Novel aerobic anoxygenic phototrophic bacterium Jannaschia pagri sp. nov., isolated from seawater around a fish farm.

The genus Jannaschia is one of the representatives of aerobic anoxygenic phototrophic (AAP) bacteria, which is a strictly aerobic bacterium, producing a photosynthetic pigment bacteriochlorophyll (BChl) a. However, a part of the genus Jannaschia members have not been confirmed the photosynthetic ability. The partly presence of the ability in the genus Jannaschia could suggest the complexity of evolutionary history for anoxygenic photosynthesis in the genus, which is expected as gene loss and/or horizontal gene transfer. Here a novel AAP bacterium designated as strain AI_62T (= DSM 115720 T = NBRC 115938 T), was isolated from coastal seawater around a fish farm in the Uwa Sea, Japan. Its closest relatives were identified as Jannaschia seohaensis SMK-146 T (95.6% identity) and J. formosa 12N15T (94.6% identity), which have been reported to produce BChl a. The genomic characteristic of strain AI_62T clearly showed the possession of the anoxygenic photosynthesis related gene sets. This could be a useful model organism to approach the evolutionary mystery of anoxygenic photosynthesis in the genus Jannaschia. Based on a comprehensive consideration of both phylogenetic and phenotypic characteristics, we propose the classification of a novel species within the genus Jannaschia, designated as Jannaschia pagri sp. nov. The type strain for this newly proposed species is AI_62T (= DSM 115720 T = NBRC 115938 T).

Microbiomic Analysis of Bacteria Associated with Rock Tripe Lichens from Alpine Areas in Eastern Alps and Equatorial Africa.

The diversity of bacteria associated with alpine lichens was profiled. Lichen samples belonging to the Umbilicariaceae family, commonly known as rock tripe lichens, were gathered from two distinct alpine fellfields: one situated on Mt. Brennkogel located in the Eastern European Alps (Austria), and the other on Mt. Stanley located in the Rwenzori mountains of equatorial Africa (Uganda). The primary aim of this research was to undertake a comparative investigation into the bacterial compositions, and diversities, identifying potential indicators and exploring their potential metabolisms, of these lichen samples. Bulk genomic DNA was extracted from the lichen samples, which was used to amplify the 18S rRNA gene by Sanger sequencing and the V3-V4 region of the 16S rRNA gene by Illumina Miseq sequencing. Examination of the fungal partner was carried out through the analysis of 18S rRNA gene sequences, belonging to the genus Umbilicaria (Ascomycota), and the algal partner affiliated with the lineage Trebouxia (Chlorophyta), constituted the symbiotic components. Analyzing the MiSeq datasets by using bioinformatics methods, operational taxonomic units (OTUs) were established based on a predetermined similarity threshold for the V3-V4 sequences, which were assigned to a total of 26 bacterial phyla that were found in both areas. Eight of the 26 phyla, i.e. Acidobacteriota, Actinomycota, Armatimonadota, Bacteroidota, Chloroflexota, Deinococcota, Planctomycetota, and Pseudomonadota, were consistently present in all samples, each accounting for more than 1% of the total read count. Distinct differences in bacterial composition emerged between lichen samples from Austria and Uganda, with the OTU frequency-based regional indicator phyla, Pseudomonadota and Armatimonadota, respectively. Despite the considerable geographic separation of approximately 5430 km between the two regions, the prediction of potential metabolic pathways based on OTU analysis revealed similar relative abundances. This similarity is possibly influenced by comparable alpine climatic conditions prevailing in both areas.

Diversity profiling of microbiomes associated with selected alpine plants and lichens from Mt. Suisho, Japan.

We report 16S rRNA gene amplicon data for the microbiomes in selected alpine plants (genera Artemisia, Parnassia, and Phyllodoce) and lichens (genera Cladonia and a mixture of Miriquidica and Rhizocarpon) from Mt. Suisho, Japan. Most of these samples were dominated by Pseudomonadota, while some contained the rarely cultivated phylum Vulcanimicrobiota (Candidatus Eremiobacterota/WPS-2).

The fatal case of "Pigmentibacter" bacteremia following aspiration pneumonia in elderly patient.

"Pigmentibacter ruber" was first reported in 2021, a novel bacterium of the family Silvanigrellaceae, isolated from human blood of the patient with aspiration pneumonia after the drowning accident in Republic of China. However, until now, there is only one report describing "P. ruber" infection, and no case of isolation from natural environment has been reported so far. Thus, the infectivity and pathogenicity of "Pigmentibacter" spp. has not been clearly understood. In this report, we described the fatal case of "Pigmentibacter" bacteremia subsequently occurred after aspiration pneumonia probably due to accidental ingestion of irrigation water in the elderly patient. Despite administration of broad-spectrum antibiotic, the patient dramatically deteriorated and eventually deceased. Whole-genome sequencing showed the strain isolated from the patient was identified as "Pigmentibacter" sp. (designated as strain Takaoka) and antimicrobial sensitivity testing showed it displayed high minimum inhibitory concentrations against various antibiotics including ß-lactam. Further studies are needed to clarify the clinical characteristics of "Pigmentibacter" and its relative's infections and their antimicrobial sensitivity; however, the present case supported the clinical characteristics of "Pigmentibacter" infection, which can lead to bacteremia following aspiration pneumonia caused by mis-swallowing contaminated water, and poor outcome potentially due to multidrug resistances.

Complete genome sequence of Opitutales bacterium strain ASA1, isolated from soil.

We present the complete genome of Opitutales bacterium ASA1, isolated from soil. The genome is 5,821,695 bp with 4,638 protein-coding sequences. The genome data suggest that this strain belongs to the class Opitutae of the phylum Verrucomicrobiota, and its genome has six unique biosynthetic gene clusters associated with secondary metabolites.

Microscopic and metatranscriptomic analyses revealed unique cross-domain parasitism between phylum Candidatus Patescibacteria/candidate phyla radiation and methanogenic archaea in anaerobic ecosystems.

To verify whether members of the phylum Candidatus Patescibacteria parasitize archaea, we applied cultivation, microscopy, metatranscriptomic, and protein structure prediction analyses on the Patescibacteria-enriched cultures derived from a methanogenic bioreactor. Amendment of cultures with exogenous methanogenic archaea, acetate, amino acids, and nucleoside monophosphates increased the relative abundance of Ca. Patescibacteria. The predominant Ca. Patescibacteria were families Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae, and the former showed positive linear relationships (r2 ≥ 0.70) Methanothrix in their relative abundances, suggesting related growth patterns. Methanothrix and Methanospirillum cells with attached Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae, respectively, had significantly lower cellular activity than those of the methanogens without Ca. Patescibacteria, as extrapolated from fluorescence in situ hybridization-based fluorescence. We also observed that parasitized methanogens often had cell surface deformations. Some Methanothrix-like filamentous cells were dented where the submicron cells were attached. Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae highly expressed extracellular enzymes, and based on structural predictions, some contained peptidoglycan-binding domains with potential involvement in host cell attachment. Collectively, we propose that the interactions of Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae with methanogenic archaea are parasitisms.IMPORTANCECulture-independent DNA sequencing approaches have explored diverse yet-to-be-cultured microorganisms and have significantly expanded the tree of life in recent years. One major lineage of the domain Bacteria, Ca. Patescibacteria (also known as candidate phyla radiation), is widely distributed in natural and engineered ecosystems and has been thought to be dependent on host bacteria due to the lack of several biosynthetic pathways and small cell/genome size. Although bacteria-parasitizing or bacteria-preying Ca. Patescibacteria have been described, our recent studies revealed that some lineages can specifically interact with archaea. In this study, we provide strong evidence that the relationship is parasitic, shedding light on overlooked roles of Ca. Patescibacteria in anaerobic habitats.

Draft genome sequences of Corallococcus strains KH5-1 and NO1, isolated from activated sludge.

Myxobacteria are known as prolific producers of secondary metabolites with a unique and wide spectrum of bioactivities. Here, we report draft genome sequences of KH5-1 and NO1, myxobacteria isolated from activated sludge, which consist of 9.89 and 9.86 Mb, both of which have G + C contents of 70.7%.

Complete Genome Sequence of Polynucleobacter sp. Strain TUM22923, Isolated from Antarctic Lake Sediment.

Here, we report the complete genome sequence of Polynucleobacter sp. strain TUM22923, isolated from Antarctic lake sediment. This strain has a genome of 1,860,127 bp, comprising 1,848 protein-coding sequences. These sequence data could contribute to the elucidation of genome streamlining and low-temperature adaptation in members of Polynucleobacter, a cosmopolitan group of ultramicrobacteria.

Whole-Genome Sequences of Three Psychrotolerant Mycolicibacterium sp. Strains Isolated from Antarctic Soil.

We report the whole-genome sequences of three psychrotolerant Mycolicibacterium strains, TUM20983, TUM20984, and TUM20985, isolated from Antarctic soils. Taxonomic analyses indicate that these strains are putative new species. These genome sequences may provide insight into the cold adaptation mechanisms of Mycolicibacterium spp. through future comparative genomic studies.

A New Species of Bisexual Milnesium (Eutardigrada: Apochela) Having Aberrant Claws from Innhovde, Dronning Maud Land, East Antarctica.

There have been several records in the last 60 years for East Antarctica for Milnesium tardigradum Doyère, 1840 sensu lato, now considered a species complex. During the 56th Japanese Antarctic Research Expedition summer operation (2014-2015), a new tardigrade species in the genus Milnesium Doyère, 1840 was found in an ice-free Innhovde area along Lützow-Holm Bay, Dronning Maud Land, East Antarctica. The new species has aberrant claws with four to seven points on each secondary claw branch, which distinguishes it from other Milnesium species. A male specimen was found in the population and evidence showed that an isolated adult female moulted twice without oviposition. This strongly suggested bisexual reproduction for this population. The new species, Milnesium rastrum sp. nov., is described with its phylogenetic position and a discussion on the reproductive strategies for the harsh environments.

Unique H2-utilizing lithotrophy in serpentinite-hosted systems.

Serpentinization of ultramafic rocks provides molecular hydrogen (H2) that can support lithotrophic metabolism of microorganisms, but also poses extremely challenging conditions, including hyperalkalinity and limited electron acceptor availability. Investigation of two serpentinization-active systems reveals that conventional H2-/CO2-dependent homoacetogenesis is thermodynamically unfavorable in situ due to picomolar CO2 levels. Through metagenomics and thermodynamics, we discover unique taxa capable of metabolism adapted to the habitat. This included a novel deep-branching phylum, "Ca. Lithacetigenota", that exclusively inhabits serpentinite-hosted systems and harbors genes encoding alternative modes of H2-utilizing lithotrophy. Rather than CO2, these putative metabolisms utilize reduced carbon compounds detected in situ presumably serpentinization-derived: formate and glycine. The former employs a partial homoacetogenesis pathway and the latter a distinct pathway mediated by a rare selenoprotein-the glycine reductase. A survey of microbiomes shows that glycine reductases are diverse and nearly ubiquitous in serpentinite-hosted environments. "Ca. Lithacetigenota" glycine reductases represent a basal lineage, suggesting that catabolic glycine reduction is an ancient bacterial innovation by Terrabacteria for gaining energy from geogenic H2 even under hyperalkaline, CO2-poor conditions. Unique non-CO2-reducing metabolisms presented here shed light on potential strategies that extremophiles may employ for overcoming a crucial obstacle in serpentinization-associated environments, features potentially relevant to primordial lithotrophy in early Earth.

Novel Cross-domain Symbiosis between Candidatus Patescibacteria and Hydrogenotrophic Methanogenic Archaea Methanospirillum Discovered in a Methanogenic Ecosystem.

To identify novel cross-domain symbiosis between Candidatus Patescibacteria and Archaea, we performed fluorescence in situ hybridization (FISH) on enrichment cultures derived from methanogenic bioreactor sludge with the newly designed 32-520-1066 probe targeting the family-level uncultured clade 32-520/UBA5633 lineage in the class Ca. Paceibacteria. All FISH-detectable 32-520/UBA5633 cells were attached to Methanospirillum, indicating high host specificity. Transmission electron microscopy observations revealed 32-520/UBA5633-like cells that were specifically adherent to the plug structure of Methanospirillum-like rod-shaped cells. The metagenome-assembled genomes of 32-520/UBA5633 encoded unique gene clusters comprising pilin signal peptides and type IV pilins. These results provide novel insights into unseen symbiosis between Ca. Patescibacteria and Archaea.

Microbiomic Analysis of Bacteria Associated with Rock Tripe Lichens in Continental and Maritime Antarctic Regions.

Increased research attention is being given to bacterial diversity associated with lichens. Rock tripe lichens (Umbilicariaceae) were collected from two distinct Antarctic biological regions, the continental region near the Japanese Antarctic station (Syowa Station) and the maritime Antarctic South Orkney Islands (Signy Island), in order to compare their bacterial floras and potential metabolism. Bulk DNA extracted from the lichen samples was used to amplify the 18S rRNA gene and the V3-V4 region of the 16S rRNA gene, whose amplicons were Sanger- and MiSeq-sequenced, respectively. The fungal and algal partners represented members of the ascomycete genus Umbilicaria and the green algal genus Trebouxia, based on 18S rRNA gene sequences. The V3-V4 sequences were grouped into operational taxonomic units (OTUs), which were assigned to eight bacterial phyla, Acidobacteriota, Actinomyceota, Armatimonadota, Bacteroidota, Cyanobacteria, Deinococcota, Pseudomonadota and the candidate phylum Saccharibacteria (also known as TM7), commonly present in all samples. The OTU floras of the two biological regions were clearly distinct, with regional biomarker genera, such as Mucilaginibacter and Gluconacetobacter, respectively. The OTU-based metabolism analysis predicted higher membrane transport activities in the maritime Antarctic OTUs, probably influenced by the sampling area's warmer maritime climatic setting.

Isolation of Aquatic Plant Growth-Promoting Bacteria for the Floating Plant Duckweed (Lemna minor).

Plant growth-promoting bacteria (PGPB) can exert beneficial growth effects on their host plants. Little is known about the phylogeny and growth-promoting mechanisms of PGPB associated with aquatic plants, although those of terrestrial PGPB have been well-studied. Here, we report four novel aquatic PGPB strains, MRB1-4 (NITE P-01645-P-01648), for duckweed Lemna minor from our rhizobacterial collection isolated from Lythrum anceps. The number of L. minor fronds during 14 days co-culture with the strains MRB1-4 increased by 2.1-3.8-fold, compared with an uninoculated control; the plant biomass and chlorophyll content in co-cultures also increased. Moreover, all strains possessed an indole-3-acetic acid production trait in common with a plant growth-promoting trait of terrestrial PGPB. Phylogenetic analysis showed that three strains, MRB-1, -3, and -4, were affiliated with known proteobacterial genera (Bradyrhizobium and Pelomonas); this report is the first to describe a plant-growth promoting activity of Pelomonas members. The gammaproteobacterial strain MRB2 was suggested to be phylogenetically novel at the genus level. Under microscopic observation, the Pelomonas strain MRB3 was epiphytic and adhered to both the root surfaces and fronds of duckweed. The duckweed PGPB obtained here could serve as a new model for understanding unforeseen mechanisms behind aquatic plant-microbe interactions.

Symbiosis between Candidatus Patescibacteria and Archaea Discovered in Wastewater-Treating Bioreactors.

Each prokaryotic domain, Bacteria and Archaea, contains a large and diverse group of organisms characterized by their ultrasmall cell size and symbiotic lifestyles (potentially commensal, mutualistic, and parasitic relationships), namely, Candidatus Patescibacteria (also known as the Candidate Phyla Radiation/CPR superphylum) and DPANN archaea, respectively. Cultivation-based approaches have revealed that Ca. Patescibacteria and DPANN symbiotically interact with bacterial and archaeal partners and hosts, respectively, but that cross-domain symbiosis and parasitism have never been observed. By amending wastewater treatment sludge samples with methanogenic archaea, we observed increased abundances of Ca. Patescibacteria (Ca. Yanofskybacteria/UBA5738) and, using fluorescence in situ hybridization (FISH), discovered that nearly all of the Ca. Yanofskybacteria/UBA5738 cells were attached to Methanothrix (95.7 ± 2.1%) and that none of the cells were attached to other lineages, implying high host dependency and specificity. Methanothrix filaments (multicellular) with Ca. Yanofskybacteria/UBA5738 attached had significantly more cells with no or low detectable ribosomal activity (based on FISH fluorescence) and often showed deformations at the sites of attachment (based on transmission electron microscopy), suggesting that the interaction is parasitic. Metagenome-assisted metabolic reconstruction showed that Ca. Yanofskybacteria/UBA5738 lacks most of the biosynthetic pathways necessary for cell growth and universally conserves three unique gene arrays that contain multiple genes with signal peptides in the metagenome-assembled genomes of the Ca. Yanofskybacteria/UBA5738 lineage. The results shed light on a novel cross-domain symbiosis and inspire potential strategies for culturing CPR and DPANN. IMPORTANCE One highly diverse phylogenetic group of Bacteria, Ca. Patescibacteria, remains poorly understood, but, from the few cultured representatives and metagenomic investigations, they are thought to live symbiotically or parasitically with other bacteria or even with eukarya. We explored the possibility of symbiotic interactions with Archaea by amending wastewater treatment sludge samples that were rich in Ca. Patescibacteria and Archaea with an isolate archaeon that is closely related to a methanogen population abundant in situ (Methanothrix). This strategic cultivation successfully established enrichment cultures that were mainly comprised of Ca. Patescibacteria (family level lineage Ca. Yanofskybacteria/UBA5738) and Methanothrix, in which we found highly specific physical interactions between the two organisms. Microscopic observations based on transmission electron microscopy, target-specific fluorescence in situ hybridization, and metagenomic analyses showed evidence that the interaction is likely parasitic. The results show a novel cross-domain parasitism between Bacteria and Archaea and suggest that the amendment of host Archaea may be an effective approach in culturing novel Ca. Patescibacteria.

Phylotypic Diversity of Bacteria Associated with Speleothems of a Silicate Cave in a Guiana Shield Tepui.

The diversity of microorganisms associated with speleological sources has mainly been studied in limestone caves, while studies in silicate caves are still under development. Here, we profiled the microbial diversity of opal speleothems from a silicate cave in Guiana Highlands. Bulk DNAs were extracted from three speleothems of two types, i.e., one soft whitish mushroom-like speleothem and two hard blackish coral-like speleothems. The extracted DNAs were amplified for sequencing the V3-V4 region of the bacterial 16S rRNA gene by MiSeq. A total of 210,309 valid reads were obtained and clustered into 3184 phylotypes or operational taxonomic units (OTUs). The OTUs from the soft whitish speleothem were mostly affiliated with Acidobacteriota, Pseudomonadota (formerly, Proteobacteria), and Chloroflexota, with the OTUs ascribed to Nitrospirota being found specifically in this speleothem. The OTUs from the hard blackish speleothems were similar to each other and were mostly affiliated with Pseudomonadota, Acidobacteriota, and Actinomycetota (formerly, Actinobacteria). These OTU compositions were generally consistent with those reported for limestone and silicate caves. The OTUs were further used to infer metabolic features by using the PICRUSt bioinformatic tool, and membrane transport and amino acid metabolism were noticeably featured. These and other featured metabolisms may influence the pH microenvironment and, consequently, the formation, weathering, and re-deposition of silicate speleothems.

Diversity of Candidatus Patescibacteria in Activated Sludge Revealed by a Size-|Fractionation Approach.

Uncultivated members of Candidatus Patescibacteria are commonly found in activated sludge treating sewage and are widely distributed in wastewater treatment plants in different regions and countries. However, the phylogenetic diversity of Ca. Patescibacteria is difficult to examine because of their low relative abundance in the environment. Since Ca. Patescibacteria members have small cell sizes, we herein collected small microorganisms from activated sludge using a filtration-based size-fractionation approach (i.e., 0.45-0.22| |µm and 0.22-0.1| |µm fractions). Fractionated samples were characterized using 16S rRNA gene amplicon and shotgun metagenomic sequence ana-lyses. The amplicon ana-lysis revealed that the relative abundance of Ca. Patescibacteria increased to 73.5% and 52.5% in the 0.45-0.22| |µm and 0.22-0.1| |µm fraction samples, respectively, from 5.8% in the unfractionated sample. The members recovered from the two size-fractionated samples included Ca. Saccharimonadia, Ca. Gracilibacteria, Ca. Paceibacteria, Ca. Microgenomatia, class-level uncultured lineage ABY1, Ca. Berkelbacteria, WS6 (Ca. Dojkabacteria), and WWE3, with Ca. Saccharimonadia being predominant in both fraction samples. The number of operational taxonomic units belonging to Ca. Patescibacteria was approximately 6-fold higher in the size-fractionated samples than in the unfractionated sample. The shotgun metagenomic ana-lysis of the 0.45-0.22| |µm fractioned sample enabled the reconstruction of 24 high-quality patescibacterial bins. The bins obtained were classified into diverse clades at the family and genus levels, some of which were rarely detected in previous activated sludge studies. Collectively, the present results suggest that the overall diversity of Ca. Patescibacteria inhabiting activated sludge is higher than previously expected.

Characterization of the First Cultured Psychrotolerant Representative of Legionella from Antarctica Reveals Its Unique Genome Structure.

Culture-independent analysis shows that Legionella spp. inhabit a wide range of low-temperature environments, but to date, no psychrotolerant or psychrophilic strains have been reported. Here, we characterized the first cultivated psychrotolerant representative, designated strain TUM19329T, isolated from an Antarctic lake using a polyphasic approach and comparative genomic analysis. A genome-wide phylogenetic tree indicated that this strain was phylogenetically separate at the species level. Strain TUM19329T shared common physiological traits (e.g., Gram-negative, limited growth on buffered charcoal-yeast extract α-ketoglutarate [BCYEα] agar with l-cysteine requirements) with its relatives, but it also showed psychrotolerant growth properties (e.g., growth at 4°C to 25°C). Moreover, this strain altered its own cellular fatty acid composition to accumulate unsaturated fatty acid at a lower temperature, which may help maintain the cell membrane fluidity. Through comparative genomic analysis, we found that this strain possessed massive mobile genetic elements compared with other species, amounting to up to 17% of the total genes. The majority of the elements were the result of the spread of only a few insertion sequences (ISs), which were spread throughout the genome by a "copy-and-paste" mechanism. Furthermore, we found metabolic genes, such as fatty acid synthesis-related genes, acquired by horizontal gene transfer (HGT). The expansion of ISs and HGT events may play a major role in shaping the phenotype and physiology of this strain. On the basis of the features presented here, we propose a new species-Legionella antarctica sp. nov.-represented by strain TUM19329T (= GTC 22699T = NCTC 14581T). IMPORTANCE This study characterized a unique cultivated representative of the genus Legionella isolated from an Antarctic lake. This psychrotolerant strain had some common properties of known Legionella species but also displayed other characteristics, such as plasticity in fatty acid composition and an enrichment of mobile genes in the genome. These remarkable properties, as well as other factors, may contribute to cold hardiness, and this first cultivated cold-tolerant strain of the genus Legionella may serve as a model bacterium for further studies. It is worth noting that environmentally derived 16S rRNA gene phylotypes closely related to the strain characterized here have been detected from diverse environments outside Antarctica, suggesting a wide distribution of psychrotolerant Legionella bacteria. Our culture- and genome-based findings may accelerate the ongoing studies of the behavior and pathogenicity of Legionella spp., which have been monitored for many years in the context of public health.

Draft Genome Sequence of Novel Filterable Rhodospirillales Bacterium Strain TMPK1, Isolated from Soil.

We report the draft genome sequence of novel Rhodospirillales bacterium strain TMPK1, isolated from a micropore-filtered soil suspension. This strain has a genome of 4,249,070 bp, comprising 4,151 protein-coding sequences. The genome sequence data further suggest that strain TMPK1 is an alphaproteobacterium capable of carotenoid production.

Internal microbial zonation during the massive growth of marimo, a lake ball of Aegagropila linnaei in Lake Akan.

Marimo (lake ball) is an uncommon ball-like aggregation of the green alga, Aegagropila linnaei. Although A. linnaei is distributed in fresh and brackish waters in the northern hemisphere, marimo colonies are found only in particular habitats. Here, we report the bacterial communities inside various sizes and aggregating structures of natural marimo collected from Lake Akan, Japan. We observed multi-layers composed of sediment particles only in the sizable radial-type marimo with >20 cm diameter and not in the tangled-type marimo. The deeper layers were enriched by Nitrospira, potential sulfur-oxidizing bacteria, and sulfate-reducing bacteria. Microorganisms of the multi-layers would form biofilms incorporating nearby sediment, which would function as microbial "seals" within large radial-type marimo. These findings provide clues to deciphering the growth of endangered marimo.