Controlled Dispersion of Activated Sludge to Enable Isolation and Cultivation of Previously Uncultured Bacteria
Author
Kucharczyk, Patrycja
Term
4. term
Education
Publication year
2026
Submitted on
2026-10-02
Abstract
Wastewater treatment plants (WWTPs) contain highly complex microbial communities, including many bacteria that have so far not been successfully grown in the laboratory. In activated sludge (AS), these microorganisms form compact clusters called flocs, where close physical contact and interactions between species can be important for their growth. This project investigated whether carefully breaking up flocs into smaller fragments or single cells, followed by cultivation in 96-well plates, could improve laboratory cultivation of these bacteria. The idea is to simplify the community while keeping small subcommunities of bacteria that originally lived closely together in the same floc, so that important interactions are preserved and can support the growth of previously uncultured taxa. Activated sludge from Aalborg West WWTP was treated with different mechanical, chemical and enzymatic dispersion methods to break up the flocs. Floc disruption was evaluated by measuring particle size, using fluorescence microscopy with LIVE/DEAD staining, and counting colony-forming units (CFU). Different bead-beating matrices and zirconium bead sizes were compared to find a treatment that effectively fragmented flocs without killing too many cultivable cells. Zirconium bead size had a strong effect, and beads of 0.5 mm provided the best balance between reducing floc size and preserving bacterial culturability. Some other matrices produced similar or slightly greater reductions in particle size, but this did not consistently lead to higher bacterial recovery. The 0.5 mm zirconium bead treatment was therefore selected for use before cultivation in 96-well plates containing activated sludge-based medium. After cultivation, the bacterial communities were characterized using 16S rRNA gene amplicon sequencing with Oxford Nanopore Technologies (ONT). The combination of floc dispersion and cultivation in separate wells of a 96-well plate successfully enriched distinct bacterial populations, with some wells dominated by one or a few taxa. However, the enrichment was still biased toward fast-growing bacteria, indicating that further optimization of the cultivation conditions is needed to recover a broader range of slow-growing and previously uncultured taxa.
Renseanlæg for spildevand (WWTPs) indeholder meget sammensatte mikrobiologiske samfund med mange bakterier, som man endnu ikke har kunnet dyrke i laboratoriet. I aktivt slam (AS) sidder disse mikroorganismer sammen i komplekse små klumper, kaldet flocs, hvor tæt kontakt og samspil mellem arter kan være vigtigt for deres vækst. I dette projekt blev der undersøgt, om en kontrolleret opsplitning af flocs til mindre dele eller enkelte celler, efterfulgt af dyrkning i 96-brønds plader, kan gøre det lettere at dyrke flere af disse bakterier. Tanken er at gøre samfundet enklere, men stadig bevare små grupper af bakterier, som oprindeligt sad tæt sammen i flocen, så de vigtige mikrobielle interaktioner bevares og kan hjælpe tidligere ukultiverede bakterier med at vokse. Aktivt slam fra Aalborg Vest renseanlæg blev behandlet med forskellige mekaniske, kemiske og enzymatiske metoder for at sprede flocs. Effekten af opsplitning blev vurderet ved at måle partikelfordeling, bruge fluorescensmikroskopi med LIVE/DEAD-farvning og tælle kolonidannende enheder (CFU). Forskellige bead-beating-matricer og størrelser af zirkoniumkugler blev sammenlignet for at finde en behandling, der effektivt fragmenterede flocs uden at dræbe for mange dyrkbare bakterier. Størrelsen på zirkoniumkuglerne havde stor betydning, og 0,5 mm kugler gav den bedste balance mellem mindre flocstørrelse og bevaret bakteriel dyrkbarhed. Nogle andre matricer gav tilsvarende eller lidt stærkere reduktion i partikelstørrelse, men dette førte ikke nødvendigvis til bedre genopdyrkning af bakterier. Behandlingen med 0,5 mm zirkoniumkugler blev derfor valgt til brug før dyrkning i 96-brønds plader med medium baseret på aktivt slam. Efter dyrkning blev bakteriesamfundene analyseret ved 16S rRNA-gen ampliconsekventering med Oxford Nanopore Technologies (ONT). Kombinationen af floc-dispersion og dyrkning i separate brønde i en 96-brønds plade gav en vellykket berigelse af forskellige bakteriesamfund, og nogle brønde var domineret af én eller få bakterietyper. Berigelsen var dog stadig skæv mod hurtigtvoksende bakterier, hvilket viser, at dyrkningsbetingelserne skal optimeres yderligere for at kunne dyrke et bredere spektrum af langsomt voksende og hidtil ukultiverede bakterier.
[This abstract has been rewritten with the help of AI based on the project's original abstract]
Keywords