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.

1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS AND DISCUSSION
    3.1. Description of Catenulispora rubraLS9-103
    3.2. Description of Cellulomonas algicola23ND14S-41
    3.3. Description of Populibacteriumcorticicola 23ND75W-61
    3.4. Description of Intrasporangium flavum21SJ05W-0M23
    3.5. Description of Ornithinimicrobiumflavum 23ND49W-34
    3.6. Description of Blastococcus aurantiacusHRW3-25
    3.7. Description of Blastococcusendophyticus 6LW2-11
    3.8. Description of Kineosporia rhamnosa22LW2-51
    3.9. Description of Arenivirga flava23ND25W-67
    3.10. Description of Cryobacterium breveLW1-R25
    3.11. Description of Actinoplanes digitatis22LW2-48
    3.12. Description of Micromonospora globbae23ND14S-8
    3.13. Description of Corynebacteriumvariabile SS-105
    3.14. Description of Gordonia sputi SJ8
    3.15. Description of Nocardia stercoris21LS8-22
    3.16. Description of Nocardia rayongensis23ND34S-94
    3.17. Description of Mycobacterium gilvumSJW1-30
    3.18. Description of Nocardioides allogilvus21LS6-111
    3.19. Description of Nocardioideslianchengensis 23ND01W-24
    3.20. Description of Nocardioides kandeliae23ND54S-44
    3.21. Description of Aestuariimicrobium soli21SJ05S-R33
    3.22. Description of Solirubrobacter pauli21SJ02S-54
4. CONCLUSION
CRediT authorship contribution statement
Declaration of Competing Interest
ACKNOWLEDGEMENTS
REFERENCES

• Soo-Yeong Lee(Biological Resources Research Department, Nakdonggang National Institute of Biological Resources (NNIBR), Sangju 37242, Republic of Korea)
• Jaeduk Goh(Biological Resources Research Department, Nakdonggang National Institute of Biological Resources (NNIBR), Sangju 37242, Republic of Korea)
• Ahyoung Choi(Biological Resources Research Department, Nakdonggang National Institute of Biological Resources (NNIBR), Sangju 37242, Republic of Korea) Corresponding author