Setting out to isolate uncultivated deep marine sediment microorganisms, we engineered and operated a methane-fed continuous-flow bioreactor system for more than 2,000 days to enrich such organisms from anaerobic marine methane-seep sediments 15 (Supplementary Note 1). We successfully enriched many phylogenetically diverse yetto-be cultured microorganisms, including Asgard archaea members (Loki-, Heimdall-and Odinarchaeota) 15. For further enrichment and isolation, samples of the bioreactor community were inoculated in glass tubes with simple substrates and basal medium. After approximately one year, we found faint cell turbidity in a culture containing casamino acids supplemented with four bacteria-suppressing antibiotics (Supplementary Note 2) that was incubated at 20 °C. Clone librarybased small subunit (SSU) rRNA gene analysis revealed a simple community that contained Halodesulfovibrio and a small population of Lokiarchaeota (Extended Data Table 1). In pursuit of this archaeon, which we designated strain MK-D1, we repeated subcultures when MK-D1 reached maximum cell densities as measured by quantitative PCR (qPCR). This approach gradually enriched the archaeon, which has an extremely slow growth rate and low cell yield (Fig. 1a). The culture consistently had a 30-60-day lag phase and required more

Microbial methanogenesis in subseafloor sediments is a key process in the carbon cycle on the Earth. However, the cultivation-dependent evidences have been poorly demonstrated. Here we report the cultivation of a methanogenic microbial consortium from subseafloor sediments using a continuous-flow-type bioreactor with polyurethane sponges as microbial habitats, called down-flow hanging sponge (DHS) reactor. We anaerobically incubated methane-rich core sediments collected from off Shimokita Peninsula, Japan, for 826 days in the reactor at 10 1C. Synthetic seawater supplemented with glucose, yeast extract, acetate and propionate as potential energy sources was provided into the reactor. After 289 days of operation, microbiological methane production became evident. Fluorescence in situ hybridization analysis revealed the presence of metabolically active microbial cells with various morphologies in the reactor. DNA-and RNA-based phylogenetic analyses targeting 16S rRNA indicated the successful growth of phylogenetically diverse microbial components during cultivation in the reactor. Most of the phylotypes in the reactor, once it made methane, were more closely related to culture sequences than to the subsurface environmental sequence. Potentially methanogenic phylotypes related to the genera Methanobacterium, Methanococcoides and Methanosarcina were predominantly detected concomitantly with methane production, while uncultured archaeal phylotypes were also detected. Using the methanogenic community enrichment as subsequent inocula, traditional batch-type cultivations led to the successful isolation of several anaerobic microbes including those methanogens. Our results substantiate that the DHS bioreactor is a useful system for the enrichment of numerous fastidious microbes from subseafloor sediments and will enable the physiological and ecological characterization of pure cultures of previously uncultivated subseafloor microbial life.

34The origin of eukaryotes remains enigmatic. Current data suggests that eukaryotes may 35 have risen from an archaeal lineage known as "Asgard archaea". Despite the eukaryote-36 like genomic features found in these archaea, the evolutionary transition from archaea to 37 eukaryotes remains unclear due to the lack of cultured representatives and corresponding 38 physiological insight. Here we report the decade-long isolation of a Lokiarchaeota-related 39Asgard archaeon from deep marine sediment. The archaeon, "Candidatus 40Prometheoarchaeum syntrophicum strain MK-D1", is an anaerobic, extremely slow-41 growing, small cocci (~550 nm), that degrades amino acids through syntrophy. Although 42 eukaryote-like intracellular complexities have been proposed for Asgard archaea, the 43 isolate has no visible organella-like structure. Ca. P. syntrophicum instead displays 44 morphological complexity -unique long, and often, branching protrusions. Based on 45 cultivation and genomics, we propose an "Entangle-Engulf-Enslave (E 3 ) model" for 46 eukaryogenesis through archaea-alphaproteobacteria symbiosis mediated by the physical 47 complexities and metabolic dependency of the hosting archaeon. 48 49 How did the first eukaryotic cell emerge? So far, among various competing evolutionary 50 models, the most widely accepted are the symbiogenetic models in which an archaeal 51 host cell and an alphaproteobacterial endosymbiont merged to become the first eukaryotic 52 cell 1-4 . Recent metagenomic discovery of Lokiarchaeota (and the Asgard archaea 53 superphylum) led to the theory that eukaryotes originated from an archaeon closely 54 related to Asgard archaea 5,6 . The Asgard archaea genomes encode a repertory of proteins 55 hitherto only found in Eukarya (eukaryotic signature proteins -ESPs), including those 56 involved in membrane trafficking, vesicle formation/transportation, ubiquitin and 57 cytoskeleton formation 6 . Subsequent metagenomic studies have suggested that Asgard 58 archaea have a wide variety of physiological properties, including hydrogen-dependent 59 anaerobic autotrophy 7 , peptide or short-chain hydrocarbon-dependent organotrophy 8-11 60 and rhodopsin-based phototrophy 12,13 . A recent study suggests that an ancient Asgard 61 archaea degraded organic substances and syntrophically handed off reducing equivalents 62 (e.g., hydrogen and electrons) to a bacterial partner, and further proposes a symbiogenetic 63 model for the origin of eukaryotes based on this interaction 14 . However, at present, no 64 single representative of the Asgard archaea has been cultivated and, thus, the physiology 65 and cell biology of this clade remains unclear. In an effort to close this knowledge gap, 66 3 we successfully isolated the first Asgard archaeon and here report the physiological 67 characteristics, potentially key insights into the evolution of eukaryotes. 68 69 Isolation of an Asgard archaeon 70Setting out to isolate uncultivated deep marine sediment microorganisms, we engineered 71 and operated a methane-fed continuous-fl...

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In situ detection of microorganisms by fluorescence in situ hybridization (FISH) is a powerful tool for environmental microbiology, but analyses can be hampered by low rRNA content in target organisms, especially in oligotrophic environments. Here, we present a non-enzymatic, hybridization chain reaction (HCR)-based signal amplified in situ whole-cell detection technique (in situ DNA-HCR). The components of the amplification buffer were optimized to polymerize DNA amplifier probes for in situ DNA-HCR. In situ hybridization of initiator probes followed by signal amplification via HCR produced bright signals with high specificity and probe permeation into cells. The detection rates for Bacteria in a seawater sample and Archaea in anaerobic sludge samples were comparable with or greater than those obtained by catalyzed reporter deposition (CARD)-FISH or standard FISH. Detection of multiple organisms (Bacteria, Archaea and Methanosaetaceae) in an anaerobic sludge sample was achieved by simultaneous in situ DNA-HCR. In summary, in situ DNA-HCR is a simple and easy technique for detecting single microbial cells and enhancing understanding of the ecology and behaviour of environmental microorganisms in situ.