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ISSN : 1226-9999(Print)
ISSN : 2287-7851(Online)

Korean J. Environ. Biol. Vol.43 No.1 pp.58-76
DOI : https://doi.org/10.11626/KJEB.2025.43.1.058

A report of 43 unrecorded bacterial species belonging to the phyla Pseudomonadota isolated from freshwater environments in Republic of Korea

Soo-Yeong Lee^*

, Ahyoung Choi, Hyangmi Kim, Ji Young Jung, Ja Young Cho, Seoni Hwang, Jaeduk Goh

Biological Resources Research Department, Nakdonggang National Institute of Biological Resources (NNIBR), Sangju 37242, Republic of Korea

^*Corresponding author Corresponding author Soo-Yeong Lee Tel. 054-530-0844 E-mail. sylee94@nnibr.re.kr

Contribution to Environmental Biology

▪ This study aims to provide a taxonomic perspective on previously unrecorded species reported abroad but not yet reported in Korea.

Received 12/12/2024 Review 05/02/2025 Accepted 17/02/2025

Abstract

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.

Key Words : unrecorded bacterial species , freshwater environments , phylum Pseudomonadota

초록

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1. INTRODUCTION

Freshwater ecosystems are rich in microbial diversity, with numerous bacterial species yet to be documented (Dudgeon et al. 2006). Recent studies in South Korea have identified several previously unrecorded bacterial species in freshwater environments. For instance, a study focusing on the Yeongsan River isolated 45 bacterial species not previously reported in Korea (Park et al. 2024). Among these microbial communities, members of the phylum Pseudomonadota, formerly known as Proteobacteria, stand out due to their ecological signifi- cance and functional diversity (Aharon 2024). Pseudomonadota are widely distributed in various habitats, from soil and oceans to freshwater systems, where they play essential roles in biogeochemical processes and the breakdown of organic compounds (Garrity et al. 2005). This phylum includes bacteria with broad metabolic capabilities, enabling them to adapt to varying environmental conditions and serve as critical players in microbial food webs (Spain et al. 2009). The phylum Pseudomonadota encompasses multiple classes, including Alpha-, Beta-, Gamma-, Delta-, and Epsilonproteobacteria, each contributing uniquely to the ecosystem functions. Gamma- and Betaproteobacteria, in particular, are highly abundant in freshwater environments and include genera such as Pseudomonas, Acinetobacter, and Legionella, which are known for their metabolic diversity and roles in nitrogen and sulfur cycling (Newton et al. 2011). These bacteria are also notable for their potential applications in bioremediation and biotechnology due to their ability to degrade various pollutants and produce bioactive compounds (Timmis et al. 2019). Despite the ecological and biotechnological importance of Pseudomonadota, the diversity and distribution of this phylum in freshwater habitats remain underexplored, especially in regions such as Korea. Most studies on Pseudomonadota have centered on marine ecosystems or soil, leaving a significant gap in our knowledge of freshwater environments (Park et al. 2020;Kim et al. 2021). The distinct physicochemical characteristics of freshwater habitats, including rivers, lakes, and reservoirs, present unique ecological niches that may support novel bacterial species or strains with potential for new biotechnological applications (Zwart et al. 2002).

In this study, we report the isolation and characterization of 43 Pseudomonadota strains from diverse freshwater environments in Korea, contributing to the understanding of microbial diversity in these ecosystems. Comparative sequence analysis confirmed that the isolates exhibited high 16S rRNA gene sequence similarity (>98.7%) with previously described species, indicating that they are not novel species but are first reported in Korea. Additionally, genetic, physiological, and morphological analyses further confirmed that these strains represent unrecorded bacterial species in Korea. By investigating the genetic and metabolic characteristics of these isolates, this research aims to shed light on their ecological roles and explore their potential in applications such as bioremediation and natural product discovery. Our findings underscore the need for continued exploration of microbial diversity in underrepresented habitats, as they may harbor previously unknown bacterial species with promising capabilities for environmental and industrial applications.

2. MATERIALS AND METHODS

A total of 43 bacterial strains were isolated from various freshwater habitats using the standard dilution plating technique across different culture media, including marine agar 2216 (MA; Difco, USA), Reasoner’s 2A (R2A; Difco, USA), 1/10-diluted R2A (1/10 R2A; Difco, USA), nutrient agar (NA; MB-cell, Korea), 1/10-diluted tryptic soy agar (1/10 TSA; Difco, USA), and Inorganic Salts Starch (ISP) Agar 4 (ISP4; Difco, USA). These media were chosen for their effectiveness in fostering the growth of a wide range of bacterial communities, particularly Pseudomonadota, which demand unique nutrient compositions. Isolates were incubated for 14 days at temperatures ranging from 15 to 25°C to support growth conditions suitable for various bacterial taxa. Following serial dilution spreading, single colonies were isolated for each strain, and the pure cultures were preserved in 20% glycerol at -80°C. Table 1 details the strain IDs, corresponding culture media, and specific incubation conditions.

For 16S rRNA sequence analysis, genomic DNA was extracted from the isolates using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The 16S rRNA gene was amplified through PCR with universal bacterial primers 27F and 1492R, and sequencing was performed via the Sanger method (Weisburg et al. 1991). Sequence identification utilized the “16S-based ID” service in EzBio Cloud (Yoon et al. 2017), with a similarity threshold set at 98.7%. Isolates with ≥98.7% similarity to known bacterial species that had not been previously recorded in Korea were categorized as unreported species.

Phylogenetic analyses were conducted by aligning the 16S rRNA sequences of the isolates with reference-type strains using the Clustal_W program, with further manual verification through EzEditor (Jeon et al. 2014). Phylogenetic trees were then constructed with the neighborjoining method (Saitou and Nei 1987) and the Kimura 2-parameter model (Kimura 1980) in MEGA 7.0 software (Kumar et al. 2016). Bootstrap analysis, with 1,000 random re-samplings, was used to confirm the robustness of the phylogenetic trees (Felsenstein 1985).

Morphological properties which cell size, cell shape, and motility of strains were observed by a transmission electron microscope (H-7650; Hitachi, Japan), using cells grown on growth medium. TEM specimens were prepared with uranyless solution (EM-stain) on a carboncoated copper grid. Gram staining was performed using a bioMérieux Gram-staining kit, and additional biochemical characteristics were examined via API 20NE galleries (bioMérieux, Marcy-I’Étoile, France) following standard protocols.

3. RESULTS AND DISCUSSION

Analysis of 16S rRNA gene sequences from approximately 1,000 bacterial strains obtained in this study revealed the presence of strains classified as novel species or species not previously reported in Korea. Among these, 43 strains showed ≥98.7% 16S rRNA gene sequence similarity to unrecorded bacterial species in Korea and were distributed across 3 classes, 13 orders, 28 families and 39 genera. The taxonomic classifications and identification results for these species are summarized in Table 1. The phylogenetic tree illustrating the relationships of the bacterial strains assigned to the phylum Pseudomonadota is presented in Figure 1 and Figure 2. The 43 strains assigned to the phylum Pseudomonadota were phylogenetically associated with various genera across different classes. Within the class Alphaproteobacteria, the strains belonged to twenty-three genera: Aestuariivirga, Ancylobacter, Asticcacaulis, Aureimonas, Azospirillum, Bradyrhizobium, Cypionkella, Donghicola, Hoeflea, Kaistia, Kandeliimicrobium, Lacibacterium, Loktanella, Neoroseomonas, Novosphingobium, Pedomicrobium, Pelagimonas, Rhizobium, Rhodobacter, Rhodospirillum, Roseomonas, Siculibacillus, Tabrizicola. Within the class Betaproteobacteria, they were affiliated with eight genera: Aromatoleum, Duganella, Herminiimonas, Inhella, Pelomonas, Rhodoferax, Vogesella, Xenophilus. Additionally, within the class Gammaproteobacteria, the strains were classified into eight genera: Arenimonas, Dyella, Hafnia, Kluyvera, Pseudomonas, Rheinheimera, Stenotrophomonas, Steroidobacter (Figs. 1, 2). These figures show the phylogenetic relationships between the isolates and their closely related species. Transmission electron microscopy images of the isolates are displayed in Figure 3.

In summary, this study identified 43 bacterial species that have not been previously reported in Korean freshwater ecosystems. The characteristics of these unreported bacterial species are detailed and described in this report.

3.1. Description of Asticcacaulis taihuensis 21LS1-75

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on 1/10 R2A are circular, convex, smooth, and ivory after incubation for 2-3 days at 20°C. Positive for urease, gelatinase and β-galactosidase, negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, esculin hydrolysis in API 20NE. D-glucose, L-arabinose, Dmannitol, N-acetyl-glucosamine are utilized. Dose not utilize D-mannose, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and Phenylacetic acid. This strain was positive for oxidase activity. Strain 21LS1-75 displays the highest 16S rRNA gene sequence similarity with Asticcacaulis taihuensis CGMCC 1.3431^T (99.5%). Strain 21LS1-75 (=NNIBR2021641BA44) was isolated from soil collected in Yangyang-si, Gangwon-do, Korea (37.952996°N, 128.768576°E). The GenBank accession number of the 16S rRNA gene sequence is OL773519.

3.2. Description of Pelagimonas varians LW3-33

Cells are Gram-stain-negative, non-flagellated, and short rod-shaped. Colonies grown on MA are circular, convex with entire edge and ivory after incubation for 3 days at 25°C. Positive for nitrate reduction, indole production, esculin hydrolysis, and β-galactosidase, but, glucose fermentation, arginine dihydrolase, urease, gelatinase in API 20NE. Does not all assimilate substrates: D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid adipic acid, malic acid, trisodium citrate, and phenylacetic acid. This strain was positive for oxidase activity. Strain LW3-33 displays the highest 16S rRNA gene sequence similarity with Pelagimonas varians SH4-1^T (99.9%). Strain LW3-33 (=NNIBR2017 301BA8) was isolated from fresh water collected in Jeodong, Gangneung, Gangwon-do, Korea (37.7970833°N, 128.8978°E). The GenBank accession number of the 16S rRNA gene sequence is MG818302.

3.3. Description of Loktanella salsilacus SJW1-31

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on NA are circular, convex, and white after incubation for 3 days at 25°C. Positive for nitrate reduction, indole production, urease, esculin hydrolysis, β-galactosidase, negative for glucose fermentation, arginine dihydrolase, and gelatinase in API 20NE. Does not all assimilate substrates: D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid adipic acid, malic acid, trisodium citrate, and phenylacetic acid. This strain showed a positive result for oxidase activity. Strain SJW1-31 displays the highest 16S rRNA gene sequence similarity with Loktanella salsilacus DSM 16199^T (100.0%). Strain SJW1-31 (=NNIBR 2017301BA10) was isolated from brackish water collected in Jinwol-myeon, Gwangyang, Jeollanam-do, Korea (35.063779°N, 127.741095°E). The GenBank accession number of the 16S rRNA gene sequence is MG818304.

3.4. Description of Donghicola mangrovi 6LW2-50

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, smooth, and cream after incubation for 3 days at 25°C. Positive for urease, esculin hydrolysis, and β-galactosidase in API 20NE, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, and gelatinase. L-arabinose, D-mannitol, and malic acid are assimilated. Does not assimilate D-glucose, D-mannose, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, trisodium citrate, and phenylacetic acid. The strain tested positive for oxidase activity. Strain 6LW2-50 displays the highest 16S rRNA gene sequence similarity with Donghicola mangrove B5-SW-15^T (99.9%). Strain 6LW2-50 (=NNIBR 2022641BA2178) was isolated from freshwater collected in Yangyang-gun, Gangwon-do, Korea (37.9534075°N, 128.7696925°E). The GenBank accession number of the 16S rRNA gene sequence is OP872589.

3.5. Description of Cypionkella sinensis 21YS10S-0N32

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, translucent, and cream after incubation for 3 days at 25°C. Positive for urease, esculin hydrolysis, and β-galactosidase in API 20NE, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, and gelatinase. D-mannose, N-acetyl-glucosamine, and D-maltose are assimilated. Does not assimilate D-glucose, L-arabinose, D-mannitol, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. This strain showed a positive result for oxidase activity. Strain 21YS10S-0N32 displays the highest 16S rRNA gene sequence similarity with Cypionkella sinensis DSM 22992^T (99.5%). Strain 21YS10S-0N32 (=NNIBR2021641BA1666) was isolated from soil collected in Yeongam-gun, Jeollanam-do, Korea (34.784111°N, 126.720166°E). The GenBank accession number of the 16S rRNA gene sequence is MZ976829.

3.6. Description of Kandeliimicrobium roseum LS9-139

Cells are Gram-stain-positive, flagellated, and short rod-shaped. Colonies grown on MA are glistening, convex, smooth with entire margin, and beige-pink after incubation for 3 days at 25°C. Positive for glucose fermentation, arginine dihydrolase, urease, gelatinase, and β-galactosidase in API 20NE, but nitrate reduction, indole production, and esculin hydrolysis. L-arabinose, D-mannose, N-acetyl-glucosamine are assimilated. Does not assimilate D-glucose, D-mannitol, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. This strain showed a positive result for oxidase activity. Strain LS9- 139 displays the highest 16S rRNA gene sequence similarity with Kandeliimicrobium roseum XY-R6^T (99.4%). Strain LS9-139 (=NNIBR2021641BA1681) was isolated from soil collected in Gangneung-si, Gangwon-do, Korea (37.911743°N, 128.806013°E). The GenBank accession number of the 16S rRNA gene sequence is OL77 3531.

3.7. Description of Tabrizicola oligotrophica 23ND62S-17

Cells are Gram-stain-positive, non-flagellated, and oval-shaped. Colonies grown on 1/10 NA are circular, raised, entire, and white after incubation for 3 days at 25°C. All negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, ure ase, esculin hydrolysis, gelatinase, and β-galactosidase in API 20NE. D-glucose, L-arabinose, D-mannose, Nacetyl- glucosamine, D-maltose, and trisodium citrate are assimilated. Does not assimilate D-mannitol, potassium gluconate, capric acid, adipic acid, malic acid, and phenylacetic acid. This strain was positive for oxidase activity. Strain 23ND62S-17 displays the highest 16S rRNA gene sequence similarity with Tabrizicola oligotrophica KMS-5^T (99.1%). Strain 23ND62S-17 (=NNIBR 2023641BA2549) was isolated from freshwater collected in Namhae-gun, Gyeongsangnam-do, Korea (34.9016°N, 127.920783°E). The GenBank accession number of the 16S rRNA gene sequence is OR945215.

3.8. Description of Rhodobacter ruber 23ND45S-58

Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on 1/10 R2A are circular, raised, entire, and white after incubation for 3 days at 25°C. Positive for esculin hydrolysis, and β-galactosidase in API 20NE, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, and gelatinase. D-glucose, D-mannose, D-maltose, and trisodium citrate are assimilated. Does not assimilate L-arabinose, D-mannitol, N-acetyl-glucosamine, potassium gluconate, capric acid, adipic acid, malic acid, and phenylacetic acid. This strain was positive for oxidase activity. Strain 23ND45S-58 displays the highest 16S rRNA gene sequence similarity with Rhodobacter ruber CCP-1^T (99.2%). Strain 23ND45S-58 (=NNIBR2023 641BA2547) was isolated from sediment collected in Sacheon-si, Gyeongsangnam-do, Korea (35.07850°N, 128.0356°E). The GenBank accession number of the 16S rRNA gene sequence is OR945213.

3.9. Description of Neoroseomonas marina 23ND67S-41

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on 1/10 R2A are circular, raised, entire, and pink after incubation for 3 days at 25°C. Positive for urease, and esculin hydrolysis in API 20NE, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, gelatinase, and β- galactosidase. N-acetyl-glucosamine, potassium gluconate, adipic acid, malic acid, and trisodium citrate are assimilated. Does not assimilate D-glucose, L-arabinose, D-mannose, D-mannitol, D-maltose, capric acid, and phenylacetic acid. This strain was positive for oxidase activity. Strain 23ND67S-41 displays the highest 16S rRNA gene sequence similarity with Neoroseomonas marina JC162^T (100.0%). Strain 23ND67S-41 (=NNIBR 2023641BA2306) was isolated from sediment collected in Jeongeup-si, Jeollabuk-do, Korea (35.74888°N, 126.889404°E). The GenBank accession number of the 16S rRNA gene sequence is OR984984.

3.10. Description of Roseomonas fluvialis 23ND27W-51

Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on 1/10 NA are circular, raised, entire, and pink after incubation for 3 days at 25°C. Positive for nitrate reduction, glucose fermentation, esculin hydrolysis in API 20NE, but indole production, arginine dihydrolase, urease, gelatinase, and β- galactosidase. D-glucose, D-maltose, and malic acid are assimilated. Does not L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, potassium gluconate, capric acid, adipic acid, trisodium citrate, and phenylacetic acid. This strain was negative for oxidase activity. Strain 23ND27W-51 displays the highest 16S rRNA gene sequence similarity with Roseomonas fluvialis S08^T (99.4%). Strain 23ND27W-51 (=NNIBR2023641BA2540) was isolated from freshwater collected in Gumi-si, Gyeongsangbuk- do, Korea (36.224972°N, 128.34094°E). The GenBank accession number of the 16S rRNA gene sequence is OR945206.

3.11. Description of Azospirillum doebereinerae 23ND38W-51

Cells are Gram-stain-negative, non-flagellated, and oval-shaped. Colonies grown on R2A are circular, raised, entire, and creamy-white after incubation for 3 days at 25°C. Positive for urease, esculin hydrolysis, and β-galactosidase in API 20NE, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, gelatinase. D-mannitol, potassium gluconate, malic acid, and trisodium citrate are assimilated. Does not D-glucose, L-arabinose, D-mannose, N-acetylglucosamine, D-maltose, capric acid, adipic acid, and phenylacetic acid. This strain showed a positive result for oxidase activity. Strain 23ND38W-51 displays the highest 16S rRNA gene sequence similarity with Azospirillum doebereinerae GSF71^T (99.3%). Strain 23ND 38W-51 (=NNIBR2023641BA1725) was isolated from freshwater collected in Taebaek-si, Gangwon-do, Korea (37.20642°N, 128.938°E). The GenBank accession number of the 16S rRNA gene sequence is OR984977.

3.12. Description of Lacibacterium aquatile 23ND68W-85

Cells are Gram-stain-negative, flagellated, and rodshaped. Colonies grown on 1/10 NA are circular, raised, entire, and creamy-white after incubation for 3 days at 25°C. Positive for only esculin hydrolysis, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, gelatinase, and β-galactosidase in API 20NE. D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, adipic acid, malic acid, trisodium citrate, and phenylacetic acid are assimilated. Does not assimilate capric acid. This strain showed a positive result for oxidase activity. Strain 23ND68W-85 displays the highest 16S rRNA gene sequence similarity with Lacibacterium aquatile LTC-2^T (99.0%). Strain 23ND 68W-85 (=NNIBR2023641BA2352) was isolated from freshwater collected in Buan-gun, Jeollabuk-do, Korea (35.59138°N, 126.583805°E). The GenBank accession number of the 16S rRNA gene sequence is OR984981.

3.13. Description of Rhodospirillum rubrum 23ND04W-104

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on 1/10 TSA are circular, raised, entire, and creamy-white after incubation for 3 days at 25°C. Positive for only esculin hydrolysis, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, gelatinase, and β- galactosidase in API 20NE. L-arabinose, D-mannose, potassium gluconate, adipic acid, malic acid, and trisodium citrate are assimilated. Does not D-glucose, Dmannitol, N-acetyl-glucosamine, D-maltose, capric acid, and phenylacetic acid. This strain showed a negative result for oxidase activity. Strain 23ND04W- 104 displays the highest 16S rRNA gene sequence similarity with Rhodospirillum rubrum ATCC 11170^T (100.0%). Strain 23ND04W-104 (=NNIBR2023641BA 213) was isolated from freshwater collected in Hamyang- gun, Gyeongsangnam-do, Korea (35.6253661°N, 127.7389866°E). The GenBank accession number of the 16S rRNA gene sequence is OR984972.

3.14. Description of Aestuariivirga litoralis 21LW3-03

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on 1/10 MA are circular, convex, smooth, translucent, and light yellow after incubation for 3 days at 20°C. Positive for only urease, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, esculin hydrolysis, gelatinase, and β-galactosidase in API 20NE. All does not assimilate D-glucose, L-arabinose, D-mannose, Dmannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. This strain showed a negative result for oxidase activity. Strain 21LW3-03 displays the highest 16S rRNA gene sequence similarity with Aestuariivirga litoralis SYSU M10001^T (99.2%). Strain 21LW3-03 (=NNIBR2021641BA56) was isolated from freshwater collected in Yangyang-gun, Gangwondo, Korea (38.037620°N, 128.700504°E). The GenBank accession number of the 16S rRNA gene sequence is OL773521.

3.15. Description of Siculibacillus lacustris 23ND46W-43

Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on 1/10 R2A are circular, raised, entire, and yellow after incubation for 3 days at 25°C. Positive for only esculin hydrolysis, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, gelatinase, and β-galactosidase in API 20NE. D-glucose, D-mannose, N-acetylglucosamine, and trisodium citrate are assimilated. Does not L-arabinose, D-mannitol, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, and phenylacetic acid. This strain was positive for oxidase activity. Strain 23ND46W-43 displays the highest 16S rRNA gene sequence similarity with Siculibacillus lacustris SA-279^T (99.1%). Strain 23ND46W-43 (=NNIBR2023641BA2548) was isolated from sediment collected in Sacheon-si, Gyeongsangnam-do, Korea (34.957527°N, 128.09116°E). The GenBank accession number of the 16S rRNA gene sequence is OR945214.

3.16. Description of Aureimonas altamirensis LS1-96

Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, smooth, and yellow after incubation for 3 days at 20°C. Positive for only urease, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, esculin hydrolysis, gelatinase, and β-galactosidase in API 20NE. D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, malic acid are assimilated. Does not capric acid, adipic acid, trisodium citrate, and phenylacetic acid. This strain was positive for oxidase activity. Strain LS1-96 displays the highest 16S rRNA gene sequence similarity with Aureimonas altamirensis DSM 21988^T (100.0%). Strain LS1-96 (=NNIBR2022641BA 614) was isolated from soil collected in Goseong-gun, Gangwon-do, Korea (38.254199°N, 128.555751°E). The GenBank accession number of the 16S rRNA gene sequence is OP872595.

3.17. Description of Pedomicrobium australicum SW3-15

Cells are Gram-stain-negative, non-flagellated, and short rod-shaped. Colonies grown on MA are circular, convex, and yellow after incubation for 3 days at 25°C. Positive for nitrate reduction and indole production, but glucose fermentation, arginine dihydrolase, urease, esculin hydrolysis, gelatinase, and β-galactosidase in API 20NE. All does not assimilate D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. This strain was positive for oxidase activity. Strain SW3-15 displays the highest 16S rRNA gene sequence similarity with Pedomicrobium australicum IFAM ST- 1306^T (99.6%). Strain SW3-15 (=NNIBR2017301BA12) was isolated from freshwater collected in Gwangyangsi, Jeollanam-do, Korea (34.996184°N, 127.777142°E). The GenBank accession number of the 16S rRNA gene sequence is MG818306.

3.18. Description of Kaistia hirudinis UTW7-4

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, and white after incubation for 3 days at 25°C. Positive for nitrate reduction, arginine dihydrolase, esculin hydrolysis, and β-galactosidase but negative for indole production, glucose fermentation, urease, and gelatinase in API 20NE. D-glucose, D-mannose, D-mannitol, N-acetyl-glucosamine, and D-maltose are assimilated. Does not L-arabinose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. This strain showed a positive result for oxidase activity. Strain UTW7-4 displays the highest 16S rRNA gene sequence similarity with Kaistia hirudinis ATCC 27833^T (100.0%). Strain UTW7-4 (=NNIBR2017 301BA19) was isolated from brackish water collected in Seongnam-dong, Jung-gu, Ulsan, Korea (35.552022°N, 129.317948°E). The GenBank accession number of the 16S rRNA gene sequence is MG818313.

3.19. Description of Bradyrhizobium oligotrophicum 23ND22S-21

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on 1/10 TSA are punctiform, flat, and creamy-white after incubation for 3 days at 20°C. Positive for urease and esculin hydrolysis, but negative for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, gelatinase, and β-galactosidase in API 20NE. D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, potassium gluconate, adipic acid, malic acid, and trisodium citrate are assimilated. Does not assimilate D-maltose, capric acid, and phenylacetic acid. This strain showed a positive result for oxidase activity. Strain 23ND22S-21 displays the highest 16S rRNA gene sequence similarity with Bradyrhizobium oligotrophicum S58^T (99.4%). Strain 23ND22S-21 (=NNIBR2023641BA332) was isolated from sediment collected in Andong-si, Gyeongsangbuk- do, Korea (36.5189113°N, 128.6744482°E). The GenBank accession number of the 16S rRNA gene sequence is OR984973.

3.20. Description of Rhizobium tumorigenes LS1-8

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on 1/10 R2A are circular, convex, smooth, and cream color after incubation for 3 days at 20°C. Positive for nitrate reduction, urease, esculin hydrolysis, and β-galactosidase, but negative for indole production, glucose fermentation, arginine dihydrolase, gelatinase, in API 20NE. D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, adipic acid, malic acid, and trisodium citrate are assimilated. Does not assimilate capric acid and phenylacetic acid. This strain was positive for oxidase activity. Strain LS1-8 displays the highest 16S rRNA gene sequence similarity with Rhizobium tumorigenes 1078^T (99.5%). Strain LS1-8 (=NNIBR2022641BA598) was isolated from soil collected in Goseong-gun, Gangwon-do, Korea (38.254199°N, 128.555751°E). The GenBank accession number of the 16S rRNA gene sequence is OP872597.

3.21. Description of Rhizobium rhizoryzae 23ND69W-100

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on MA are circular, raised, entire, umbonate, and creamy-white after incubation for 3 days at 25°C. Positive for urease, esculin hydrolysis, and β-galactosidase, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, gelatinase in API 20NE. D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, Dmaltose, capric acid, and malic acid are assimilated. Does not assimilate potassium gluconate, adipic acid, trisodium citrate, and phenylacetic acid. This strain was positive for oxidase activity. Strain 23ND69W-100 displays the highest 16S rRNA gene sequence similarity with Rhizobium rhizoryzae J3-AN59^T (99.8%). Strain 23ND69W-100 (=NNIBR2023641BA2426) was isolated from freshwater collected in Sinsang-dong, Dong-gu, Daejeon, Korea (36.3516388°N, 127.4914722°E). The GenBank accession number of the 16S rRNA gene sequence is OR984982.

3.22. Description of Rhizobium azooxidifex 23ND70S-60

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, raised, entire, and white after incubation for 3 days at 25°C. Positive for nitrate reduction, urease, and esculin hydrolysis, but indole production, glucose fermentation, arginine dihydrolase, gelatinase, and β-galactosidase in API 20NE. D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, and malic acid are assimilated. Does not capric acid, adipic acid, trisodium citrate, and phenylacetic acid. This strain was positive for oxidase activity. Strain 23ND70S-60 displays the highest 16S rRNA gene sequence similarity with Rhizobium azooxidifex Po 20/26^T (99.8%). Strain 23ND70S-60 (=NNIBR2023641BA 2558) was isolated from sediment collected in Uljin-gun, Gyeongsangbuk-do, Korea (36.73268°N, 129.47122°E). The GenBank accession number of the 16S rRNA gene sequence is OR984987.

3.23. Description of Hoeflea marina LW1-19

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on NA are circular, convex, smooth, and white after incubation for 3 days at 25°C. Positive for nitrate reduction, indole production, urease, esculin hydrolysis, and β-galactosidase, but glucose fermentation, arginine dihydrolase, and gelatinase in API 20NE. D-glucose, L-arabinose, D-mannose, D-mannitol, and malic acid are assimilated. Does not N-acetylglucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, trisodium citrate, and phenylacetic acid. This strain was positive for oxidase activity. Strain LW1-19 displays the highest 16S rRNA gene sequence similarity with Hoeflea marina LMG 128^T (99.4%). Strain LW1-19 (=NNIBR2017301BA7) was isolated from freshwater collected in Jeo-dong, Gangneung, Korea (37.7970833°N, 128.89788°E). The GenBank accession number of the 16S rRNA gene sequence is MG818 301.

3.24. Description of Hoeflea olei 22LW2-114

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, smooth, translucent, entire margin, and dark pinkish beige colored after incubation for 3 days at 25°C. Positive for urease, esculin hydrolysis, and β-galactosidase, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, and gelatinase in API 20NE. D-glucose, L-arabinose, D-mannose, Dmannitol, D-maltose, malic acid, and trisodium citrate are assimilated. Does not N-acetyl-glucosamine, potassium gluconate, capric acid, adipic acid, and phenylacetic acid. This strain showed a positive result for oxidase activity. Strain 22LW2-114 displays the highest 16S rRNA gene sequence similarity with Hoeflea olei JC234^T (99.2%). Strain 22LW2-114 (=NNIBR2022641BA2285) was isolated from freshwater collected in Yangyang-gun, Gangwon-do, Korea (37.952990°N, 128.768982°E). The GenBank accession number of the 16S rRNA gene sequence is OP999351.

3.25. Description of Angulomicrobium tetraedrale 23ND67S-26

Cells are Gram-stain-negative, non-flagellated, and oval-shaped. Colonies grown on 1/10 NA are circular, raised, entire, and creamy-white after incubation for 3 days at 25°C. Positive for urease, esculin hydrolysis, and β-galactosidase, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, and gelatinase in API 20NE. D-glucose, L-arabinose, Dmannitol, potassium gluconate, and malic acid are assimilated. Does not D-mannose, N-acetyl-glucosamine, D-maltose, capric acid, adipic acid, trisodium citrate and phenylacetic acid. This strain showed a positive result for oxidase activity. Strain 23ND67S-26 displays the highest 16S rRNA gene sequence similarity with Angulomicrobium tetraedrale DSM 5895^T (99.6%). Strain 23ND67S-26 (=NNIBR2023641BA2300) was isolated from sediment collected in Jeongeup-si, Jeollabukdo, Korea (35.74888°N, 126.889404°E). The GenBank accession number of the 16S rRNA gene sequence is OR984980.

3.26. Description of Novosphingobium arvoryzae 21LS1-37

Cells are Gram-stain-positive, non-flagellated, and long rod-shaped. Colonies grown on 1/10 NA are circular, convex, smooth with entire margin, and yellowish orange after incubation for 3 days at 20°C. Positive for urease and β-galactosidase, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, esculin hydrolysis, and gelatinase in API 20NE. D-glucose and L-arabinose are assimilated. Does not D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. This strain showed a positive result for oxidase activity. Strain 21LS1-37 displays the highest 16S rRNA gene sequence similarity with Novosphingobium arvoryzae Jyi-02^T (99.0%). Strain 21LS1-37 (=NNIBR2021641 BA34) was isolated from soil collected in Yangyanggun, Gangwon-do, Korea (37.952547°N, 128.768976°E). The GenBank accession number of the 16S rRNA gene sequence is OL773518.

3.27. Description of Vogesella mureinivorans UTW7-1

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, smooth, and ivory after incubation for 3 days at 25°C. Positive for only esculin hydrolysis, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, gelatinase, and β-galactosidase in API 20NE. D-glucose, N-acetyl-glucosamine, Dmaltose, and potassium gluconate are assimilated. Does not L-arabinose, D-mannose, D-mannitol, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. This strain was positive for oxidase activity. Strain UTW7-1 displays the highest 16S rRNA gene sequence similarity with Vogesella mureinivorans 389^T (99.9%). Strain UTW7-1 (=NNIBR2017301BA20) was isolated from brackish water collected in Seongnamdong, Jung-gu, Ulsan, Korea (35.552022°N, 129.317948 °E). The GenBank accession number of the 16S rRNA gene sequence is MG818314.

3.28. Description of Duganella sacchari 23ND41W-8

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on 1/10 MA are circular, raised, entire, and creamy-white after incubation for 3 days at 25°C. Positive for esculin hydrolysis, gelatinase, and β-galactosidase, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease in API 20NE. D-glucose, L-arabinose, D-mannose, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, malic acid, trisodium citrate, and phenylacetic acid are assimilated. Does not assimilate D-mannitol and adipic acid. This strain was positive for oxidase activity. Strain 23ND41W-8 displays the highest 16S rRNA gene sequence similarity with Duganella sacchari Sac-22^T (98.7%). Strain 23ND41W-8 (=NNIBR2023641BA1804) was isolated from freshwater collected in Jeongseon-gun, Gangwon-do, Korea (37.22695°N, 128.81398°E). The GenBank accession number of the 16S rRNA gene sequence is OR984979.

3.29. Description of Herminiimonas aquatilis 21LW1-65

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on 1/10 TSA are circular, convex, smooth, translucent, and light yellow after incubation for 3 days at 20°C. Positive for arginine dihydrolase, urease, but nitrate reduction, indole production, glucose fermentation, esculin hydrolysis, gelatinase, and β-galactosidase in API 20NE. Adipic acid is assimilated. Does not assimilate D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, malic acid, trisodium citrate, and phenylacetic acid. This strain was positive for oxidase activity. Strain 21LW1-65 displays the highest 16S rRNA gene sequence similarity with Herminiimonas aquatilis CCUG 36956^T (99.9%). Strain 21LW1-65 (=NNIBR2021641BA18) was isolated from soil collected in Yangyang-gun, Gangwon-do, Korea (37.952547°N, 128.768976°E). The GenBank accession number of the 16S rRNA gene sequence is OL773520.

3.30. Description of Rhodoferax antarcticus HRW3-6

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, and ivory after incubation for 3 days at 25°C. Positive for nitrate reduction, urease, esculin hydrolysis, and β-galactosidase, but indole production, glucose fermentation, arginine dihydrolase, and gelatinase in API 20NE. Does not all assimilate substrates: D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid adipic acid, malic acid, trisodium citrate, and phenylacetic acid. This strain was positive for oxidase activity. Strain HRW3-6 displays the highest 16S rRNA gene sequence similarity with Rhodoferax antarcticus ANT. BR^T (98.6%). Strain HRW3-6 (=NNIBR2017301BA2) was isolated from freshwater collected in Hanam-si, Gyeonggi-do, Korea (37.542801°N, 127.239583°E). The GenBank accession number of the 16S rRNA gene sequence is MG818296.

3.31. Description of Xenophilus arseniciresistens CS-27

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on NA are circular, convex, and yellow after incubation for 7 days at 25°C. Positive for glucose fermentation, but nitrate reduction, indole production, arginine dihydrolase, urease, esculin hydrolysis, gelatinase, and β-galactosidase in API 20NE. Malic acid is assimilated. Does not assimilate D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid adipic acid, trisodium citrate, and phenylacetic acid. This strain showed a positive result for oxidase activity. Strain CS-27 displays the highest 16S rRNA gene sequence similarity with Xenophilus arseniciresistens YW8^T (99.5%). Strain CS-27 (=NNIBR2022641BA2314) was isolated from sediment collected in Sinsang-dong, Dong-gu, Daejeon, Korea (36.3515833°N, 127.4911°E). The GenBank accession number of the 16S rRNA gene sequence is OQ073741.

3.32. Description of Inhella crocodyli ADW1-35

Cells are Gram-stain-negative, non-flagellated, and short rod-shaped. Colonies grown on R2A are circular, convex, entire, and yellow after incubation for 2 days at 25°C. Positive for nitrate reduction, esculin hydrolysis, and β-galactosidase, but indole production, glucose fermentation, arginine dihydrolase, urease, and gelatinase in API 20NE. D-mannose and trisodium citrate are assimilated. Does not assimilate D-glucose, L-arabinose, Dmannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, and phenylacetic acid. This strain showed a positive result for oxidase activity. Strain ADW1-35 displays the highest 16S rRNA gene sequence similarity with Inhella crocodyli CCP-18^T (99.2%). Strain ADW1-35 (=NNIBR2019 641BA241) was isolated from freshwater collected in Andong-si, Gyeongsangbuk-do, Korea (36.689694°N, 128.851278°E). The GenBank accession number of the 16S rRNA gene sequence is MN755856.

3.33. Description of Pelomonas aquatic 23ND37W-67

Cells are Gram-stain-negative, non-flagellated, and oval-shaped. Colonies grown on R2A are circular, raised, entire, and creamy-white after incubation for 2 days at 25°C. Positive for esculin hydrolysis and β- galactosidase, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, and gelatinase in API 20NE. L-arabinose is assimilated. Does not assimilate D-glucose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. This strain showed a positive result for oxidase activity. Strain 23ND37W-67 displays the highest 16S rRNA gene sequence similarity with Pelomonas aquatic CCUG 52575^T (98.8%). Strain 23ND 37W-67 (=NNIBR2023641BA1695) was isolated from freshwater collected in Taebaek-si, Gangwon-do, Korea (37.207844°N, 128.93227°E). The GenBank accession number of the 16S rRNA gene sequence is OR984976.

3.34. Description of Aromatoleum tolulyticum ADS2-16

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, entire, and white after incubation for 2 days at 25°C. Positive for nitrate reduction and esculin hydrolysis, but indole production, glucose fermentation, arginine dihydrolase, urease, gelatinase, and β-galactosidase in API 20NE. D-glucose, D-maltose adipic acid, and malic acid are assimilated. Does not assimilate L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, potassium gluconate, capric acid, trisodium citrate, and phenylacetic acid. This strain showed a negative result for oxidase activity. Strain ADS2-16 displays the highest 16S rRNA gene sequence similarity with Aromatoleum tolulyticum ATCC 51758^T (99.9%). Strain ADS2-16 (=NNIBR2019641BA242) was isolated from freshwater collected in Andong-si, Gyeongsangbuk-do, Korea (36.576861°N, 128.781389°E). The GenBank accession number of the 16S rRNA gene sequence is MN759325.

3.35. Description of Rheinheimera riviphila 21SJ05W-0M51

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, entire, and white after incubation for 2 days at 20°C. Positive for esculin hydrolysis, gelatinase, and β-galactosidase, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, and urease in API 20NE. Does not all assimilate substrates: D-glucose, L-arabinose, D-mannose, D-mannitol, Nacetyl- glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. This strain showed a positive result for oxidase activity. Strain 21SJ05W-0M51 displays the highest 16S rRNA gene sequence similarity with Rheinheimera riviphila KYPC3^T (99.6%). Strain 21SJ05W- 0M51 (=NNIBR2021641BA1534) was isolated from freshwater collected in Imsil-gun, Jeollabuk-do, Korea (35.496203°N, 127.190908°E). The GenBank accession number of the 16S rRNA gene sequence is MZ976828.

3.36. Description of Kluyvera sichuanensis 23ND70W-93

Cells are Gram-stain-negative, non-flagellated, and oval-shaped. Colonies grown on R2A are circular, raised, entire, punctiform, and creamy-white after incubation for 2 days at 25°C. Positive for indole production, glucose fermentation, esculin hydrolysis, and β-galactosidase, but nitrate reduction, arginine dihydrolase, urease, and gelatinase in API 20NE. D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, malic acid, trisodium citrate, and phenylacetic acid are assimilated. Adipic acid is not assimilated. This strain showed a negative result for oxidase activity. Strain 23ND70W-93 displays the highest 16S rRNA gene sequence similarity with Kluyvera sichuanensis SCKS 090646^T (99.3%). Strain 23ND70W-93 (=NNIBR2023 641BA2556) was isolated from freshwater collected in Uljin-gun, Gyeongsangbuk-do, Korea (36.73268°N, 129.47122°E). The GenBank accession number of the 16S rRNA gene sequence is OR984986.

3.37. Description of Hafnia psychrotolerans 22LW2-120

Cells are Gram-stain-positive, non-flagellated, and rod-shaped. Colonies grown on R2A are circular, convex, opaque, entire margin, and white after incubation for 2 days at 15°C. Positive for nitrate reduction, glucose fermentation, and urease, but indole production, arginine dihydrolase, esculin hydrolysis, gelatinase, and β-galactosidase in API 20NE. D-glucose, D-mannitol, D-maltose, potassium gluconate, and malic acid are assimilated. L-arabinose, D-mannose, N-acetyl-glucosamine, capric acid, adipic acid, trisodium citrate, and phenylacetic acid are not assimilated. This strain was positive for oxidase activity. Strain 22LW2-120 displays the highest 16S rRNA gene sequence similarity with Hafnia psychrotolerans DJC1-1^T (99.4%). Strain 22LW2- 120 (=NNIBR2022641BA2286) was isolated from freshwater collected in Yangyang-gun, Gangwon-do, Korea (37.952990°N, 128.768982°E). The GenBank accession number of the 16S rRNA gene sequence is OP999352.

3.38. Description of Arenimonas metalli 23ND71S-1

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on R2A are filamentous, flat, and creamy-white after incubation for 2 days at 25°C. Positive for esculin hydrolysis, gelatinase, and β-galactosidase in API 20NE, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, and urease. D-glucose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, adipic acid, malic acid, trisodium citrate, and phenylacetic acid are assimilated. L-arabinose and capric acid are not assimilated. This strain was positive for oxidase activity. Strain 23ND71S-1 displays the highest 16S rRNA gene sequence similarity with Arenimonas metalli CF5-1^T (99.0%). Strain 23ND71S-1 (=NNIBR2023641BA 2559) was isolated from sediment collected in Uljin-gun, Gyeongsangbuk-do, Korea (36.67597°N, 129.44344°E). The GenBank accession number of the 16S rRNA gene sequence is OR984988.

3.39. Description of Stenotrophomonas terrae HRW4-14

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on NA are circular, convex with entire edge, and ivory after incubation for 3 days at 25°C. Positive for nitrate reduction, indole production, esculin hydrolysis, and gelatinase, but glucose fermentation, arginine dihydrolase, urease, and β-galactosidase in API 20NE. D-glucose, D-mannose, N-acetyl-glucosamine, D-maltose, and trisodium citrate are assimilated. L-arabinose, D-mannitol, potassium gluconate, capric acid, adipic acid, malic acid, and phenylacetic acid are not assimilated. This strain was negative for oxidase activity. Strain HRW4-14 displays the highest 16S rRNA gene sequence similarity with Stenotrophomonas terrae DSM 18941^T (99.7%). Strain HRW4-14 (=NNIBR 2017301BA3) was isolated from freshwater collected in Jamsil-dong, Songpa-gu, Seoul, Korea (37.518432°N, 127.081634°E). The GenBank accession number of the 16S rRNA gene sequence is MG818297.

3.40. Description of Dyella amyloliquefaciens 21LS8-45

Cells are Gram-stain-positive, flagellated, and rodshaped. Colonies grown on 1/10 NA are circular, convex, and yellowish ivory after incubation for 3 days at 25°C. Positive for urease, esculin hydrolysis, gelatinase, β-galactosidase in API 20NE, but nitrate reduction, indole production, glucose fermentation, and arginine dihydrolase. L-arabinose, D-mannitol, and N-acetyl-glucosamine are assimilated. D-glucose, D-mannose, Dmaltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid are not assimilated. This strain was positive for oxidase activity. Strain 21LS8-45 displays the highest 16S rRNA gene sequence similarity with Dyella amyloliquefaciens DHC06^T (99.5%). Strain 21LS8-45 (=NNIBR2021641 BA1248) was isolated from soil collected in Goseonggun, Gangwon-do, Korea (38.253831°N, 128.555642°E). The GenBank accession number of the 16S rRNA gene sequence is OL773525.

3.41. Description of Steroidobacter soli DCSS-21

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on 1/10 NA are circular, convex, and yellow after incubation for 7 days at 25°C. Positive for esculin hydrolysis and β-galactosidase in API 20NE, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, gelatinase. Does not all assimilate substrates: D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid. This strain was positive for oxidase activity. Strain DCSS-21 displays the highest 16S rRNA gene sequence similarity with Steroidobacter soli JW-3^T (98.7%). Strain DCSS-21 (=NNIBR2022641BA2358) was isolated from freshwater collected in Sinsang-dong, Dong-gu, Daejeon, Korea (36.3515833°N, 127.4911°E). The GenBank accession number of the 16S rRNA gene sequence is OQ073745.

3.42. Description of Pseudomonas wadenswilerensis 21SJ07S-11

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on TSA are circular, convex, and yellow after incubation for 2 days at 25°C. Positive for nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, and gelatinase, but esculin hydrolysis and β-galactosidase in API 20NE. D-mannose, N-acetyl-glucosamine, D-maltose, capric acid, adipic acid, and trisodium citrate are assimilated. Does not assimilate D-glucose, L-arabinose, Dmannitol, potassium gluconate, malic acid, and phenylacetic acid. This strain was positive for oxidase activity. Strain 21SJ07S-11 displays the highest 16S rRNA gene sequence similarity with Pseudomonas wadenswilerensis CCOS 864^T (99.9%). Strain 21SJ07S-11 (=NNIBR2021 641BA1638) was isolated from soil collected in Goseonggun, Jeollanam-do, Korea (35.193180°N, 127.375249°E). The GenBank accession number of the 16S rRNA gene sequence is OL872217.

3.43. Description of Pseudomonas capsici 23ND69W-112

Cells are Gram-stain-negative, non-flagellated, and rod-shaped. Colonies grown on ISP4 are filamentous, and creamy-white after incubation for 2 days at 25°C. Positive for esculin hydrolysis, but nitrate reduction, indole production, glucose fermentation, arginine dihydrolase, urease, gelatinase, and β-galactosidase in API 20NE. D-glucose, L-arabinose, D-mannose, D-mannitol, D-maltose, potassium gluconate, capric acid, malic acid, and trisodium citrate are assimilated. Does not assimilate N-acetyl-glucosamine, adipic acid, and phenylacetic acid. This strain was positive for oxidase activity. Strain 23ND69W-112 displays the highest 16S rRNA gene sequence similarity with Pseudomonas capsici Pc19-1^T (98.7%). Strain 23ND69W-112 (=NNIBR2023 641BA2431) was isolated from freshwater collected in Sinsang-dong, Dong-gu, Daejeon, Korea (36.3516388°N, 127.4914722°E). The GenBank accession number of the 16S rRNA gene sequence is OR984983.

ACKNOWLEDGEMENTS

This study was supported by a grant from the Nakdonggang National Institute of Biological Resources (NNIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NNIBR20251102).

CRediT authorship contribution statement

SY Lee: Project administration, Funding acquisition, Conceptualization, Writing-Original draft, Writing- Review and editing. A Choi: Funding acquisition, Investigation, Data curation. H Kim: Investigation, Data curation. JY Jung: Investigation, Data curation. JY Cho: Investigation, Data curation. S Hwang: Data curation. J Goh: Supervision.

Declaration of Competing Interest

The authors declare no conflict of interest.

Figure

Fig. 1.

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing relationships of 26 strains isolated in this study and their relatives in the class Alphaproteobacteria, phylum Pseudomonadota. Acidipila rosea AP8^T (GenBank accession no. AB561884) was used as an outgroup. Bootstrap values (>70%) are shown at nodes. Bar, 0.02 substitutions per nucleotide position.

Fig. 2.

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing relationships of 17 strains isolated in this study and their relatives in classes Betaproteobacteria and Gammaproteobacteria, phylum Pseudomonadota. Acidipila rosea AP8^T (GenBank accession no. AB561884) was used as an outgroup. Bootstrap values (>70%) are shown at nodes. Bar, 0.02 substitutions per nucleotide position.

Fig. 3.

Transmission electron micrographs for cells of strains isolated in this study. The scale bar size for each image is provided alongside corresponding strain number as follows: Strain 1, 21LS1-75, 0.5 μm; Strain 2, LW3-33, 0.2 μm; Strain 3, SJW1-31, 0.2 μm; Strain 4, 6LW2- 50, 1 μm; Strain 5, 21YS10S-0N32, 0.5 μm; Strain 6, LS9-139, 0.5 μm; Strain 7, 23ND62S-17, 0.2 μm; Strain 8, 23ND45S-58, 0.5 μm; Strain 9, 23ND67S-41, 0.5 μm; Strain 10, 23ND27W-51, 0.5 μm; Strain 11, 23ND38W-51, 0.1 μm; Strain 12, 23ND68W-85, 0.5 μm; Strain 13, 23ND04W-104, 0.1 μm; Strain 14, 21LW3-03, 1 μm; Strain 15, 23ND46W-43, 0.5 μm; Strain 16, LS1-96, 0.5 μm; Strain 17, SW3-15, 0.2 μm; Strain 18, UTW7-4, 0.2 μm; Strain 19, 23ND22S-21, 0.5 μm; Strain 20, LS1-8, 1 μm; Strain 21, 23ND69W-100, 0.5 μm; Strain 22, 23ND70S-60, 0.5 μm; Strain 23, LW1-19, 0.2 μm; Strain 24, 22LW2-114, 1 μm; Strain 25, 23ND67S-26, 0.5 μm; Strain 26, 21LS1-37, 0.2 μm; Strain 27, UTW7- 1, 0.5 μm; Strain 28, 23ND41W-8, 0.5 μm; Strain 29, 21LW1-65, 0.2 μm; Strain 30, HRW3-6, 0.2 μm; Strain 31, CS-27, 0.5 μm; Strain 32, ADW1-35, 0.5 μm; Strain 33, 23ND37W-67, 0.5 μm; Strain 34, ADS2-16, 0.5 μm; Strain 35, 21SJ05W-0M51, 0.5 μm; Strain 36, 23ND70W-93, 0.5 μm; Strain 37, 22LW2-120, 0.5 μm; Strain 38, 23ND71S-1, 0.5 μm; Strain 39, HRW4-14, 0.2 μm; Strain 40, 21LS8-45, 0.5 μm; Strain 41, DCSS-21, 0.5 μm; Strain 42, 21SJ07S-11, 0.5 μm; Strain 43, 23ND69W-112, 0.5 μm.

Table

Table 1.

Summary of 43 isolates belonging to the phyla Pseudomonadota and their taxonomic affiliations, isolation sources, and culture conditions

Phylum	Class	Order	Family	Strain ID	NNIBR ID	Most closely related species	Similarity (%)	Isolation source	Medium	Incubation conditions

Pseudomonadota	Alphaproteobacteria	Caulobacterales	Caulobacteraceae	21LS1-75	NNIBR2021641BA44	Asticcacaulis taihuensis	99.5	Soil	1/10 R2A	2-3 d, 20°C, pH 7

		Rhodobacterales	Roseobacteraceae	LW3-33	NNIBR2017301BA8	Pelagimonas varians	99.9	Freshwater	MA	3 d, 25°C
				SJW1-31	NNIBR2017301BA537	Loktanella salsilacus	100.0	Brackish water	NA	3 d, 25°C
				6LW2-50	NNIBR2022641BA2178	Donghicola mangrovi	99.9	Freshwater	R2A	25°C, pH 7

			Rhodobacteraceae	21YS10S-0N32	NNIBR2021641BA1666	Cypionkella sinensis	99.5	Soil	R2A	3 d, 25°C, pH 7
				LS9-139	NNIBR2021641BA1681	Kandeliimicrobium roseum	99.4	Soil	MA	3 d, 25°C, pH 7
				23ND62S-17	NNIBR2023641BA2549	Tabrizicola oligotrophica	99.1	Freshwater	1/10 NA	2-3 d, 25°C, pH 7
				23ND45S-58	NNIBR2023641BA2547	Rhodobacter ruber	99.2	Sediment	1/10 R2A	2-3 d, 25°C, pH 7

		Rhodospirillales	Acetobacteraceae	23ND67S-41	NNIBR2023641BA2306	Neoroseomonas marina	100.0	Sediment	1/10 R2A	2-3 d, 25°C, pH 7
			Acetobacteraceae	23ND27W-51	NNIBR2023641BA2540	Roseomonas fluvialis	99.4	Freshwater	1/10 NA	2-3 d, 25°C, pH 7

			Azospirillaceae	23ND38W-51	NNIBR2023641BA1725	Azospirillum doebereinerae	99.3	Freshwater	R2A	2-3 d, 25°C, pH 7
			Azospirillaceae	23ND68W-85	NNIBR2023641BA2352	Lacibacterium aquatile	99.0	Freshwater	1/10 NA	2-3 d, 25°C, pH 7

			Rhodospirillaceae	23ND04W-104	NNIBR2023641BA213	Rhodospirillum rubrum	100.0	Freshwater	1/10TSA	2-3 d, 25°C, pH 7

		Rhizobiales	Aestuariivirgaceae	21LW3-03	NNIBR2021641BA56	Aestuariivirga litoralis	99.2	Freshwater	0.1 MA	2-3 d, 20°C, pH 7

			Ancalomicrobiaceae	23ND46W-43	NNIBR2023641BA2548	Siculibacillus lacustris	99.1	Sediment	1/10 R2A	2-3 d, 25°C, pH 7

			Aurantimonadaceae	LS1-96	NNIBR2022641BA614	Aureimonas altamirensis	100.0	Soil	R2A	20°C, pH 7

			Hyphomicrobiaceae	SW3-15	NNIBR2017301BA12	Pedomicrobium australicum	99.6	Freshwater	MA	3 d, 25°C

			Kaistiaceae	UTW7-4	NNIBR2017301BA19	Kaistia hirudinis	100.0	Brackish water	R2A	3 d, 25°C

			Nitrobacteraceae	23ND22S-21	NNIBR2023641BA332	Bradyrhizobium oligotrophicum	99.4	Sediment	1/10TSA	2-3 d, 20°C, pH 7

			Rhizobiaceae	LS1-8	NNIBR2022641BA598	Rhizobium tumorigenes	99.5	Soil	R2A	20°C, pH 7
				23ND69W-100	NNIBR2023641BA2426	Rhizobium rhizoryzae	99.8	Freshwater	MA	2-3 d, 25°C, pH 7
				23ND70S-60	NNIBR2023641BA2558	Rhizobium azooxidifex	99.8	Sediment	R2A	2-3 d, 25°C, pH 7

			Phyllobacteriaceae	LW1-19	NNIBR2017301BA7	Hoeflea marina	99.4	Freshwater	NA	3 d, 25°C
			Phyllobacteriaceae	22LW2-114	NNIBR2022641BA2285	Hoeflea olei	99.2	Freshwater	R2A	2-3 d, 25°C, pH 7

			Xanthobacteraceae	23ND67S-26	NNIBR2023641BA2300	Angulomicrobium tetraedrale	99.6	Sediment	1/10 NA	2-3 d, 25°C, pH 7

		Sphingomonadales	Sphingomonadaceae	21LS1-37	NNIBR2021641BA34	Novosphingobium arvoryzae	99.0	Soil	1/10 NA	2-3 d, 20°C, pH 7

	Betaproteobacteria	Neisseriales	Chromobacteriaceae	UTW7-1	NNIBR2017301BA20	Vogesella mureinivorans	99.9	Brackish water	R2A	3 d, 25°C

		Burkholderiales	Oxalobacteraceae	23ND41W-8	NNIBR2023641BA1804	Duganella sacchari	98.7	Freshwater	1/10 MA	2-3 d, 25°C, pH 7
			Oxalobacteraceae	21LW1-65	NNIBR2021641BA18	Herminiimonas aquatilis	99.9	Soil	1/10TSA	2-3 d, 20°C, pH 7

			Comamonadaceae	HRW3-6	NNIBR2017301BA645	Rhodoferax antarcticus	98.6	Freshwater	R2A	3 d, 25°C
			Comamonadaceae	CS-27	NNIBR2022641BA2314	Xenophilus arseniciresistens	99.5	Sediment	NA	7 d, 25°C, pH 7

			Sphaerotilaceae	ADW1-35	NNIBR2019641BA241	Inhella crocodyli	99.2	Freshwater	R2A	2 d, 25°C, pH 7
			Sphaerotilaceae	23ND37W-67	NNIBR2023641BA1695	Pelomonas aquatica	98.8	Freshwater	R2A	2-3 d, 25°C, pH 7

		Rhodocyclales	Zoogloeaceae	ADS2-16	NNIBR2019641BA242	Aromatoleum tolulyticum	99.9	Freshwater	R2A	2 d, 25°C, pH 7

	Gammaproteobacteria	Chromatiales	Chromatiaceae	21SJ05W-0M51	NNIBR2021641BA1534	Rheinheimera riviphila	99.6	Freshwater	R2A	2-3 d, 20°C, pH 7

		Enterobacterales	Enterobacteriaceae	23ND70W-93	NNIBR2023641BA2556	Kluyvera sichuanensis	99.3	Freshwater	R2A	2-3 d, 25°C, pH 7
		Enterobacterales	Hafniaceae	22LW2-120	NNIBR2022641BA2286	Hafnia psychrotolerans	99.4	Freshwater	R2A	2-3 d, 15°C, pH 7

		Lysobacterales	Lysobacteraceae	23ND71S-1	NNIBR2023641BA2559	Arenimonas metalli	99.0	Sediment	R2A	2-3 d, 25°C, pH 7
			Lysobacteraceae	HRW4-14	NNIBR2017301BA662	Stenotrophomonas terrae	99.7	Freshwater	NA	3 d, 25°C

			Rhodanobacteraceae	21LS8-45	NNIBR2021641BA1248	Dyella amyloliquefaciens	99.5	Soil	1/10 NA	25°C, pH 7

Steroidobacterales		Steroidobacteraceae	DCSS-21	NNIBR2022641BA2358	Steroidobacter soli	98.7	Freshwater	1/10 NA	7 d, 25°C, pH 7

Pseudomonadales		Pseudomonadaceae	21SJ07S-11	NNIBR2021641BA1638	Pseudomonas wadenswilerensis	99.9	Soil	TSA	2 d, 25°C, pH 7
Pseudomonadales		Pseudomonadaceae	23ND69W-112	NNIBR2023641BA2431	Pseudomonas capsici	98.7	Freshwater	ISP4	2-3 d, 25°C, pH 7

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Vol. 40 No. 4 (2022.12)

Journal Abbreviation 'Korean J. Environ. Biol.'
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Doi Prefix 10.11626/KJEB.
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