant R-M enzymes from microbial dark matter.

In situ devices can culture the microbial dark matter of corals

Most microorganisms found in environmental samples have never been cultured and can often only be explored through molecular or microscopic approaches. Here, we adapt the use of in situ diffusion-based devices to culture "yet-to-be-cultured" microorganisms associated with coral mucus and compare this with a traditional culturing method. The culturability of microorganisms associated with mucus of the coral Pocillopora damicornis increased by 420% and 570% with diffusion growth chambers and microwell chip devices, respectively, compared with the traditional method tested. The obtained cultures represent up to 64.4% of the total diversity of amplicon sequence variants (ASVs) found in the mucus of the coral P.damicornis. In addition, some previously uncultured microorganisms, such as members of the family Nitrosopumilaceae and halophilic/halotolerant bacteria were cultured. Our results validate alternative microbialculturing strategies to culture coral-associated microorganisms, while significantly increasing the culturability of previous microbial dark matter.

A genomic catalogue of soil microbiomes boosts mining of biodiversity and genetic resources

Soil harbors a vast expanse of unidentified microbes, termed as microbial dark matter, presenting an untapped reservoir of microbial biodiversity and genetic resources, but has yet to be fully explored. In this study, we conduct a large-scale excavation of soil microbial dark matter by reconstructing 40,039 metagenome-assembled genome bins (the SMAG catalogue) from 3304 soil metagenomes. We identify 16,530 of 21,077 species-level genome bins (SGBs) as unknown SGBs (uSGBs), which expand archaeal and bacterial diversity across the tree of life. We also illustrate the pivotal role of uSGBs in augmenting soil microbiome’s functional landscape and intra-species genome diversity, providing large proportions of the 43,169 biosynthetic gene clusters and 8545 CRISPR-Cas genes. Additionally, we determine that uSGBs contributed 84.6% of previously unexplored viral-host associations from the SMAG catalogue. The SMAG catalogue provides an useful genomic resource for further studies investigating soil microbial biodiversity and genetic resources.

Microbial dark matter sequences verification in amplicon sequencing and environmental metagenomics data.

Although microorganisms constitute the most diverse and abundant life form on Earth, in many environments, the vast majority of them remain uncultured. As it is based on information gleaned mainly from cultivated microorganisms, our current body of knowledge regarding microbial life is partial and does not reflect actual microbial diversity. That diversity is hidden in the uncultured microbial majority, termed by microbiologists as "microbial dark matter" (MDM), a term borrowed from astrophysics. Metagenomic sequencing analysis techniques (both 16S rRNA gene and shotgun sequencing) compare gene sequences to reference databases, each of which represents only a small fraction of the existing microorganisms. Unaligned sequences lead to groups of "unknown microorganisms" that are usually ignored and rarefied from diversity analysis. To address this knowledge gap, we analyzed the 16S rRNA gene sequences of microbial communities from four different environments-a living organism, a desert environment, a natural aquatic environment, and a membrane bioreactor for wastewater treatment. From those datasets, we chose representative sequences of potentially unknown bacteria for additional examination as "microbial dark matter sequences" (MDMS). Sequence existence was validated by specific amplification and re-sequencing. These sequences were screened against databases and aligned to the Genome Taxonomy Database to build a comprehensive phylogenetic tree for additional sequence classification, revealing potentially new candidate phyla and other lineages. These putative MDMS were also screened against metagenome-assembled genomes from the explored environments for additional validation and for taxonomic and metabolic characterizations. This study shows the immense importance of MDMS in environmental metataxonomic analyses of 16S rRNA gene sequences and provides a simple and readily available methodology for the examination of MDM hidden behind amplicon sequencing results.

Unraveling the functional dark matter through global metagenomics.

Metagenomes encode an enormous diversity of proteins, reflecting a multiplicity of functions and activities1,2. Exploration of this vast sequence space has been limited to a comparative analysis against reference microbial genomes and protein families derived from those genomes. Here, to examine the scale of yet untapped functional diversity beyond what is currently possible through the lens of reference genomes, we develop a computational approach to generate reference-free protein families from the sequence space in metagenomes. We analyse 26,931 metagenomes and identify 1.17 billion protein sequences longer than 35 amino acids with no similarity to any sequences from 102,491 reference genomes or the Pfam database3. Using massively parallel graph-based clustering, we group these proteins into 106,198 novel sequence clusters with more than 100 members, doubling the number of protein families obtained from the reference genomes clustered using the same approach. We annotate these families on the basis of their taxonomic, habitat, geographical and gene neighbourhood distributions and, where sufficient sequence diversity is available, predict protein three-dimensional models, revealing novel structures. Overall, our results uncover an enormously diverse functional space, highlighting the importance of further exploring the microbial functional dark matter.

Capturing the microbial dark matter in desert soils using culturomics-based metagenomics and high-resolution analysis

Deserts occupy one-third of the Earth’s terrestrial surface and represent a potentially significant reservoir of microbial biodiversity, yet the majority of desert microorganisms remain uncharacterized and are seen as “microbial dark matter”. Here, we introduce a multi-omics strategy, culturomics-based metagenomics (CBM) that integrates large-scale cultivation, full-length 16S rRNA gene amplicon, and shotgun metagenomic sequencing. The results showed that CBM captured a significant amount of taxonomic and functional diversity missed in direct sequencing by increasing the recovery of amplicon sequence variants (ASVs) and high/medium-quality metagenome-assembled genomes (MAGs). Importantly, CBM allowed the post hoc recovery of microbes of interest (e.g., novel or specific taxa), even those with extremely low abundance in the culture. Furthermore, strain-level analyses based on CBM and direct sequencing revealed that the desert soils harbored a considerable number of novel bacterial candidates (1941, 51.4%), of which 1095 (from CBM) were culturable. However, CBM would not exactly reflect the relative abundance of true microbial composition and functional pathways in the in situ environment, and its use coupled with direct metagenomic sequencing could provide greater insight into desert microbiomes. Overall, this study exemplifies the CBM strategy with high-resolution is an ideal way to deeply explore the untapped novel bacterial resources in desert soils, and substantially expands our knowledge on the microbial dark matter hidden in the vast expanse of deserts.

Metagenomic analysis unveils the underexplored roles of prokaryotic viruses in a full-scale landfill leachate treatment plant

Enormous viral populations have been identified in activated sludge systems, but their ecological and biochemical roles in landfill leachate treatment plants remain poorly understood. To address this knowledge gap, we conducted an in-depth analysis using 36 metagenomic datasets that we collected and sequenced during a half-year time-series sampling campaign at six sites in a full-scale landfill leachate treatment plant (LLTP), elucidating viral distribution, virus‒host dynamics, virus-encoded auxiliary metabolic genes (AMGs), and viral contributions to the spread of virulence and antibiotic resistance genes. Our findings demonstrated that viral and prokaryotic communities differed widely among different treatment units, with stability over time. LLTP viruses were linked to various prokaryotic hosts, spanning 35 bacterial phyla and one archaeal phylum, which included the core microbes involved in biological treatments, as well as some of the less well-characterized microbial dark matter phyla. By encoding 2364 auxiliary metabolic genes (AMGs), viruses harbored the potential to regulate microbial nucleotide metabolism, facilitate the biodegradation of complex organic matter, and enhance flocculation and settling in biological treatment plants. The abundance distribution of AMGs varied considerably across treatment units and showed a lifestyle-dependent pattern with temperate virus-associated AMGs exhibiting a higher average abundance in downstream biological treatment units and effluent water. Meanwhile, temperate viruses tended to carry a higher load of virulence factor genes (VFGs), antibiotic resistance genes (ARGs), and biotic and metal resistance genes (BMRGs), and engaged in more frequent gene exchanges with prokaryotes than lytic viruses, thus acting as a pivotal contributor to the dissemination of pathogenicity and resistance genes in downstream LLTP units. This study provided a comprehensive profile of viral and prokaryotic communities in the LLTP and unveiled the varying roles of different-lifestyle viruses in biochemical processes and water quality safety.

Genetic manipulation of Patescibacteria provides mechanistic insights into microbial dark matter and the epibiotic lifestyle

Patescibacteria, also known as the candidate phyla radiation (CPR), are a diverse group of bacteria that constitute a disproportionately large fraction of microbial dark matter. Its few cultivated members, belonging mostly to Saccharibacteria, grow as epibionts on host Actinobacteria. Due to a lack of suitable tools, the genetic basis of this lifestyle and other unique features of Patescibacteira remain unexplored. Here, we show that Saccharibacteria exhibit natural competence, and we exploit this property for their genetic manipulation. Imaging of fluorescent protein-labeled Saccharibacteria provides high spatiotemporal resolution of phenomena accompanying epibiotic growth, and a transposon-insertion sequencing (Tn-seq) genome-wide screen reveals the contribution of enigmatic Saccharibacterial genes to growth on their hosts. Finally, we leverage metagenomic data to provide cutting-edge protein structure-based bioinformatic resources that support the strain Southlakia epibionticum and its corresponding host, Actinomyces israelii, as a model system for unlocking the molecular underpinnings of the epibiotic lifestyle.

Integration of multi-omics data to elucidate keystone unknown taxa within microbialite-forming ecosystems.

Microbes continually shape Earth's biochemical and physical landscapes by inhabiting diverse metabolic niches. Despite the important role microbes play in ecosystem functioning, most microbial species remain unknown highlighting a gap in our understanding of structured complex ecosystems. To elucidate the relevance of these unknown taxa, often referred to as "microbial dark matter," the integration of multiple high throughput sequencing technologies was used to evaluate the co-occurrence and connectivity of all microbes within the community. Since there are no standard methodologies for multi-omics integration of microbiome data, we evaluated the abundance of "microbial dark matter" in microbialite-forming communities using different types meta-omic datasets: amplicon, metagenomic, and metatranscriptomic sequencing previously generated for this ecosystem. Our goal was to compare the community structure and abundances of unknown taxa within the different data types rather than to perform a functional characterization of the data. Metagenomic and metatranscriptomic data were input into SortMeRNA to extract 16S rRNA gene reads. The output, as well as amplicon sequences, were processed through QIIME2 for taxonomy analysis. The R package mdmnets was utilized to build co-occurrence networks. Most hubs presented unknown classifications, even at the phyla level. Comparisons of the highest scoring hubs of each data type using sequence similarity networks allowed the identification of the most relevant hubs within the microbialite-forming communities. This work highlights the importance of unknown taxa in community structure and proposes that ecosystem network construction can be used on several types of data to identify keystone taxa and their potential function within microbial ecosystems.

Influence of soil nutrients on the presence and distribution of CPR bacteria in a long-term crop rotation experiment.

Bacteria affiliated with the Candidate Phyla Radiation (CPR) are a hyper-diverse group of ultra-small bacteria with versatile yet sparse metabolisms. However, most insights into this group come from a surprisingly small number of environments, and recovery of CPR bacteria from soils has been hindered due to their extremely low abundance within complex microbial assemblages. In this study we enriched soil samples from 14 different soil fertility treatments for ultra-small (<0.45 μm) bacteria in order to study rare soil CPR. 42 samples were sequenced, enabling the reconstruction of 27 quality CPR metagenome-assembled genomes (MAGs) further classified as Parcubacteria/Paceibacteria, Saccharibacteria/Saccharimonadia and ABY1, in addition to representative genomes from Gemmatimonadetes, Dependentiae and Chlamydae phyla. These genomes were fully annotated and used to reconstruct the CPR community across all 14 plots. Additionally, for five of these plots, the entire microbiota was reconstructed using 16S amplification, showing that specific soil CPR may form symbiotic relationships with a varied and circumstantial range of hosts. Cullars CPR had a prevalence of enzymes predicted to degrade plant-derived carbohydrates, which suggests they have a role in plant biomass degradation. Parcubacteria appear to be more apt at microfauna necromass degradation. Cullars Saccharibacteria and a Parcubacteria group were shown to carry a possible aerotolerance mechanism coupled with potential for aerobic respiration, which appear to be a unique adaptation to the oxic soil environment. Reconstruction of CPR communities across treatment plots showed that they were not impacted by changes in nutrient levels or microbiota composition, being only impacted by extreme conditions, causing some CPR to dominate the community. These findings corroborate the understanding that soil-dwelling CPR bacteria have a very broad symbiont range and have metabolic capabilities associated to soil environments which allows them to scavenge resources and form resilient communities. The contributions of these microbial dark matter species to soil ecology and plant interactions will be of significant interest in future studies.

Evolutionary patterns of archaea predominant in acidic environment

BackgroundArchaea of the order Thermoplasmatales are widely distributed in natural acidic areas and are amongst the most acidophilic prokaryotic organisms known so far. These organisms are difficult to culture, with currently only six genera validly published since the discovery of Thermoplasma acidophilum in 1970. Moreover, known great diversity of uncultured Thermoplasmatales represents microbial dark matter and underlines the necessity of efforts in cultivation and study of these archaea. Organisms from the order Thermoplasmatales affiliated with the so-called “alphabet-plasmas”, and collectively dubbed “E-plasma”, were the focus of this study. These archaea were found predominantly in the hyperacidic site PM4 of Parys Mountain, Wales, UK, making up to 58% of total metagenomic reads. However, these archaea escaped all cultivation attempts.ResultsTheir genome-based metabolism revealed its peptidolytic potential, in line with the physiology of the previously studied Thermoplasmatales isolates. Analyses of the genome and evolutionary history reconstruction have shown both the gain and loss of genes, that may have contributed to the success of the “E-plasma” in hyperacidic environment compared to their community neighbours. Notable genes among them are involved in the following molecular processes: signal transduction, stress response and glyoxylate shunt, as well as multiple copies of genes associated with various cellular functions; from energy production and conversion, replication, recombination, and repair, to cell wall/membrane/envelope biogenesis and archaella production. History events reconstruction shows that these genes, acquired by putative common ancestors, may determine the evolutionary and functional divergences of “E-plasma”, which is much more developed than other representatives of the order Thermoplasmatales. In addition, the ancestral hereditary reconstruction strongly indicates the placement of Thermogymnomonas acidicola close to the root of the Thermoplasmatales.ConclusionsThis study has analysed the metagenome-assembled genome of “E-plasma”, which denotes the basis of their predominance in Parys Mountain environmental microbiome, their global ubiquity, and points into the right direction of further cultivation attempts. The results suggest distinct evolutionary trajectories of organisms comprising the order Thermoplasmatales, which is important for the understanding of their evolution and lifestyle.

Large-Scale Integration of Amplicon Data Reveals Massive Diversity within Saprospirales, Mostly Originating from Saline Environments.

The order Saprospirales, a group of bacteria involved in complex degradation pathways, comprises three officially described families: Saprospiraceae, Lewinellaceae, and Haliscomenobacteraceae. These collectively contain 17 genera and 31 species. The current knowledge on Saprospirales diversity is the product of traditional isolation methods, with the inherited limitations of culture-based approaches. This study utilized the extensive information available in public sequence repositories combined with recent analytical tools to evaluate the global evidence-based diversity of the Saprospirales order. Our analysis resulted in 1183 novel molecular families, 15,033 novel molecular genera, and 188 K novel molecular species. Of those, 7 novel families, 464 novel genera, and 1565 species appeared in abundances at ≥0.1%. Saprospirales were detected in various environments, such as saline water, freshwater, soil, various hosts, wastewater treatment plants, and other bioreactors. Overall, saline water was the environment showing the highest prevalence of Saprospirales, with bioreactors and wastewater treatment plants being the environments where they occurred with the highest abundance. Lewinellaceae was the family containing the majority of the most prevalent species detected, while Saprospiraceae was the family with the majority of the most abundant species found. This analysis should prime researchers to further explore, in a more targeted way, the Saprospirales proportion of microbial dark matter.

Meta-analysis of the microbial diversity cultured in bioreactors simulating the gut microbiome: another key to unlocking the microbial dark matter

Meta-analysis of the microbial diversity cultured in bioreactors simulating the gut microbiome: another key to unlocking the microbial dark matter