Isolation of contaminant-degraders from the UGent lab

Bioremediation – the use of living organisms such as bacteria to reduce or remove contamination – has emerged as a cost-effective and environmentally-friendly alternative to traditional remediation techniques. This application requires isolation experiments to obtain cultures of degrading bacteria, which are therefore an indispensable element of the MIBIREM toolbox.

Culturomics

As early as 1985, researchers observed a major discrepancy between the bacterial cell numbers visible under a microscope, and the number of cultures that could be grown on agar plates, a phenomenon that became known as “the great plate count anomaly” and that gave rise to the belief that much less than 1% of all bacteria were “cultivable”.

Because microorganisms are adapted to their habitats, they often have specific growth requirements – such as pH, temperature or nutrients available in this environment– leading to the inability to grow them using standard laboratory conditions and conventional cultivation methods. As a result, many bacterial species are considered uncultivable, while they more correctly should be considered “uncultivated”, as the use of novel and creative cultivation conditions may facilitate their isolation.

Today, the use of such novel approaches to cultivation, combined with high-throughput picking, dereplication and identification of isolates, allows for the isolation of many bacteria that were earlier considered “uncultivable”. The term “culturomics” denotes such large-scale cultivation experiments combined with high-throughput analysis of isolates using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) (Lagier et al., 2015, 2016).

Isolation of contaminant-degraders

Within the MIBIREM project, isolation efforts target bacteria that degrade  hexachlorocyclohexane (HCH), petroleum hydrocarbons (HCH) or cyanides (CYA). The isolation process starts with a soil or groundwater sample, prepared by one of our project partners and enriched with a contaminant-degrading bacterial community to increase the likelihood of isolating contaminant-degrading or -tolerant bacteria.

Samples are inoculated onto a set of isolation media that target both contaminant-degrading and contaminant-tolerant bacteria. To select for contaminant-degrading bacteria, the HCH or PHC contaminant is added as the sole carbon and energy source, while CYA is added as the sole nitrogen source, allowing only bacteria that can metabolize the contaminant to grow, multiply and form colonies. To also isolate “supporting” bacteria – that do not necessarily degrade the contaminant but that can tolerate it – a general medium supplemented with the contaminant is used.

Per sample, >200 bacterial colonies are picked from the different media, purified into axenic cultures, and stored as glycerol stocks. Each culture is analysed using MALDI-TOF MS, which generates a unique protein fingerprint that can be used for both dereplication – the grouping of redundant isolates – and identification through comparison with a fingerprint library. Isolates for which no identification is obtained this way, are further identified using comparative 16S rRNA gene sequence analysis.

By now (April 2025), samples have been processed from eleven contaminated sites spread across seven countries, and resulted in a diverse collection of >4300 bioremediation isolates, including 1435 isolates from CYA-contaminated samples, 1558 isolates from PHC-contaminated samples, and 1338 isolates from HCH-contaminated samples.

Helping hands

Much of this work was made possible through the help of students, who dedicated a semester to this isolation campaign. An MSc student in Biochemistry and Biotechnology from Ghent University is doing her master’s thesis on the isolation of HCH-degrading bacteria, while two BSc students in Biomedical Laboratory Technology from the Odisee University College are doing their internship on PHC, CYA and HCH-degrading bacteria.

Each student is responsible for their own set of samples, and gains hands-on experience with every step of the process. The full-time involvement of students significantly increases our throughput capacity, and allows the simultaneous processing of multiple samples.

What is next?

After completing the isolation campaign, representative isolates will be selected for whole genome sequence analysis and for public deposit in the BCCM/LMG Bacteria Collection, as part of the MIBIREM Toolbox, along with all metadata. Within the project, their contaminant-degradation capabilities will be tested and some will be applied in field tests to evaluate their performance.

 

Author: Joni Elsmoortel,

Researcher, Ghent University