Microbial Dark Matter Research Articles

Machine Learning-Assisted Single-Cell Raman Fingerprinting for <i>in Situ<i> and Nondestructive Prokaryotic Classification in Complex Microbial Populations</i></i>

Accessing enormous uncultivated microorganisms (known as microbial dark matter) in a variety of Earth environments requires accurate, nondestructive classification and molecular understanding of the microorganisms in situ and at the single-cell level, which are not feasible with the current genomics approaches. Here we demonstrate a combined approach of random forest (RF) machine learning and less-invasive, single-cell Raman microspectroscopy for accurate classification of phylogenetically diverse prokaryotes (three bacterial and three archaeal species from different phyla). Our RF classifier achieved 98.8±1.9% classification accuracy among the six species in pure populations and 98.4% for three species in an artificially mixed population. Feature importance scores against each wavenumber in the Raman spectrum reveal that, in addition to protein and DNA/RNA abundances, the presence of carotenoids and structure of membrane lipids play key roles in distinguishing the prokaryotic species. We also find unique Raman markers for an ammonia-oxidizing archaeon. Our approach with moderate data pretreatment and intuitive visualization of feature importance is easy to use for non-spectroscopists and thus offers microbiologists a new single-cell tool for shedding light on microbial dark matter.

Microbial dark matter coming to light: challenges and opportunities.

Microbes are the most abundant and diverse cellular life forms on Earth and colonize a wide range of environmental niches. However, more than 99% of bacterial and archaeal species have not been obtained in pure culture [1] and we have only glimpsed the surface of this mysterious microbial world. This is so-called Microbial Dark Matter (MDM): the enormous diversity of yet-uncultivated microbes that microbiologists can only study by using cultivation-independent techniques. Recently, a number of international projects have dramatically increased our understanding of the extent and distribution of microbial diversity, including the Global Catalogue of Microorganisms (GCM), the Genomic Encyclopedia of Bacteria and Archaea (GEBA), the Earth Microbiome Project (EMP), the Genomic Encyclopedia of Bacteria and Archaea-Microbial Dark Matter (GEBA-MDM) and several primate microbiome projects; however, the functional diversity of MDM is still mysterious. This perspective addresses why MDM deserves scientific effort and illustrates challenges and opportunities in the future study of these enigmas.

Probing periodontal microbial dark matter using metataxonomics and metagenomics.

Our view of the periodontal microbial community has been shaped by a century or more of cultivation-based and microscopic investigations. While these studies firmly established the infection-mediated etiology of periodontal diseases, it was apparent from the very early days that periodontal microbiology suffered from what Staley and Konopka described as the "great plate count anomaly", in that these culturable bacteria were only a minor part of what was visible under the microscope. For nearly a century, much effort has been devoted to finding the right tools to investigate this uncultivated majority, also known as "microbial dark matter". The discovery that DNA was an effective tool to "see" microbial dark matter was a significant breakthrough in environmental microbiology, and oral microbiologists were among the earliest to capitalize on these advances. By identifying the order in which nucleotides are arranged in a stretch of DNA (DNA sequencing) and creating a repository of these sequences, sequence databases were created. Computational tools that used probability-driven analysis of these sequences enabled the discovery of new and unsuspected species and ascribed novel functions to these species. This review will trace the development of DNA sequencing as a quantitative, open-ended, comprehensive approach to characterize microbial communities in their native environments, and explore how this technology has shifted traditional dogmas on how the oral microbiome promotes health and its role in disease causation and perpetuation.

Genome-Centric Metagenomic Insights into the Impact of Alkaline/Acid and Thermal Sludge Pretreatment on the Microbiome in Digestion Sludge.

Pretreatment of waste-activated sludge (WAS) is an effective way to destabilize sludge floc structure and release organic matter for improving sludge digestion efficiency. Nonetheless, information on the impact of WAS pretreatment on digestion sludge microbiomes, as well as mechanistic insights into how sludge pretreatment improves digestion performance, remains elusive. In this study, a genome-centric metagenomic approach was employed to investigate the digestion sludge microbiome in four sludge digesters with different types of feeding sludge: WAS pretreated with 0.25 mol/liter alkaline/acid (APAD), WAS pretreated with 0.8 mol/liter alkaline/acid (HS-APAD), thermally pretreated WAS (thermal-AD), and fresh WAS (control-AD). We retrieved 254 metagenome-assembled genomes (MAGs) to identify the key functional populations involved in the methanogenic digestion process. These MAGs span 28 phyla, including 69 yet-to-be-cultivated lineages, and 30 novel lineages were characterized with metabolic potential associated with hydrolysis and fermentation. Interestingly, functional populations involving carbohydrate digestion were enriched in APAD and HS-APAD, while lineages related to protein and lipid fermentation were enriched in thermal-AD, corroborating the idea that different substrates are released from alkaline/acid and thermal pretreatments. Among the major functional populations (i.e., fermenters, syntrophic acetogens, and methanogens), significant correlations between genome sizes and abundance of the fermenters were observed, particularly in APAD and HS-APAD, which had improved digestion performance.IMPORTANCE Wastewater treatment generates large amounts of waste-activated sludge (WAS), which consists mainly of recalcitrant microbial cells and particulate organic matter. Though WAS pretreatment is an effective way to release sludge organic matter for subsequent digestion, detailed information on the impact of the sludge pretreatment on the digestion sludge microbiome remains scarce. Our study provides unprecedented genome-centric metagenomic insights into how WAS pretreatments change the digestion sludge microbiomes, as well as their metabolic networks. Moreover, digestion sludge microbiomes could be a unique source for exploring microbial dark matter. These results may inform future optimization of methanogenic sludge digestion and resource recovery.

The Problem of Microbial Dark Matter in Neonatal Sepsis.

Neonatal sepsis (NS) kills 750,000 infants every year. Effectively treating NS requires timely diagnosis and antimicrobial therapy matched to the causative pathogens, but most blood cultures for suspected NS do not recover a causative pathogen. We refer to these suspected but unidentified pathogens as microbial dark matter. Given these low culture recovery rates, many non–culture-based technologies are being explored to diagnose NS, including PCR, 16S amplicon sequencing, and whole metagenomic sequencing. However, few of these newer technologies are scalable or sustainable globally. To reduce worldwide deaths from NS, one possibility may be performing population-wide pathogen discovery. Because pathogen transmission patterns can vary across space and time, computational models can be built to predict the pathogens responsible for NS by region and season. This approach could help to optimally treat patients, decreasing deaths from NS and increasing antimicrobial stewardship until effective diagnostics that are scalable become available globally.

FACS-iChip: a high-efficiency iChip system for microbial 'dark matter' mining.

The isolation chip method (iChip) provides a novel approach for culturing previously uncultivable microorganisms; this method is currently limited by the user being unable to ensure single-cell loading within individual wells. To address this limitation, we integrated flow cytometry-based fluorescence-activated cell sorting with a modified iChip (FACS-iChip) to effectively mine microbial dark matter in soils. This method was used for paddy soils with the aim of mining uncultivable microorganisms and making preliminary comparisons between the cultured microorganisms and the bulk soil via 16S rRNA gene sequencing. Results showed that the FACS-iChip achieved a culture recovery rate of almost 40% and a culture retrieval rate of 25%. Although nearly 500 strains were cultured from 19 genera with 8 FACS-iChip plates, only six genera could be identified via 16S rRNA gene amplification. This result suggests that the FACS-iChip is capable of detecting strains in the currently dead spaces of PCR-based sequencing technology. We, therefore, conclude that the FACS-iChip system provides a highly efficient and readily available approach for microbial 'dark matter' mining.

A network approach to elucidate and prioritize microbial dark matter in microbial communities

Microbes compose most of the biomass on the planet, yet the majority of taxa remain uncharacterized. These unknown microbes, often referred to as “microbial dark matter,” represent a major challenge for biology. To understand the ecological contributions of these Unknown taxa, it is essential to first understand the relationship between unknown species, neighboring microbes, and their respective environment. Here, we establish a method to study the ecological significance of “microbial dark matter” by building microbial co-occurrence networks from publicly available 16S rRNA gene sequencing data of four extreme aquatic habitats. For each environment, we constructed networks including and excluding unknown organisms at multiple taxonomic levels and used network centrality measures to quantitatively compare networks. When the Unknown taxa were excluded from the networks, a significant reduction in degree and betweenness was observed for all environments. Strikingly, Unknown taxa occurred as top hubs in all environments, suggesting that “microbial dark matter” play necessary ecological roles within their respective communities. In addition, novel adaptation-related genes were detected after using 16S rRNA gene sequences from top-scoring hub taxa as probes to blast metagenome databases. This work demonstrates the broad applicability of network metrics to identify and prioritize key Unknown taxa and improve understanding of ecosystem structure across diverse habitats.

Microbial dark matter filling the niche in hypersaline microbial mats

BackgroundShark Bay, Australia, harbours one of the most extensive and diverse systems of living microbial mats that are proposed to be analogs of some of the earliest ecosystems on Earth. These ecosystems have been shown to possess a substantial abundance of uncultivable microorganisms. These enigmatic microbes, jointly coined as ‘microbial dark matter’ (MDM), are hypothesised to play key roles in modern microbial mats.ResultsWe reconstructed 115 metagenome-assembled genomes (MAGs) affiliated to MDM, spanning 42 phyla. This study reports for the first time novel microorganisms (Zixibacterial order GN15) putatively taking part in dissimilatory sulfate reduction in surface hypersaline settings, as well as novel eukaryote signature proteins in the Asgard archaea. Despite possessing reduced-size genomes, the MDM MAGs are capable of fermenting and degrading organic carbon, suggesting a role in recycling organic carbon. Several forms of RuBisCo were identified, allowing putative CO2 incorporation into nucleotide salvaging pathways, which may act as an alternative carbon and phosphorus source. High capacity of hydrogen production was found among Shark Bay MDM. Putative schizorhodopsins were also identified in Parcubacteria, Asgard archaea, DPANN archaea, and Bathyarchaeota, allowing these members to potentially capture light energy. Diversity-generating retroelements were prominent in DPANN archaea that likely facilitate the adaptation to a dynamic, host-dependent lifestyle.ConclusionsThis is the first study to reconstruct and describe in detail metagenome-assembled genomes (MAGs) affiliated with microbial dark matter in hypersaline microbial mats. Our data suggests that these microbial groups are major players in these systems. In light of our findings, we propose H2, ribose and CO/CO2 as the main energy currencies of the MDM community in these mat systems.Cmbfp_aj5R9yxCxBzxBrnsVideo

Targeted Cell Sorting Combined With Single Cell Genomics Captures Low Abundant Microbial Dark Matter With Higher Sensitivity Than Metagenomics.

Rare members of environmental microbial communities are often overlooked and unexplored, primarily due to the lack of techniques capable of acquiring their genomes. Chloroflexi belong to one of the most understudied phyla, even though many of its members are ubiquitous in the environment and some play important roles in biochemical cycles or biotechnological applications. We here used a targeted cell-sorting approach, which enables the selection of specific taxa by fluorescent labeling and is compatible with subsequent single-cell genomics, to enrich for rare Chloroflexi species from a wastewater-treatment plant and obtain their genomes. The combined workflow was able to retrieve a substantially higher number of novel Chloroflexi draft genomes with much greater phylogenetical diversity when compared to a metagenomics approach from the same sample. The method offers an opportunity to access genetic information from rare biosphere members which would have otherwise stayed hidden as microbial dark matter and can therefore serve as an essential complement to cultivation-based, metagenomics, and microbial community-focused research approaches.

Metagenomic analysis of gut microbiome and resistome of diarrheal fecal samples from Kolkata, India, reveals the core and variable microbiota including signatures of microbial dark matter

BackgroundMetagenomic analysis of the gut microbiome and resistome is instrumental for understanding the dynamics of diarrheal pathogenesis and antimicrobial resistance transmission (AMR). Metagenomic sequencing of 20 diarrheal fecal samples from Kolkata was conducted to understand the core and variable gut microbiota. Five of these samples were used for resistome analysis. The pilot study was conducted to determine a microbiota signature and the source of antimicrobial resistance genes (ARGs) in the diarrheal gut.Results16S rRNA amplicon sequencing was performed using Illumina MiSeq platform and analysed using the MGnify pipeline. The Genome Taxonomy Database (GTDB-Tk) was used for bacterial taxonomic identification. Diarrheal etiology was determined by culture method. Phylum Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were consistently present in 20 samples. Firmicutes was the most abundant phylum in 11 samples. The Bacteroidetes/Firmicutes ratio was less than 1 in 18 samples. 584 genera were observed. 18 of these were present in all the 20 samples. Proteobacteria was the dominant phylum in 6 samples associated with Vibrio cholerae infection. Conservation of operational taxonomic units (OTUs) among all the samples indicated the existence of a core microbiome. Asymptomatic carriage of pathogens like Vibrio cholerae and Helicobacter pylori was found. Signature of Candidate phyla or “microbial dark matter” occurred. Significant correlation of relative abundance of bacterial families of commensals and pathogens were found. Whole-genome sequencing (WGS) on Illumina MiSeq system and assembly of raw reads using metaSPAdes v3.9.1 was performed to study the resistome of 5 samples. ABRicate was used to assign ARG function. 491 resistance determinants were identified. In 80% of the samples tetracycline resistance was the most abundant resistance determinant. High abundance of ARGs against β-lactams, aminoglycosides, quinolones and macrolides was found. Eschericia sp. was the major contributor of ARGs.ConclusionsThis is the first comparative study of the gut microbiome associated with different diarrheal pathogens. It presents the first catalogue of different bacterial taxa representing the core and variable microbiome in acute diarrheal patients. The study helped to define a trend in the gut microbiota signature associated with diarrhea and revealed which ARGs are abundantly present and the metagenome-assembled genomes (MAGs) contributing to AMR.

A multiproducer microbiome generates chemical diversity in the marine sponge Mycale hentscheli

Bacterial specialized metabolites are increasingly recognized as important factors in animal-microbiome interactions: for example, by providing the host with chemical defenses. Even in chemically rich animals, such compounds have been found to originate from individual members of more diverse microbiomes. Here, we identified a remarkable case of a moderately complex microbiome in the sponge host Mycale hentscheli in which multiple symbionts jointly generate chemical diversity. In addition to bacterial pathways for three distinct polyketide families comprising microtubule-inhibiting peloruside drug candidates, mycalamide-type contact poisons, and the eukaryotic translation-inhibiting pateamines, we identified extensive biosynthetic potential distributed among a broad phylogenetic range of bacteria. Biochemical data on one of the orphan pathways suggest a previously unknown member of the rare polytheonamide-type cytotoxin family as its product. Other than supporting a scenario of cooperative symbiosis based on bacterial metabolites, the data provide a rationale for the chemical variability of M. hentscheli and could pave the way toward biotechnological peloruside production. Most bacterial lineages in the compositionally unusual sponge microbiome were not known to synthesize bioactive metabolites, supporting the concept that microbial dark matter harbors diverse producer taxa with as yet unrecognized drug discovery potential.

Salt tolerance of Cressa cretica and its rhizosphere microbiota

The dwarf shrub Cressa cretica is a thermocosmopolitan halophilic species. Different mechanisms confer salt stress tolerance such as tissue and osmotic tolerance and ion exclusion, as well as the associated microbiota. The aims were (i) to investigate the best conditions for C. cretica seed germination and to examine the tolerance of germinated seeds and seedlings to different salt concentrations and (ii) to characterize the rhizosphere and bulk soil microbiota. Germination and growth experiments were conducted to address plant salt tolerance, and with Illumina sequencing the microbiota of rhizosphere and bulk soil was investigated. While high salt concentrations (600, 800, and 1000 mM NaCl) inhibited C.cretica seed germination, recovery of ungerminated seeds under non-saline conditions was high, indicating osmotic rather than toxic effects of high salt concentrations. The microbiota found in rhizosphere and bulk soil showed high similarity with that found in previous studies on halophyte-associated microbiota, among the Planctomyces, Halomonas and Jeotgalibacillus. Concluding, salt stress responses on the plant level were shown, as has the involvement of associated halotolerant bacteria. Still, the potential role for mitigating saline stress by the bacterial associates of C. cretica, most of them microbial dark matter, will have to be investigated, as will the contributions of archaea and fungi.

Microbial diversity and related secondary metabolite gene assortment at an estuarine mangrove ecosystem

Mangrove ecosystems are a major biodiversity hotspot of the world. The world heritage site Sundarbans is an archipelago with an estuarine mangrove ecosystem. It is assumed that each individual Island of the Sundarbans possess a unique biodiversity due to the seasonal variation of physicochemical parameters, along with tidal dynamics of Bay of Bengal. This study unfolds a time series analysis of microbial diversity using high-resolution 16S rRNA tag pyrosequencing. Eight soil samples were collected over three years at a pristine temperate estuarine site located at the sea mangrove interface at the island of Kalash. Apart from the predominant phyla such as Proteobacteria [31.6%], Chloroflexi [10.7%], Firmicutes [7%] Bacteroidetes [7%] and Actinobacteria [1.2%], soil from Kalash also contained a significant amount of rare phyla NC10 and WS3 categorized as microbial dark matter (MDM). Variation in bacterial community composition occurs mainly as a result of the seasonal changes and consequent alterations of physicochemical parameters leading to microbial adaptations. Further we performed functional metagenomics to estimate the possibility of the synthesis of new natural compounds by diverse Polyketide Synthase (PKS). The data indicated diverse microbial origins of keto synthase sequences from polyketide synthase genes. Presence of this unique keto synthases confirmed the existence of a potential microbiome which may be capable of producing novel small molecules. This is the pioneering report to correlate the microbial community structure of Sundarbans with their potential to produce essential bioactive compounds.

Cultivation and functional characterization of 79 planctomycetes uncovers their unique biology.

When it comes to the discovery and analysis of yet uncharted bacterial traits, pure cultures are essential as only these allow detailed morphological and physiological characterization as well as genetic manipulation. However, microbiologists are struggling to isolate and maintain the majority of bacterial strains, as mimicking their native environmental niches adequately can be a challenging task. Here, we report the diversity-driven cultivation, characterization and genome sequencing of 79 bacterial strains from all major taxonomic clades of the conspicuous bacterial phylum Planctomycetes. The samples were derived from different aquatic environments but close relatives could be isolated from geographically distinct regions and structurally diverse habitats, implying that 'everything is everywhere'. With the discovery of lateral budding in 'Kolteria novifilia' and the capability of the members of the Saltatorellus clade to divide by binary fission as well as budding, we identified previously unknown modes of bacterial cell division. Alongside unobserved aspects of cell signalling and small-molecule production, our findings demonstrate that exploration beyond the well-established model organisms has the potential to increase our knowledge of bacterial diversity. We illustrate how 'microbial dark matter' can be accessed by cultivation techniques, expanding the organismic background for small-molecule research and drug-target detection.

Microbiome Big-Data Mining and Applications Using Single-Cell Technologies and Metagenomics Approaches Toward Precision Medicine.

With the development of high-throughput sequencing technologies as well as various bioinformatics analytic tools, microbiome is not a “microbial dark matter” anymore. In this review, we first summarized the current analytical strategies used for big-data mining such as single-cell sequencing and metagenomics. We then provided insights into the integration of these strategies, showing significant advantages in fully describing microbiome from multiple aspects. Moreover, we discussed the correlation between gut microbiome with host organs and diseases, confirming the importance of big-data mining in clinical practices. We finally proposed new ideas about the trend of big-data mining in microbiome using multi-omics approaches and single-cell sequencing. The integration of multi-omics approaches and single-cell sequencing can provide full understanding of microbiome at both macroscopic level and microscopic level, thus contributing to precision medicine.

Genome mining- and synthetic biology-enabled production of hypermodified peptides.

SummaryThe polytheonamides are among the most complex and biosynthetically distinctive natural products known to date. These potent peptide cytotoxins are derived from a ribosomal precursor processed by 49 mostly non-canonical posttranslational modifications. Since the producer is a "microbial dark matter" bacterium only distantly related to any cultivated organism, >70-step chemical syntheses have been developed to access these unique compounds. Here we mined prokaryotic diversity to establish a synthetic platform based on the new host Microvirgula aerodenitrificans that produces hypermodified peptides within two days. Using this system, we generated the aeronamides, new polytheonamide-type compounds with near-picomolar cytotoxicity. Aeronamides, as well as the polygeonamides produced from deep-rock biosphere DNA, contain the highest numbers of d-amino acids in known biomolecules. With increasing bacterial genomes being sequenced, similar host mining strategies might become feasible to access further elusive natural products from uncultivated life.

Two intracellular and cell type-specific bacterial symbionts in the placozoan Trichoplax H2

Placozoa is an enigmatic phylum of simple, microscopic, marine metazoans1,2. Although intracellular bacteria have been found in all members of this phylum, almost nothing is known about their identity, location and interactions with their host3–6. We used metagenomic and metatranscriptomic sequencing of single host individuals, plus metaproteomic and imaging analyses, to show that the placozoan Trichoplax sp. H2 lives in symbiosis with two intracellular bacteria. One symbiont forms an undescribed genus in the Midichloriaceae (Rickettsiales)7,8 and has a genomic repertoire similar to that of rickettsial parasites9,10, but does not seem to express key genes for energy parasitism. Correlative image analyses and three-dimensional electron tomography revealed that this symbiont resides in the rough endoplasmic reticulum of its host’s internal fibre cells. The second symbiont belongs to the Margulisbacteria, a phylum without cultured representatives and not known to form intracellular associations11–13. This symbiont lives in the ventral epithelial cells of Trichoplax, probably metabolizes algal lipids digested by its host and has the capacity to supplement the placozoan’s nutrition. Our study shows that one of the simplest animals has evolved highly specific and intimate associations with symbiotic, intracellular bacteria and highlights that symbioses can provide access to otherwise elusive microbial dark matter.

Human Skin Microbiome: Integrating bacterial, fungal and viral communities

The varied topography of human skin offers a unique opportunity to study how the body's microenvironments influence the functional and taxonomic composition of microbial communities. The general accessibility of skin also enables longitudinal clinical studies throughout the course of disease manifestations. Metagenomic analysis of diverse body sites in healthy humans defined the skin microbiome as shaped by local biogeography yet marked by strong individuality. We identified strain‐level variation of dominant species as heterogeneous and multiphyletic. Re‐sampling months and years later revealed that despite the skin's exposure to the external environment, its bacterial, fungal, and viral communities were largely stable over time. Strain and single nucleotide variant level analysis showed that individuals maintain, rather than reacquire prevalent microbes from the environment.Longitudinal stability of skin microbial communities generates hypotheses about colonization resistance and empowers clinical studies exploring alterations observed in disease states, such as the inflammatory skin disorder atopic dermatitis (AD; commonly known as eczema). Integrating shotgun metagenomic sequencing, culturing, and animal models, we explored a model whereby staphylococcal strains contribute to AD disease exacerbation. This delineation of highly individualized skin microbiomes with patient‐specific strains underscores the individuality of the disease course and therapeutic response in AD patients and may represent an opportunity for precision medicine.Although landmark studies have shown that microbiota activate and educate host immunity, how the immune system shapes microbial communities and contributes to disease is less‐well characterized. We explore the skin microbiome of patients with primary immune deficiencies to address this question and to expand our understanding of microbes that colonize human skin. Dedicator of cytokinesis 8 (DOCK8) deficiency is a rare primary human immunodeficiency characterized by recurrent cutaneous and systemic infections, as well as atopy and cancer susceptibility5. DOCK8, encoding a guanine nucleotide exchange factor highly expressed in lymphocytes, regulates actin cytoskeleton, which is critical for migration through collagen‐dense tissues, such as skin6. Analyzing deep metagenomic sequencing data from DOCK8‐deficient skin samples demonstrated a notable increase in eukaryotic viral representation and diversity as compared to healthy volunteers. De novo assembly approaches identified hundreds of novel human papillomavirus genomes, illuminating microbial dark matter. Expansion of the skin virome in DOCK8‐deficient patients underscores the importance of immune surveillance in controlling eukaryotic viral colonization and infection.Support or Funding InformationNHGRI, NIAMS, NCI, NIAID, NIH Intramural Research ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Marine fungal dark matter in the global ocean

Marine fungi are a major part of ‘microbial dark matter’, with most organisms known from sequence data and currently not in culture. Interest in marine fungi has substantially increased over the past decade, and studies using culture independent methods have indicated that fungal diversity in the oceans may be greater than previously estimates based on cultivation alone. There remains much to learn about the true diversity of marine fungi in the global oceans and the ecological roles that they could play. The Tara Oceans expedition has allowed for significant advancements in our understanding of the global diversity of planktonic microorganisms. Interrogation of the Tara Oceans 18S rRNA gene dataset for fungal sequences shows that fungi are found throughout the global oceans, appearing in all marine regions covered by the Tara Oceans expedition. In the survey, Ascomycota and Basidiomycota were common, while some locations had increased abundances of Chytridiomycota. Differences in community composition were observed between oceanic regions and, although clear signals were not apparent due to the nature of the sampling, there was some indication of community variation between upwelling, coastal, shelf and gyral provinces. Different size sampling fractions appeared to capture different portions of the pelagic fungal community. These findings highlight a number of ecological questions: How important are oceanic currents in determining fungal biogeography? What is the relationship between marine fungi and biogeochemical processes? What is actually there? By targeting these questions directly, we will be able to bring the dark matter of marine fungi into the light.

Shining a light on microbial dark matter: a role for the forgotten B vitamins in marine algal communities?

The ocean is home to a huge diversity of life that perform a wide range of functions key to earth systems. Marine algae communities contribute a significant proportion of net carbon fixation of the globe as well as forming the basis of the food webs for every trophic level above their own. Despite their importance, many of the taxa that make up these microalgal communities are relatively unknown and understudied, due to their recalcitrance to lab culturing. This concept is known as microbial dark matter and applies in particular to many non-photosynthetic lineages which have been long overlooked by the research community. To investigate whether dependencies of these taxa on certain B vitamins may play a role in this unculturability, data sets produced form the Tara oceans expedition will be analysed. The Stramenopile lineage will be analysed in greater detail for their metabolic potential to synthesise the B vitamins, and/or whether they may dependent on an external source. Both Single-Amplified Genomes (SAG) and metatrascriptomic data sets will be analysed to determine the complement of genes present in different Stramenopile lineages. This will then be correlated with the global distribution data attainable from analysing Tara data sets. By this means, we hope to understand more about the metabolic contribution of this taxonomic group to the community, how this supports the community as a whole and whether dependence on B vitamins is a contributing factor to unculturability.