INTRODUCTION

Freshwater ecosystems such as rivers, lakes and wetlands are home to a wide variety of species and while they constitute approximately <1% of the Earth̓s surface, they are inhabited by 10% of earth’s species including a third of all vertebrate species (Strayer and Dudgeon 2010). Threats to freshwater ecosystems include pollution, exploitation, invasive species, land-use and climate change. The cumulative impact in recent decades of these threats has significantly reduced freshwater biodiversity (Dudgeon et al., 2006), though in turn has promoted bacterial diversity (Li 2017;Rodríguez et al. 2018;Tanentzap et al. 2019). The study of microbial diversity is conducted generally with next-generation sequencing (NGS). However, isolating microbes, particularly any new species and culturing them, needs to be a priority (Han et al. 2020) because the majority of microorganisms remain uncultured and thus poorly characterized (Lewis et al. 2021), without much data on their roles and functions, nor their ecological impact and industrial applications.

As a part of the research program ‘Survey of freshwater organisms and specimen collection’ by the Nakdonggang Institute of Biological Resources (NNIBR), we collected freshwater samples from the Geum River basin to report the taxonomic information and phenotypic characterization of any previously unrecorded and unidentified bacterial strains.

MATERIALS AND METHODS

Freshwater environmental samples were collected from the Geum River basin in Republic of Korea for bacterial specimens (Table 1). The samples were inoculated on to Reasoner’s 2A agar (R2A; BD), tryptic soy agar (TSA; BD), 1/10-diluted TSA, marine agar 2216 (MA; BD), 1/10 diluted MA and nutrient agar (NA) using a standard dilution plating technique and plates were incubated at 25°C for 3 days. The pure cultures of bacterial isolates were preserved at -80°C as a suspension in 20% (w/v) glycerol. The 31 bacterial species designations, isolation sources, medium and incubation conditions are summarized in Table 1.

The genomic DNA of isolates were extracted using the DNeasy Blood and Tissue kit (Qiagen, Germany) and the almost complete 16S rRNA gene was sequenced using the universal primers 27F and 1492R (Weisburg et al. 1991) by Macrogen (Republic of Korea). The complete sequence was compared to sequences obtained from the EzBioCloud server (Yoon et al. 2017) and a cutoff value of 98.7% sequence similarity was applied for bacterial species determination. Bacterial strains showing 98.7% or higher sequence similarity with type strains unreported in Republic of Korea were identified as unreported bacterial species. Phylogenetic trees based on the neighbor-joining (Saitou and Nei 1987), maximum-parsimony (Fitch 1971) and maximum-likelihood (Felsenstein 1981) algorithms, with 1,000-replicate bootstrap values were constructed using MEGA11 software (Tamura et al. 2021).

Cellular morphology and size were examined by transmission electron microscopy. Gram staining was performed using a Gram-staining kit (bioMérieux, France). Biochemical characteristics were assessed using API 20NE (bioMérieux) according to the manufacturer’s instructions. Oxidase activity was measured using oxidase reagent (bioMérieux).

RESULTS AND DISCUSSION

Based on the phylogenetic analyses using the 16S rRNA gene sequences, the 31 strains (NCS-2-32, KCS-47, NSS- 12, MYG-2-5, GAP-35, JU-13, KMU-11, MS-33, IBC-2- 120, 20LEG-88, KCS-46, MBC-2-58, 20LEG-73, 9S-12, KCS-3, JKS-1, MND-3-64, 20HNW-03, IND-2-6, JU-40, 7J1-06, 7J3-01, JU-31, NSW-11, JJ1S-14, 20CSW-3, 9W- 13, YDC-53, 9S-10, 9W-02 and KKMS-29) isolated from Geum River were assigned to the phyla Actinobacteria (Fig. 1), Bacteroidetes (Fig. 2), Firmicutes (Fig. 3) and Proteobacteria (Fig. 4). The strains represented 31 unrecorded species in Korea, belonging to 8 classes, 18 orders, 21 families. The taxonomic identification of the isolates is summarized in Table 1. Transmission electron micrographs of the isolates are provided in Fig. 5. The morphological, physiological and biochemical properties of isolated strains are described in detail as below.

Description of Kineococcus radiotolerans NCS-2-32

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are circular, opaque, rough and faint orange colored after 2 days on NA at 25°C. Positive for glucose fermentation, esculin hydrolysis, arginine dihydrolase, urease and β-galactosidase, but negative for oxidase, gelatin hydrolysis, nitrate reduction and indole production. D-Glucose, L-Arabinose, D-Mannose, D-Mannitol, N-acetyl-glucosamine, D-Maltose, potassium gluconate, malic acid and trisodium citrate are utilized. Does not utilize capric acid, adipic acid and phenylacetic acid. Strain NCS-2-32 (=NNIBR2020641BA782) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MW367770.

Description of Pedococcus aerophilus KCS-47

Cells are Gram-staining-positive, non-flagellated, rodshaped and aerobic. Colonies are circular, convex, entire and white colored after 3 days on R2A at 25°C. Positive for esculin hydrolysis, gelatin hydrolysis and β-galactosidase, but negative for oxidase, nitrate reduction, glucose fermentation, indole production, arginine dihydrolase and urease. D-Glucose, D-Mannitol, potassium gluconate and malic acid are utilized. Does not utilize L-Arabinose, D-Mannose, N-acetyl-glucosamine, D-Maltose, capric acid, adipic acid, trisodium citrate and phenylacetic acid. Strain KCS- 47 (=NNIBR2019641BA240) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MN782285.

Description of Pedococcus bigeumensis NSS-12

Cells are Gram-staining-positive, non-flagellated, rodshaped and aerobic. Colonies are circular, convex, entire and white colored after 3 days on R2A at 25°C. Positive for oxidase, nitrate reduction, esculin hydrolysis, gelatin hydrolysis and β-galactosidase, but negative for glucose fermentation, indole production and urease. D-Glucose, D-Mannitol, D-Maltose and potassium gluconate are utilized. Does not utilize L-Arabinose, D-Mannose, N-acetyl- glucosamine, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Strain NSS-12 (=NNIBR2020641BA815) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MW293919.

Description of Pedococcus cremeus MYG-2-5

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are circular, smooth and cream colored after 3 days on MA at 25°C. Positive for oxidase, esculin hydrolysis, glucose fermentation and β-galactosidase, but negative for nitrate reduction, gelatin hydrolysis, indole production, arginine dihydrolase and urease. D-Glucose and potassium gluconate are utilized. Does not utilize L-Arabinose, D-Mannose, D-Mannitol, N-acetyl- glucosamine, D-Maltose, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Strain MYG- 2-5 (=NNIBR2020641BA789) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MW367771.

Description of Rhodoluna limnophila GAP-35

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are circular, convex with entire edge and orange colored after 3 days on R2A at 25°C. Positive for nitrate reduction, esculin hydrolysis and β-galactosidase, but negative for oxidase, gelatin, glucose fermentation, indole production, arginine dihydrolase and urease. D-Glucose, L-Arabinose, D-Mannose, D-Mannitol, N-acetyl-glucosamine, malic acid and trisodium citrate, D-Maltose, potassium gluconate, capric acid, adipic acid and phenylacetic acid are not utilized. GAP-35 (=NNIBR2019641BA25) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MN559417.

Description of Salinibacterium xinjiangense JU-13

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are circular, are circular, convex with entire edge and yellow colored after 3 days on R2A at 25°C. Positive for esculin hydrolysis and β-galactosidase, but negative for oxidase, gelatin hydrolysis, nitrate reduction, glucose fermentation, indole production, arginine dihydrolase and urease. D-Glucose, L-Arabinose, D-Mannose, D-Mannitol and D-Maltose are utilized. Does not utilize N-acetyl-glucosamine, malic acid, trisodium citrate, potassium gluconate, capric acid, adipic acid and phenylacetic acid. Strain JU-13 (=NNIBR2019641BA26) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MN559418.

Description of Rhodoluna lacicola KMU-11

Cells are Gram-staining-negative, non-flagellated, filament- shaped and aerobic. Colonies are circular, convex, smooth and red colored after 3 days on R2A at 25°C. Positive for oxidase and esculin hydrolysis, but negative for gelatin hydrolysis, nitrate reduction, glucose fermentation, indole production, β-galactosidase, arginine dihydrolase and urease. D-Glucose, L-Arabinose, D-Mannose, D-Mannitol, N-acetyl-glucosamine, malic acid, trisodium citrate, D-Maltose, potassium gluconate, capric acid, adipic acid and phenylacetic acid are not utilized. Strain KMU-11 (=NNIBR2020641BA794) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MT893382.

Description of Arthrobacter flavus MS-33

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are circular, convex, smooth and yellow colored after 3 days on R2A at 25°C. Positive for esculin hydrolysis, β-galactosidase and urease, but negative for oxidase, gelatin hydrolysis, nitrate reduction, glucose fermentation, indole production, arginine dihydrolase. D-Glucose, L-Arabinose, D-Mannose, D-Mannitol, N-acetyl-glucosamine, malic acid, trisodium citrate, D-Maltose, potassium gluconate, capric acid, adipic acid and phenylacetic acid are not utilized. Strain MS-33 (=NNIBR2020641BA795) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MT893383.

Description of Williamsia herbipolensis IBC-2-120

Cells are Gram-staining-positive, non-flagellated, coccid or rod-shaped and aerobic. Colonies are circular, convex with entire edge, opaque, smooth and light orange colored after 3 days on R2A at 25°C. Positive for glucose fermentation, urease and β-galactosidase (week) but negative for oxidase, esculin hydrolysis, gelatin hydrolysis, nitrate reduction, indole production and arginine dihydrolase. D-Glucose, D-Mannose, D-Mannitol, malic acid and trisodium citrate are utilized. Does not utilize L-Arabinose, N-acetyl-glucosamine, D-Maltose, potassium gluconate, capric acid, adipic acid and phenylacetic acid. Strain IBC- 2-120 (=NNIBR2020641BA788) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MW367775.

Description of Nakamurella deserti 20LEG-88

Cells are Gram-staining-positive, non-flagellated, coccus- shaped and aerobic. Colonies are irregular, dry, undulate and ivory colored after 3 days on 0.1TSA at 25°C. Positive for esculin hydolysis, urease and β-galactosidase, but negative for oxidase, gelatin hydrolysis, nitrate reduction, glucose fermentation, indole production and arginine dihydrolase. D-Glucose, L-Arabinose, D-Mannitol, N-acetyl- glucosamine, D-Maltose, potassium gluconate and malic acid are utilized. Does not utilize D-Mannose, capric acid, adipic acid, trisodium citrate and phenylacetic acid. Strain 20LEG-88 (=NNIBR2020641BA804) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MW295517.

Description of Nocardioides zeicaulis KCS-46

Cells are Gram-staining-positive, non-flagellated, rodshaped and aerobic. Colonies are circular, convex, entire and yellow colored after 3 days on R2A at 25°C. Positive for esculin hydrolysis and β-galactosidase, but negative for oxidase, gelatin hydrolysis, nitrate reduction, glucose fermentation, indole production, arginine dihydrolase and urease. D-Glucose and D-Mannitol are utilized. Does not utilize L-Arabinose, D-Mannose, N-acetyl-glucosamine, D-Maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Strain KCS-46 (=NNIBR2019641BA239) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MN749884.

Description of Nocardioides taihuensis MBC-2-58

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are convex, opaque with entire edge and white colored after 4 days on MA at 25°C. Positive for glucose fermentation and β-galactosidase, but negative for oxidase, esculin hydrolysis and gelatin hydrolysis, nitrate reduction, indole production, arginine dihydrolase and urease. D-Glucose, L-Arabinose, adipic acid are utilized. Does not utilize D-Mannose, D-Mannitol, N-acetyl- glucosamine, D-Maltose, potassium gluconate, capric acid, malic acid, trisodium citrate and phenylacetic acid. Strain MBC-2-58 (=NNIBR2020641BA787) was isolated from freshwater of the Geum River, Korea. The Gen- Bank accession number for the 16S rRNA gene sequence is MW367773.

Description of Patulibacter americanus 20LEG-73

Cells are Gram-staining-positive, non-flagellated, rodsshaped and aerobic. Colonies are circular, convex, smooth and yellowish ivory colored after 3 days on TSA at 25°C. Positive for oxidase, esculin hydrolysis, gelatin hydrolysis and arginine dihydrolase, but negative for nitrate reduction, glucose fermentation, indole production, urease and β-galactosidase. D-Glucose and malic acid are utilized. Does not utilize L-Arabinose, D-Mannose, D-Mannitol, N-acetyl- glucosamine, D-Maltose, potassium gluconate, capric acid, adipic acid, trisodium citrate and phenylacetic acid. Strain 20LEG-73 (=NNIBR2020641BA803) was isolated from freshwater of the Geum River, Korea. The Gen- Bank accession number for the 16S rRNA gene sequence is MW295516.

Description of Pontibacter populi 9S-12

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are circular, convex, shiny and pink colored after 3 days on R2A at 25°C. Positive for oxidase, but negative for esculin hydrolysis, gelatin hydrolysis, nitrate reduction, glucose fermentation, indole production, arginine dihydrolase, β-galactosidase and urease. D-Glucose, D-Mannose, N-acetyl-glucosamine, D-Maltose (weakly) and potassium gluconate are utilized. Does not utilize L-Arabinose, D-Mannitol, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Strain 9S-10 (=NNIBR2019641BA1) was isolated from sediment of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MN602460.

Description of Hymenobacter perfusus KCS-3

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are circular, convex, entire and red colored after 3 days on R2A at 25°C. Positive for esculin hydrolysis and gelatin hydrolysis, but negative for oxidase, nitrate reduction, glucose fermentation, indole production, arginine dihydrolase, urease and β-galactosidase. D-Mannose, D-Mannitol and trisodium citrate are utilized. Does not utilize D-Glucose, L-Arabinose, N-acetyl-glucosamine, D-Maltose, potassium gluconate, capric acid, adipic acid, malic acid and phenylacetic acid. Strain KCS-3 (=NNIBR2019641BA238) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MN749883.

Description of Flavobacterium riviphilum JKS-1

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are circular, convex, entire and yellow colored after 3 days on R2A at 25°C. Positive for esculin hydrolysis, but negative for oxidase, gelatin hydrolysis, nitrate reduction, glucose fermentation, indole production, arginine dihydrolase, urease and β-galactosidase. D-Maltose are utilized. Does not utilize D-Glucose, L-Arabinose, D-Mannose, D-Mannitol, N-acetyl-glucosamine, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Strain JKS-1 (=NNIBR2019641BA244) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MN749893.

Description of Geomicrobium sediminis MND-3-64

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are circular, convex with entire edge, smooth and beige colored after 2 days on MA at 25°C. Positive for oxidase, esculin hydrolysis, gelatin hydrolysis arginine dihydrolase and urease, but negative for nitrate reduction, glucose fermentation, indole production and β-galactosidase. 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 are not utilized. Strain MND- 3-64 (=NNIBR2020641BA791) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MW367772.

Description of Brevundimonas balnearis 20HNW-03

Cells are Gram-staining-negative, non-flagellated, rods shaped and aerobic. Colonies are circular, convex, smooth and yellow colored after 3 days on 0.1 MA at 25°C. Positive for esculin hydrolysis and β-galactosidase, but negative for oxidase, gelatin hydrolysis, nitrate reduction, glucose fermentation, indole production, arginine dihydrolase and urease. 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 are not utilized. Strain 20HNW-03 (=NNIBR2020641BA805) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MW295518.

Description of Gellertiella hungarica IND-2-6

Cells are Gram-staining-negative, flagellated, rod-shaped and aerobic. Colonies are mucoid, irregular, opaque, convex and ivory colored after 2 days on R2A at 25°C. Positive for oxidase, esculin hydrolysis, β-galactosidase, arginine dihydrolase and urease, but negative for gelatin hydrolysis, nitrate reduction, glucose fermentation and indole production. D-Glucose, L-Arabinose, D-Mannose, D-Mannitol, N-acetyl-glucosamine and D-Maltose are utilized. Does not utilize potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Strain IND-2- 6 (=NNIBR2020641BA790) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MW367774.

Description of Rhizobium ipomoeae JU-40

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are circular, convex with entire edge and ivory colored after 3 days on R2A at 25°C. Positive for oxidase, esculin hydrolysis, nitrate reduction and β-galactosidase, but negative for gelatin hydrolysis, glucose fermentation, indole production, arginine dihydrolase and urease. D-Glucose, L-Arabinose, D-Mannose, D-Mannitol, N-acetyl-glucosamine, D-Maltose and trisodium citrate are utilized. Does not utilize potassium gluconate, capric acid, adipic acid, malic acid and phenylacetic acid. Strain JU-40 (=NNIBR2019641BA29) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MN559421.

Description of Paracoccus salipaludis 7J1-06

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are circular, smooth, flat and yellow-brown colored after 3 days on R2A at 25°C. Positive for oxidase, but negative for esculin hydrolysis, gelatin hydrolysis, nitrate reduction, glucose fermentation, indole production, arginine dihydrolase, β-galactosidase and urease. L-Arabinose are utilized. Does not utilize D-Glucose, D-Mannose, D-Mannitol, N-acetyl-glucosamine, D-Maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Strain 7J1-06 (=NNIBR2019641BA4) was isolated from water of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MN602458.

Description of Taonella mepensis 7J3-01

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are circular, convex, smooth and cream white colored after 3 days on R2A at 25°C. Positive for oxidase, gelatin hydrolysis, nitrate reduction, arginine dihydrolase and urease, but negative for esculin hydrolysis, glucose fermentation, indole production and β-galactosidase. D-Glucose, L-Arabinose, D-Mannose, D-Mannitol, N-acetyl-glucosamine, D-Maltose, potassium gluconate, malic acid are utilized. Does not utilize capric acid, adipic acid, trisodium citrate and phenylacetic acid. Strain 7J3-01 (=NNIBR2019641BA5) was isolated from water of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MN602464.

Description of Sphingomonas psychrolutea JU-31

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are circular, convex, smooth and yellow colored after 3 days on R2A at 25°C. Positive for esculin hydrolysis and β-galactosidase, but negative for oxidase, gelatin hydrolysis, nitrate reduction, glucose fermentation, indole production, arginine dihydrolase and urease. D-Glucose, L-Arabinose, D-Mannose, D-Mannitol, N-acetyl-glucosamine, malic acid, trisodium citrate, D-Maltose, potassium gluconate, capric acid, adipic acid and phenylacetic acid are not utilized. Strain JU-31 (=NNIBR2020641BA793) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MN559425.

Description of Burkholderia gladioli NSW-11

Cells are Gram-staining-negative, non-flagellated, short rod-shaped and aerobic. Colonies are circular, convex, entire and white colored after 3 days on R2A at 25°C. Positive for oxidase, esculin hydrolysis, gelatin hydrolysis, nitrate reduction and β-galactosidase, but negative for glucose fermentation, indole production, arginine dihydrolase and urease. D-Glucose, L-Arabinose, D-Mannose, D-Mannitol, N-acetyl-glucosamine, potassium gluconate, capric acid, malic acid and trisodium citrate are utilized. Does not utilize D-Maltose, adipic acid and phenylacetic acid. Strain NSW- 11 (=NNIBR2020641BA816) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MW293920.

Description of Polaromonas eurypsychrophila JJ1S-14

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are circular, convex, smooth and white colored after 3 days on R2A at 25°C. Positive for esculin hydrolysis and nitrate reduction, but negative for oxidase, gelatin hydrolysis, glucose fermentation, indole production, arginine dihydrolase, urease and β-galactosidase. D-Maltose and adipic acid are utilized. Does not utilize D-Glucose, L-Arabinose, D-Mannose, D-Mannitol, N-acetyl- glucosamine, potassium gluconate, capric acid, malic acid, trisodium citrate and phenylacetic acid. Strain JJ1S-14 (=NNIBR2019641BA236) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MN749850.

Description of Hydrogenophaga laconesensis 20CSW-3

Cells are Gram-staining-negative, non-flagellated, rodsshaped and aerobic. Colonies are circular, convex, smooth, translucent and red colored after 3 days on 0.1 TSA at 25°C. Positive for esculin hydrolysis, but negative for oxidase, gelatin hydrolysis, nitrate reduction, glucose fermentation, indole production, arginine dihydrolase, urease and β-galactosidase. 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 are not utilized. Strain 20CSW- 3 (=NNIBR2020641BA802) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MW295515.

Description of Chitinilyticum litopenaei 9W-13

Cells are Gram-staining-positive, flagellated, rod-shaped and aerobic. Colonies are circular, convex, smooth and cream white colored after 3 days on R2A at 25°C. Positive for oxidase. nitrate reduction and glucose fermentation, but negative for esculin hydrolysis, gelatin hydrolysis, indole production, arginine dihydrolase, β-galactosidase and urease. D-Glucose, D-Mannose, N-acetyl-glucosamine and potassium gluconate are utilized. Does not utilize L-Arabinose, D-Mannitol, D-Maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Strain 9W-13 (=NNIBR2019641BA3) was isolated from water of the Geum River, Korea. The Gen- Bank accession number for the 16S rRNA gene sequence is MN602461.

Description of Azospira oryzae YDC-53

Cells are Gram-staining-negative, single-flagellated, rodshaped and aerobic. Colonies are circular, convex, opaque and cream colored after 3 days on R2A at 25°C. Positive for esculin hydrolysis and β-galactosidase, but negative for oxidase, gelatin hydrolysis, nitrate reduction, glucose fermentation, indole production, arginine dihydrolase and urease. Malic acid are utilized. Does not utilize D-Glucose, L-Arabinose, D-Mannose, D-Mannitol, N-acetyl-glucosamine, trisodium citrate, D-Maltose, potassium gluconate, capric acid, adipic acid and phenylacetic acid. Strain YDC- 53 (=NNIBR2020641BA796) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MT893384.

Description of Zoogloea resiniphila 9S-10

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are circular, convex, smooth and cream white colored after 3 days on R2A at 25°C. Positive for oxidase, but negative for esculin hydrolysis, gelatin hydrolysis, nitrate reduction, glucose fermentation, indole production, arginine dihydrolase, β-galactosidase and urease. N-acetyl-glucosamine and malic acid are utilized. Does not utilize D-Glucose, L-Arabinose, D-Mannose, D-Mannitol, D-Maltose, potassium gluconate, capric acid, adipic acid, trisodium citrate and phenylacetic acid. Strain 9S-10 (=NNIBR2019641BA2) was isolated from sediment of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MN602459.

Description of Zoogloea oryzae 9W-02

Cells are Gram-staining-negative, non-flagellated, rodshaped and aerobic. Colonies are translucent, convex and bright yellow colored after 3 days on R2W at 25°C. Positive for oxidase, gelatin hydrolysis, nitrate reduction and urease, but negative for esculin hydrolysis, glucose fermentation, indole production, arginine dihydrolase and β-galactosidase. D-Glucose, D-Mannitol and D-Maltose are utilized. Does not utilize L-Arabinose, D-Mannose, N-acetyl-glucosamine, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate and phenylacetic acid. Strain 9W-02 (=NNIBR2019654BA3) was isolated from water of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MN696219.

Description of Pseudomonas furukawaii KKMS-29

Cells are Gram-staining-negative, non-flagellated, short rod-shaped and aerobic. Colonies are circular, convex, entire and white colored after 3 days on R2A at 25°C. Positive for arginine dihydrolase, but negative for oxidase, esculin hydrolysis, gelatin hydrolysis, nitrate reduction, indole production, glucose fermentation, β-galactosidase and urease. D-Glucose, capric acid and phenylacetic acid are utilized. Does not utilize L-Arabinose, D-Mannose, D-Mannitol, N-acetyl-glucosamine, D-Maltose, potassium gluconate, adipic acid, malic acid and trisodium citrate. Strain KKMS- 29 (=NNIBR2019641BA237) was isolated from freshwater of the Geum River, Korea. The GenBank accession number for the 16S rRNA gene sequence is MN749851.