Isolation of an archaeon at the prokaryote-eukaryote interface.

The origin of eukaryotes remains unclear1-4. Current data suggest that eukaryotes may have emerged from an archaeal lineage known as 'Asgard' archaea5,6. Despite the eukaryote-like genomic features that are found in these archaea, the evolutionary transition from archaea to eukaryotes remains unclear, owing to the lack of cultured representatives and corresponding physiological insights. Here we report the decade-long isolation of an Asgard archaeon related to Lokiarchaeota from deep marine sediment. The archaeon-'Candidatus Prometheoarchaeum syntrophicum' strain MK-D1-is an anaerobic, extremely slow-growing, small coccus (around 550 nm in diameter) that degrades amino acids through syntrophy. Although eukaryote-like intracellular complexes have been proposed for Asgard archaea6, the isolate has no visible organelle-like structure. Instead, Ca. P. syntrophicum is morphologically complex and has unique protrusions that are long and often branching. On the basis of the available data obtained from cultivation and genomics, and reasoned interpretations of the existing literature, we propose a hypothetical model for eukaryogenesis, termed the entangle-engulf-endogenize (also known as E3) model.

Aggregatilinea lenta gen. nov., sp. nov., a slow-growing, facultatively anaerobic bacterium isolated from subseafloor sediment, and proposal of the new order Aggregatilineales ord. nov. within the class Anaerolineae of the phylum Chloroflexi.

A novel slow-growing, facultatively anaerobic, filamentous bacterium, strain MO-CFX2T, was isolated from a methanogenic microbial community in a continuous-flow bioreactor that was established from subseafloor sediment collected off the Shimokita Peninsula of Japan. Cells were multicellular filamentous, non-motile and Gram-stain-negative. The filaments were generally more than 20 µm (up to approximately 200 µm) long and 0.5-0.6 µm wide. Cells possessed pili-like structures on the cell surface and a multilayer structure in the cytoplasm. Growth of the strain was observed at 20-37 °C (optimum, 30 °C), pH 5.5-8.0 (pH 6.5-7.0), and 0-30 g l-1 NaCl (5 g l-1 NaCl). Under optimum growth conditions, doubling time and maximum cell density were estimated to be approximately 19 days and ~105 cells ml-1, respectively. Strain MO-CFX2T grew chemoorganotrophically on a limited range of organic substrates in anaerobic conditions. The major cellular fatty acids were saturated C16 : 0 (47.9 %) and C18 : 0 (36.9 %), and unsaturated C18 : 1ω9c (6.0 %) and C16 : 1ω7 (5.1 %). The G+C content of genomic DNA was 63.2 mol%. 16S rRNA gene-based phylogenetic analysis showed that strain MO-CFX2T shares a notably low sequence identity with its closest relatives, which were Thermanaerothrix daxensis GNS-1T and Thermomarinilinea lacunifontana SW7T (both 85.8 % sequence identity). Based on these phenotypic and genomic properties, we propose the name Aggregatilinea lenta gen. nov., sp. nov. for strain MO-CFX2T (=KCTC 15625T, =JCM 32065T). In addition, we also propose the associated family and order as Aggregatilineaceae fam. nov. and Aggregatilineales ord. nov., respectively.

Nitrogen and Oxygen Isotope Effects of Ammonia Oxidation by Thermophilic Thaumarchaeota from a Geothermal Water Stream.

UNLABELLED: Ammonia oxidation regulates the balance of reduced and oxidized nitrogen pools in nature. Although ammonia-oxidizing archaea have been recently recognized to often outnumber ammonia-oxidizing bacteria in various environments, the contribution of ammonia-oxidizing archaea is still uncertain due to difficulties in the in situ quantification of ammonia oxidation activity. Nitrogen and oxygen isotope ratios of nitrite (δ(15)NNO2- and δ(18)ONO2-, respectively) are geochemical tracers for evaluating the sources and the in situ rate of nitrite turnover determined from the activities of nitrification and denitrification; however, the isotope ratios of nitrite from archaeal ammonia oxidation have been characterized only for a few marine species. We first report the isotope effects of ammonia oxidation at 70°C by thermophilic Thaumarchaeota populations composed almost entirely of "Candidatus Nitrosocaldus." The nitrogen isotope effect of ammonia oxidation varied with ambient pH (25‰ to 32‰) and strongly suggests the oxidation of ammonia, not ammonium. The δ(18)O value of nitrite produced from ammonia oxidation varied with the δ(18)O value of water in the medium but was lower than the isotopic equilibrium value in water. Because experiments have shown that the half-life of abiotic oxygen isotope exchange between nitrite and water is longer than 33 h at 70°C and pH ≥6.6, the rate of ammonia oxidation by thermophilic Thaumarchaeota could be estimated using δ(18)ONO2- in geothermal environments, where the biological nitrite turnover is likely faster than 33 h. This study extended the range of application of nitrite isotopes as a geochemical clock of the ammonia oxidation activity to high-temperature environments. IMPORTANCE: Because ammonia oxidation is generally the rate-limiting step in nitrification that regulates the balance of reduced and oxidized nitrogen pools in nature, it is important to understand the biological and environmental factors underlying the regulation of the rate of ammonia oxidation. The discovery of ammonia-oxidizing archaea (AOA) in marine and terrestrial environments has transformed the concept that ammonia oxidation is operated only by bacterial species, suggesting that AOA play a significant role in the global nitrogen cycle. However, the archaeal contribution to ammonia oxidation in the global biosphere is not yet completely understood. This study successfully identified key factors controlling nitrogen and oxygen isotopic ratios of nitrite produced from thermophilic Thaumarchaeota and elucidated the applicability and its limit of nitrite isotopes as a geochemical clock of ammonia oxidation rate in nature. Oxygen isotope analysis in this study also provided new biochemical information on archaeal ammonia oxidation.

Community composition of known and uncultured archaeal lineages in anaerobic or anoxic wastewater treatment sludge.

Microbial systems are widely used to treat different types of wastewater from domestic, agricultural, and industrial sources. Community composition is an important factor in determining the successful performance of microbial treatment systems; however, a variety of uncultured and unknown lineages exist in sludge that requires identification and characterization. The present study examined the archaeal community composition in methanogenic, denitrifying, and nitrogen-/phosphate-removing wastewater treatment sludge by Archaea-specific 16S rRNA gene sequencing analysis using Illumina sequencing technology. Phylotypes belonging to Euryarchaeota, including methanogens, were most abundant in all samples except for nitrogen-/phosphate-removing wastewater treatment sludge. High levels of Deep Sea Hydrothermal Vent Group 6 (DHVEG-6), WSA2, Terrestrial Miscellaneous Euryarchaeotal Group, and Miscellaneous Crenarchaeotic Group were also detected. Interestingly, DHVEG-6 was dominant in nitrogen-/phosphate-removing wastewater treatment sludge, indicating that unclear lineages of Archaea still exist in the anaerobic wastewater treatment sludges. These results reveal a previously unknown diversity of Archaea in sludge that can potentially be exploited for the development of more efficient wastewater treatment strategies.

Microbial community structure of a simultaneous nitrogen and phosphorus removal reactor following treatment in a UASB-DHS system.

The anaerobic-anoxic sequence batch reactor (A2SBR) was applied to achieve nitrogen and phosphorus removal in an energy-saving sewage treatment system involving an up-flow anaerobic sludge blanket combined with a down-flow hanging sponge reactor to treat municipal sewage. After sludge acclimation, the A2SBR showed satisfactory denitrification and phosphorus removal performance with total phosphate and nitrate concentrations of the effluent of 8.4 ± 3.4 mg-N L⁻¹ and 0.9 ± 0.6 mg-P L⁻¹, respectively. 16S rRNA gene sequence and fluorescence in situ hybridization analyses revealed that 'Candidatus Accumulibacter phosphatis' was the dominant phosphate-accumulating micro-organism. Although a competitive bacterium for polyphosphate-accumulating organisms, 'Ca. Competibacter phosphatis', was not detected, Dechloromonas spp. were abundant. The ppk1 gene sequence analysis showed that the type II lineage of 'Ca. Accumulibacter' was dominant. The results suggest that denitrification and phosphorus removal in the A2SBR could be achieved by cooperative activity of 'Ca. Accumulibacter' and nitrate-reducing bacteria.

Atrimonas thermophila gen. nov., sp. nov., a novel anaerobic thermophilic bacterium of the phylum Atribacterota isolated from deep subsurface gas field and proposal of Atrimonadaceae fam. nov. within the class Atribacteria in the phylum Atribacterota.

A novel anaerobic, thermophilic bacterium of the class Atribacteria, strain M15T, was isolated from a high-temperature gas reservoir, Japan. Cells of strain M15T were gram-negative, short oval-shaped, and lacked flagella. Growth occurred at 45-75 °C (optimum 70-75 °C) and pH 6.5-8.5 (optimum pH 7.5-8.0) and was fast under optimal conditions (doubling time 11.4 h). Yeast extract was required for growth. Fermentative growth with glucose, arabinose, xylose, and cellobiose was observed. The major fermentative end products of glucose were acetate and hydrogen. The major cellular fatty acids were C16:0, iso-C15:0, and C18:0. The genomic G + C content was 46.0 mol%. Fluorescence and electron microscopy observations revealed the intracellular localization of genomic DNA surrounded by a membrane in the cells of strain M15T as reported in a sole validly described species of the class Atribacteria in the phylum Atribacterota, Atribacter laminatus strain RT761T, suggesting that the unique morphological traits are widely shared in this class. Phylogenetic analyses indicated that strain M15T belongs to a distinct family-level lineage in the class Atribacteria and shows low similarities to Atribacter laminatus strain RT761T (16S rRNA gene sequence identity of 90.1 %, average nucleotide identity [ANI] of 66.1 %, average amino acid identity [AAI] of 55.8 %). Phenotypic traits of strain M15T (thermophilic, fast-growing, relatively high G + C content, etc.) were clearly distinct from A. laminatus. Based on these phenotypic and genomic properties, we propose a novel genus and species, Atrimonas thermophila gen. nov., sp. nov. for strain M15T (=JCM39389T, =KCTC25731T) representing a novel family Atrimonadaceae fam., nov. in the class Atribacteria.

Metagenome-Assembled Genome Sequence of Marine Rhizobiaceae sp. Strain MnEN-MB40S, Obtained from Manganese-Oxidizing Enrichment Culture.

Here, we report a new metagenome-assembled genome (MAG) from a marine Rhizobiaceae species. The MnEN-MB40S genome was assembled from a manganese-oxidizing enrichment culture metagenome. A 4.1-Mb MAG comprising 26 contigs, with a GC content of 60.0%, was obtained. This MAG contributes to the genomic information regarding the family Rhizobiaceae.

High-rate cotreatment of purified terephthalate and dimethyl terephthalate manufacturing wastewater by a mesophilic upflow anaerobic sludge blanket reactor and the microbial ecology relevant to aromatic compound degradation.

Polyethylene terephthalate (PET) is produced worldwide, mainly as material for plastic drink bottles. PET is produced by polymerization of purified terephthalate (PTA) or dimethyl terephthalate (DMT) with ethylene glycol. During the synthetic manufacturing processes of PTA and DMT, high organic loading wastewater is produced, which is typically treated separately by anaerobic wastewater treatment technologies. Given the high demand for PET, manufacturing plants are expanding globally, which will result in an increase in the amounts of PTA and DMT wastewater in need of treatment. In terms of effective treatment, the cotreatment of PTA and DMT wastewater has several advantages, including lower area and energy requirements. In this study, we examined the performance of an upflow anaerobic sludge blanket (UASB) reactor in cotreating PTA and DMT wastewater with high organic loading, evaluating its removal characteristics after 518 days of continuous operation. In addition, we performed a microbiome analysis of the UASB granular sludge to uncover the microbial interactions and metabolic functions within the reactor. By continuous operation, we achieved an organic removal rate of 6.6 kg m-3 day-1. In addition, we confirmed that aromatic compounds in the complex wastewater from the PTA and DMT manufacturing processes are biodegradable in the following order: benzoate > orthophthalate > terephthalate > isophthalate > p-toluic acid. 16S rRNA gene-based network analysis shows that anaerobic Woesearchaeales belonging to phylum Nanoarchaeota has a positive correlation with Methanoregula, Candidatus Methanofastidiosum, and Methanosarcina, suggesting a symbiotic relationship with methanogens in granular sludge. Shotgun metagenomic analysis revealed that terephthalate, isophthalate/orthophthalate, and benzoate were degraded by different members of Pelotomaculaceae and Syntrophorhabdaceae. According to the genomic information, we propose two new possible routes for orthophthalate degradation by the Syntrophorhabdaceae organism.

16S rRNA gene-based comprehensive analysis of microbial community compositions in a full-scale leachate treatment system.

In this study, we performed a comprehensive analysis of microbial community compositions in leachate and leachate treatment system (14 processes) during dry and rainy seasons (from February to September and from October to January, respectively), at Khanh Son landfill site, Danang City, Vietnam. In this study, raw leachate in dry and rainy seasons was predominated by Arcobacter, Clostridia, Thermotogales, Methanobacteriaceae, and Methanosaeta. During the two seasons, the system had different microbial community compositions. Orders Methanobacteriales, Clostridiales, MBA08 (order-level clone cluster), and Thermotogales predominated the influent, anaerobic pond, and anoxic pond during the dry season, while Campylobacterales and Pseudomonadales orders were predominant in the anaerobic/anoxic systems during the rainy season. In the facultative pond, aerated ponds, sediment tanks, and polishing ponds, predominant orders during the dry season included Actinomycetales, "Saprospirales", Flavobacteriales, Rhizobiales, Rhodospirillales, Burkholderiales, and Alteromonadales; during the rainy season: Sphingobacteriales, Rickettsiales, Sphingomonadales, and Pseudomonadales. In the final post treatment (polishing ponds with vegetation), significant removal of organic matter, total nitrogen, and colour occurred, while nitrogen-fixing and root-associated or related organisms predominated. This suggested that the vegetation in the ponds was essential to achieve the sufficient leachate treatment.

High organic loading treatment for industrial molasses wastewater and microbial community shifts corresponding to system development.

Molasses wastewater contains high levels of organic compounds, cations, and anions, causing operational problems for anaerobic biological treatment. To establish a high organic loading treatment system for industrial molasses wastewater, this study designed a combined system comprising an acidification tank, a thermophilic multi-stage (MS)-upflow anaerobic sludge blanket (UASB) reactor, mesophilic UASB reactor, and down-flow hanging sponge reactor. The average total chemical oxygen demand (COD) and biochemical oxygen demand removal rates were 85%±3% and 95%±2%, respectively, at an organic loading rate of 42kgCODcrm(-3)d(-1) in the MS-UASB reactor. By installation of the acidification tank, the MS-UASB reactor achieved low H2-partial pressure. The abundance of syntrophs such as fatty acid-degrading bacteria increased in the MS-UASB and 2nd-UASB reactors. Thus, the acidification tank contributed to maintaining a favorable environment for syntrophic associations. This study provides new information regarding microbial community composition in a molasses wastewater treatment system.