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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

Isolation, Enrichment and Analysis of Aerobic, Anaerobic, Pathogen-Free and Non-Resistant Cellulose-Degrading Microbial Populations from Methanogenic Bioreactor

by
Lyudmila Dimitrova
1,*,
Yana Ilieva
1,
Dilnora Gouliamova
2,
Vesselin Kussovski
1,
Venelin Hubenov
3,
Yordan Georgiev
1,
Tsveta Bratanova
1,
Mila Kaleva
1,
Maya M. Zaharieva
1 and
Hristo Najdenski
1
1
Department of Infectious Microbiology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
2
Department of General Microbiology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
3
Department of Biotechnology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
*
Author to whom correspondence should be addressed.
Genes 2025, 16(5), 551; https://doi.org/10.3390/genes16050551
Submission received: 11 April 2025 / Revised: 29 April 2025 / Accepted: 29 April 2025 / Published: 30 April 2025
(This article belongs to the Section Genes & Environments)
Versions Notes

Abstract

Background: Nowadays, the microbial degradation of cellulose represents a new perspective for reducing cellulose waste from industry and households and at the same time obtaining energy sources. Methods: We isolated and enriched two aerobic (at 37 °C and 50 °C) and one anaerobic microbial consortium from an anaerobic bioreactor for biogas production by continuous subculturing on peptone cellulose solution (PCS) medium supplemented with 0.3% treated or untreated Whatman filter paper under static conditions. Samples were taken every 7 days until day 21 to determine the percentage of cellulose biodegradation. We determined the antimicrobial resistance of aerobic and anaerobic consortia and some single colonies by disc diffusion method, against 42 clinically applied antibiotics. PCR analyses were performed to search for the presence of eight genes for cellulolytic activity and nine genes for antibiotic resistance. By metagenomics analysis, the bacterial and fungal genus distributions in the studied populations were determined. Results: Aerobes cultured at 50 °C degraded cellulose to the greatest extent (47%), followed by anaerobes (24–38%) and aerobes (8%) cultured at 37 °C. The bacterial sequence analysis showed that the dominant phyla are Bacillota and Bacteroidetes and genera—Paraclostridium, Defluvitalea, Anaerobacillus, Acetivibrio, Lysinibacillus, Paenibacillus, Romboutsia, Terrisporobacter, Clostridium, Sporanaerobacter, Lentimicrobium, etc. in a different ratio depending on the cultivation conditions and the stage of the process. Some of these representatives are cellulolytic and hemicellulolytic microorganisms. We performed lyophilization and proved that it is suitable for long-term storage of the most active consortium, which degrades even after the 10th re-inoculation for a period of one year. We proved the presence of ssrA, ssrA BS and blaTEM genes. Conclusions: Our findings demonstrated the potential utility of the microbial consortium of anaerobes in the degradation of waste lignocellulose biomass.
Keywords: microbial consortia; biodegradation of cellulose; antimicrobial resistance; long-term storage; PCR; metagenomic analysis microbial consortia; biodegradation of cellulose; antimicrobial resistance; long-term storage; PCR; metagenomic analysis

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MDPI and ACS Style

Dimitrova, L.; Ilieva, Y.; Gouliamova, D.; Kussovski, V.; Hubenov, V.; Georgiev, Y.; Bratanova, T.; Kaleva, M.; Zaharieva, M.M.; Najdenski, H. Isolation, Enrichment and Analysis of Aerobic, Anaerobic, Pathogen-Free and Non-Resistant Cellulose-Degrading Microbial Populations from Methanogenic Bioreactor. Genes 2025, 16, 551. https://doi.org/10.3390/genes16050551

AMA Style

Dimitrova L, Ilieva Y, Gouliamova D, Kussovski V, Hubenov V, Georgiev Y, Bratanova T, Kaleva M, Zaharieva MM, Najdenski H. Isolation, Enrichment and Analysis of Aerobic, Anaerobic, Pathogen-Free and Non-Resistant Cellulose-Degrading Microbial Populations from Methanogenic Bioreactor. Genes. 2025; 16(5):551. https://doi.org/10.3390/genes16050551

Chicago/Turabian Style

Dimitrova, Lyudmila, Yana Ilieva, Dilnora Gouliamova, Vesselin Kussovski, Venelin Hubenov, Yordan Georgiev, Tsveta Bratanova, Mila Kaleva, Maya M. Zaharieva, and Hristo Najdenski. 2025. "Isolation, Enrichment and Analysis of Aerobic, Anaerobic, Pathogen-Free and Non-Resistant Cellulose-Degrading Microbial Populations from Methanogenic Bioreactor" Genes 16, no. 5: 551. https://doi.org/10.3390/genes16050551

APA Style

Dimitrova, L., Ilieva, Y., Gouliamova, D., Kussovski, V., Hubenov, V., Georgiev, Y., Bratanova, T., Kaleva, M., Zaharieva, M. M., & Najdenski, H. (2025). Isolation, Enrichment and Analysis of Aerobic, Anaerobic, Pathogen-Free and Non-Resistant Cellulose-Degrading Microbial Populations from Methanogenic Bioreactor. Genes, 16(5), 551. https://doi.org/10.3390/genes16050551

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