Reduced-Complexity Cultivation of Human Oral Anaerobic Bacteria Using a Microdroplet-Based Culturomics Platform.
6 affiliations
- 1Integrated Biomedical and Life Science, Graduate School, Korea University, Seoul 02841, Republic of Korea.
- 2Interdisciplinary Program in Precision Public Health, Korea University, Seoul 02841, Republic of Korea.
- 3Department of Bioengineering, Korea University, Seoul 02841, Republic of Korea.
- 4Institute for Biomaterials, Korea University, Seoul 02841, Republic of Korea.
- 5School of Biomedical Engineering, Korea University, Seoul 02841, Republic of Korea.
- 6School of Biosystems and Biomedical Sciences, Korea University, Seoul 02841, Republic of Korea.
Abstract
The human oral microbiota contains fastidious anaerobes that remain uncultivated due to resource competition and overgrowth by dominant species in conventional plating. Culturomics approaches leveraging droplet-based microfluidics offer an alternative by physically compartmentalizing microbial cells. We established a polydimethylsiloxane-based microdroplet platform to isolate anaerobic bacteria from human oral rinse samples. Droplet generation conditions were optimized using fluorescent beads, and bacterial loading at the collection-drop level was evaluated using Escherichia coli. Two downstream isolation strategies-Direct-plating (Method 1) and Enrichment (Method 2)-were compared against conventional serial dilution across various media compositions. Isolated strains were identified via 16S rRNA gene sequencing. At optimized flow rates (oil: 1.5 mL/h, core: 0.3 mL/h), the system produced uniform microdroplets with an average occupancy (λ) of 0.010-0.012 cells/droplet, corresponding to a high theoretical probability of single-cell occupancy among non-empty microdroplets. Each macro-collection drop delivered approximately 50-60 cells into recipient wells, serving as a reduced-complexity cultivation unit. Under the matched solid-medium condition, Method 1 achieved the highest taxonomic yield (14.3%), representing a 3.5-fold enhancement over conventional serial dilution (4.1%). Method 2 yielded 4.7%, indicating that liquid enrichment reintroduced growth bias toward fast-growing taxa. Among the tested media, unsupplemented Tryptic Soy Agar preserved the highest species diversity, whereas 5% sheep blood supplementation led to monoculture dominance by fast-growing taxa. Overall, the platform isolated diverse oral anaerobic species including fastidious taxa like Capnocytophaga, Cardiobacterium, and Eikenella. The microdroplet-based reduced-complexity platform may limit the dominance of rapidly growing taxa, providing a complementary culturomics tool for expanding human oral anaerobic bacterial resources.
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BibTeX
@article{kang2026reducedcomplexity,
title = {{Reduced-Complexity Cultivation of Human Oral Anaerobic Bacteria Using a Microdroplet-Based Culturomics Platform.}},
author = {Chaeeun Kang and You-Jeong Kim and Min-Gyung Baek and Aram J Chung and Hana Yi},
journal = {Journal of microbiology and biotechnology},
year = {2026},
month = oct,
doi = {10.4014/jmb.2607.07042},
url = {https://europepmc.org/article/MED/42894962},
note = {CC BY},
}
RIS
TY - JOUR
TI - Reduced-Complexity Cultivation of Human Oral Anaerobic Bacteria Using a Microdroplet-Based Culturomics Platform.
AU - Chaeeun Kang
AU - You-Jeong Kim
AU - Min-Gyung Baek
AU - Aram J Chung
AU - Hana Yi
PY - 2026
DA - 2026/10/08
JO - Journal of microbiology and biotechnology
DO - 10.4014/jmb.2607.07042
UR - https://europepmc.org/article/MED/42894962
AB - The human oral microbiota contains fastidious anaerobes that remain uncultivated due to resource competition and overgrowth by dominant species in conventional plating. Culturomics approaches leveraging droplet-based microfluidics offer an alternative by physically compartmentalizing microbial cells. We established a polydimethylsiloxane-based microdroplet platform to isolate anaerobic bacteria from human oral rinse samples. Droplet generation conditions were optimized using fluorescent beads, and bacterial loading at the collection-drop level was evaluated using Escherichia coli. Two downstream isolation strategies-Direct-plating (Method 1) and Enrichment (Method 2)-were compared against conventional serial dilution across various media compositions. Isolated strains were identified via 16S rRNA gene sequencing. At optimized flow rates (oil: 1.5 mL/h, core: 0.3 mL/h), the system produced uniform microdroplets with an average occupancy (λ) of 0.010-0.012 cells/droplet, corresponding to a high theoretical probability of single-cell occupancy among non-empty microdroplets. Each macro-collection drop delivered approximately 50-60 cells into recipient wells, serving as a reduced-complexity cultivation unit. Under the matched solid-medium condition, Method 1 achieved the highest taxonomic yield (14.3%), representing a 3.5-fold enhancement over conventional serial dilution (4.1%). Method 2 yielded 4.7%, indicating that liquid enrichment reintroduced growth bias toward fast-growing taxa. Among the tested media, unsupplemented Tryptic Soy Agar preserved the highest species diversity, whereas 5% sheep blood supplementation led to monoculture dominance by fast-growing taxa. Overall, the platform isolated diverse oral anaerobic species including fastidious taxa like Capnocytophaga, Cardiobacterium, and Eikenella. The microdroplet-based reduced-complexity platform may limit the dominance of rapidly growing taxa, providing a complementary culturomics tool for expanding human oral anaerobic bacterial resources.
SN - 1017-7825
SN - 1738-8872
ER -