Microbial community fingerprinting to predict pathogen outgrowth in engineered aquatic systems

Props Ruben

Promoter

Boon Nico, (UGent), nico.boon@ugent.be

SCK•CEN Mentor

Monsieurs Pieter
pieter.monsieurs@sckcen.be
+32 14 33 21 08

SCK•CEN Co-mentor

Leys Natalie
natalie.leys@sckcen.be
+32 14 33 27 26

Expert group

Microbiology

PhD started

2014-10-13

Short project description

Cooling towers are known to be a potential source for harbouring, amplifying and disseminating microbial human pathogens, such as Legionella and Mycobacterium species. According to a European directive (90/679/EEG) and the Belgian legislation (CODEX Welzijn op het werk, KB Biologische Agentia) it is required to monitor for such biological agents in cooling towers, including those of nuclear plants. Aside from the health risk, the microbes encountered in cooling water circuits can also pose operational and structural problems via biofouling and biofilm formation, or even biodegradation or biocorrosion of the tubes and pipes, condenser materials, or concrete walls.  

Until now, traditional culture-based methods (i.e. growing bacterial cultures in the lab) are used for characterising the microbiota of the cooling water and for detecting and quantifying specific hazardous bacteria. However, this technique severely underestimates cell densities (by 10% to 60%), and only allows the measurement of a limited subset of pathogens. For example, disinfection treatments can create viable but non culturable cells (VBNC) which are still harmful but will not be detected via such culture-based methods. In addition, the factors contributing to the occurrence, proliferation and persistence of potential human pathogenic organism in the microbiota of cooling water circuits are still unclear.

A better understanding of these microbial communities, as well as their evolution under diverse operational conditions of the nuclear plants, could help to develop better monitoring, and potential also preventive and counteractive measures.

Objective

The aim of this project is to investigate the microbiological diversity and community dynamics in cooling tower water using the newest cell-based and DNA-based technologies, such as flow cytometry and metagenomics. The main advantage of these new methodologies is that they are not requiring culturing and are thus not limiting the results to specific pathogenic species or those microbes that are cultivable under lab conditions.

Therefore this project will combine the expertise of the microbiology research teams MIC of SCK•CEN and LabMET of UGent in Belgium. The Microbiology research unit of SCK•CEN, has a large expertise regarding the genetic characterization of microbial populations of cooling, drinking and ground water by metagenomic and bioinformatic analysis. The Laboratory of Microbial Ecology and Technology of UGent, has developed a sophisticated flow cytometry based approach for a fast and objective comparison of the physiological state of microbial communities based on cellular features of the single cells.

In this project, the flow cytometry method will be tested and optimised as a quick fingerprinting method of the microbial communities. This method consists of two main parts: (1) the generation of fingerprint data by flow cytometry, and (2) the analysis of flow cytometry data by a novel statistical pipeline. It will be evaluated if this advanced flow cytometry can be used as a tool to detect shifts within the cooling water communities, in response to operational factors like water temperature, chemical composition, and pH, and disinfection treatments. The focus will be on the interrelationship between the evenness (i.e. the relative abundance of species in a community), richness, diversity, uniformity, invasion, and stability of the microbial communities in response to the environmental changes (including disinfection treatments), as detected by flow cytometry. The aim is to identify a set of parameters that can be used as key indicators of community biodiversity, functionality and recovery under environmental changing conditions.

In parallel, the metagenomics analysis will provide a more detailed identification of the total microbial community in the cooling water and its dynamics in response to the environmental conditions. Such an analysis will hopefully lead to the identification of one or more (non-pathogenic) reporter organisms (e.g. Amoeba), able to predict the outbreak of a hazardous microbial processes or species. Moreover, one can search for alternative marker genes that might be used as a criterion to quantify the presence of the biologic agents in the water. This unique combination of advanced flow cytometry with metagenomics, allows characterizing the community and its dynamics in detail and to confirm and explain the overall community changes observed by flow cytometry.  

The project will use the BR2 cooling water system and tower, as unique test facility at SCK•CEN. Water of the final (non-active) BR2 cooling water circuit and tower will be sampled, at different locations and during different operational conditions, and analysed for its microbiota in respect to the recorded operational parameters (such as temperature, chemical composition, and pH). In addition, it is aimed to compare the results obtained for BR2 with data obtained by the UGent team for water samples of other cooling water facilities and drinking water facilities.

Ideally this research project will lead to the development of a flow cytometry protocol providing a straightforward, fast and cost-efficient tool for accurately monitoring and estimating the risk of biofouling and/or pathogens release in the environment via cooling towers.