During an investigation of indigenous prokaryotic species in the Republic of Korea, 17 bacterial strains belonging to the phylum Bacteroidota and 4 strains belonging to the phylum Bacillota were isolated from freshwater environments. These isolated bacterial strains formed a robust phylogenetic clade with type strains of the closest related bacterial species, with 16S rRNA gene sequences sharing similarities of higher than 98.7%. To date, there have been no official report of these 21 isolates in the Republic of Korea. At the genus level, these unreported species were affiliated with Mucilaginibacter and Pedobacter of class Sphingobacteriia, Flavobacterium and Gillisia of class Flavobacteriia, Hymenobacter of class Cytophagia, and Paenibacillus and Planococcus of class Bacilli. These species were further examined by performing Gram staining, analyzing their colonies and cell morphologies, and determining their basic biochemical characteristics and phylogenetic positions. Detailed descriptions of the 21 previously unreported species are provided.

Freshwater environments serve as crucial habitats for diverse microorganisms, playing essential roles alongside plants and animals within the ecosystem. Bacteria in these environments are particularly important for maintaining ecosystem functions. Among them, members of the phylum Pseudomonadota are known to be involved in processes such as nitrogen fixation, ammonia oxidation, and biofilm formation. Pseudomonadota comprises six classes: Acidithiobacillia, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Hydrogenophilia, and Zetaproteobacteria. This study reports 43 previously unrecorded species belonging to the phylum Pseudomonadota. They were isolated from various freshwater habitats in Republic of Korea. These strains were isolated and cultured using nine different standard media, with classification based on Gram staining, cell morphology, and biochemical characteristics. Species identity was determined using 16S rRNA gene sequencing, with a similarity threshold of 98.7% for designation as unreported species. Phylogenetic analysis revealed that these 43 strains were distributed across three classes, 13 orders, 28 families, and 39 genera. This research emphasizes microbial diversity in freshwater ecosystems and provides valuable insights into ecological roles and potential novel functions of unreported bacterial species in Korea.

As part of the 2024 research initiative, “Investigation and Discovery of Prokaryotes in Freshwater Systems,” samples were collected from diverse freshwater habitats, including both water and soil environments. Approximately 2,000 bacterial strains were isolated as single colonies and identified through 16S rRNA gene sequence analysis. Among these, 38 strains shared ≥98.7% 16S rRNA gene sequence similarities with those of known bacterial species not previously reported in Korea. These strains were thus categorized as newly recorded bacterial species in Korea. These 38 bacterial strains displayed significant phylogenetic diversities, spanning 2 phyla, 4 classes, 15 orders, 24 families, and 34 genera. These unrecorded species were classified into the following classes: Actinomycetia (with genera including Microcella, Conyzicola, Curtobacterium, Leucobacter, Microbacterium, Frigoribacterium, Lysinibacter, Streptomyces, Nonomuraea, Actinocorallia, Ruania, and Actinoplanes), Alphaproteobacteria (Paracoccus, Youngimonas, Loktanella, Corticibacterium, Neorhizobium, Onobrychidicola, Ferranicluibacter, Aureimonas, Asticcacaulis, and Novosphingobium), Betaproteobacteria (Rhodoferax, Rugamonas, and Cupriavidus), and Gammaproteobacteria (Rheinheimera, Shewanella, Kosakonia, Leclercia, Hafnia, Yersinia, Pseudomonas, Lysobacter, and Acinetobacter ). Further characterization included assessment of Gram reaction, colony and cell morphology, biochemical properties, and phylogenetic relationships. This report presents detailed phylogenetic and phenotypic characteristics of these bacterial species.

Freshwater environments are rich ecosystems that support diverse microbial communities, including members of the phylum Actinomycetota critical for nutrient cycling, organic matter decomposition, and water quality maintenance. Actinomycetota known to produce numerous bioactive secondary metabolites are valuable in biotechnology, medicine, and agriculture. Despite their significance, the diversity and distribution of Actinomycetota in freshwater habitats, especially in the Republic of Korea, are underexplored. This study aimed to report the isolation and characterization of 22 previously unrecorded bacterial species of Actinomycetota from various freshwater environments in Korea. Using standard dilution plating techniques on six different culture media, 22 bacterial strains were isolated, incubated, and characterized based on colony and cellular morphologies, Gram staining, and biochemical properties. Genomic DNA was extracted and the 16S rRNA gene was sequenced to determine species identity using the EzBioCloud service with a cutoff of 98.7% sequence similarity for classification as unreported species. These strains were phylogenetically diverse, belonging to two classes, ten orders, and eighteen genera. This study enhances our understanding of bacterial diversity in freshwater ecosystems and underscores the importance of exploring microbial diversity in underexplored habitats, potentially leading to discovery of novel bioactive compounds. Findings of this study contribute valuable insights into ecological roles and biotechnological potential of Actinomycetota in freshwater environments.

As part of the research program “Freshwater Prokaryotic Organisms Research and Discovery,” freshwater samples were collected from the Nakdonggang River. After plating the samples on several culture media and incubating aerobically, approximately 900 bacterial strains were isolated and identified using 16S rRNA gene sequences. Among the bacterial isolates showing higher than 98.7% 16S rRNA gene sequence similarity with those of already confirmed bacterial species previously unreported in Korea, 29 strains were selected. These strains were phylogenetically diverse and belonged to 3 phyla, 6 classes, 13 orders, and 21 genera. At the genus level, these previously unreported species were found to be affiliated with Novosphingobium, Sphingomonas, Polymorphobacter, Croceibacterium, Devosia, Endobacterium, Agaricicola, Bradyrhizobium, Paracoccus, and Pseudotabrizicola of the class Alphaproteobacteria; Undibacterium, Azonexus, and Dechloromonas of the class Betaproteobacteria; Acinetobacter and Budvicia of the class Gammaproteobacteria; Streptomyces, Nocardioides, Mycobacterium, and Cellulomonas of the phylum Actinomycetota; Flavobacterium and Pedobacter of the phylum Bacteroidota. These species were further characterized by examining their Gram reaction, colony and cell morphologies, biochemical properties, and phylogenetic positions. Detailed descriptions of these 29 previously unreported species are provided.