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Nucleating the growth of biofilms for biocatalysis with polymer chemistry

Primary Supervisor: Dr Paco Fernandez-Trillo (PFT) Secondary supervisor: Tim W Overton (TWO) and Prof Mark Simmons (MS)

PhD project title: Nucleating the growth of biofilms for biocatalysis with polymer chemistry.

University of Registration: University of Birmingham

Project outline:

Bacteria often attach to surfaces in the form of biofilms. Biofilms represent a problem in many medical and industrial settings as they are physically tough and hard to kill or remove from surfaces. However, these characteristics make biofilms a potentially very useful platform for biocatalysis, where bacteria are used to make chemicals for a variety of purposes. This project will focus on controlling the way in which bacteria attach to surfaces, so we can specify when and where biofilms form.

In this project we will investigate the use of synthetic polymers to nucleate the growth of biofilms that will be utilised as a platform for biocatalysis. Previous work in FFT’s laboratory has demonstrated the potential of polymers to induce the clustering of bacteria and dictate the phenotype of the formed biofilm (F. Fernandez-Trillo et al. bioRxiv 2016). This expertise will be coupled with TWO’s expertise in the use of biofilms as a platform for biocatalysis. We will develop a high-throughput platform to evaluate how polymer length and composition determines the phenotype of the formed biofilms and their potential to produce relevant metabolites. This work has relevance in the production of pharmaceuticals, and potential applications for synthesis of fine chemicals.

Research Objectives: Our high-throughput strategy is based on the modification of a polymer scaffold (P1) to give us access to a library of modulated polymers (P2) with a broad range of chemical functionalities in highly efficient way (Scheme). Chemical functionality will be selected from a range of moieties known to mediate bacterial adhesion to surfaces and hosts, such as cationic residues, carbohydrates and peptides. These functional polymers will be then incubated with non-pathogenic strains of Escherichia coli, and the characteristics of the formed biofilms evaluated. Detailed objectives are:

O1. Synthesis of Polymer Scaffolds and Modulated Polymers: We have recently reported the use of poly(acryloyl hydrazide) (P1) for the rapid screening of siRNA delivery agents. Here, we will employ modulate P1 functionality with a series of aldehydes carrying relevant moieties for bacterial binding (Scheme). All new compounds will be characterised by NMR, HRMS, SEC and IR. The ability of these 1st generation “modulated” polymers (P2) to induce bacterial clustering will be initially assessed by a simple aggregation assay, that will identify lead candidates that can induce biofilm formation.

O2. Biofilm characterization, growth and catalysis: E. coli biofilms generated using the developed polymer scaffolds will be then characterised using a series of imaging techniques to determine morphology, composition (e.g. fluorescently-labelled lectins and SYTOX dyes for extracellular polysaccharides and nucleic acids respectively) and viability (LIVE/DEAD® BacLight). Bacterial localization will be monitored using laser scanning confocal microscopy. In addition to the surface techniques described, samples of the colonised surfaces will be taken and viability of the bacteria will be evaluated using standard techniques (i.e. CFUs, flow cytometry). Finally biocatalysis will be measured at the level of reaction rate using HPLC or comparable techniques (depending upon reaction) and spatially using confocal Raman microscopy.

We will employ an iterative process so that biofilm morphology and biocatalysis performance will inform “modulated” polymer synthesis and the choice of aldehydes. Overall, we aim to identify “modulated” polymers that can nucleate the growth of robust biofilms with a high catalytic activity.

O3. Lab-scale Bioreactor Design and Development: Lead candidates developed in O1-O2 will be used to build a flow-through bioreactor in packed bed, trickle bed or monolith configurations. Support geometry and size will be varied along with flow rate, retention time and other bioreactor operational parameters.

BBSRC Strategic Research Priority: Renewable Resources and Clean Growth: Industrial Biotechnology

Techniques that will be undertaken during the project:

Due to the multidisciplinary approach, the PhD student will develop a wide range of experimental skill described in the following training objectives:

Training 1-Microbiology:

  • Microbial culture: Post-graduate will receive training in standard techniques for microbial culture and identification. Post graduate will also receive training in flow cytometry and molecular microbiology methods. This expertise will be key for the completion of Objective 2-3.

  • Biofim Growth and Analysis: Post-graduate will receive training in techniques for biofilm generation and analysis, including Fluorescence and Raman Confocal Microscopy, Electron Microscopy. This expertise will be key for the completion of Objective 2-3.

  • Biocatalysis: Post-graduate will receive training in techniques to analysise biocatalyst performance including HPLC, and other assays as determined by the biocatalytic systems used. This expertise will be key for the completion of Objectives 2-3.

Training 2-Statistics and Data analysis:

The skills developed as part of the Foundation Skills Modules (i.e.: Large Dataset Handling, Quantitative Biology) will be supported by training in the following software packages:

  • GraphPad Prism: This software combines scientific graphing, curve fitting, statistics, and data organisation, and is specially designed for experimental biologists. This software will be key throughout the project, Objectives 1-3.

  • Design of Experiments: Post-graduate will receive training in R, a language and environment for statistical computing and graphics. Training in this package will allow student to implement a design of experiments approach and use surface response methodology to rapidly optimise experimental conditions, Objectives 1- 3.

Training 3-(Polymer) synthesis and Characterisation: Controlled polymerisation (i.e. free radical), combinatorial chemistry, (bio)conjugation using efficient chemistries (i.e. hydrazine-aldehyde condensation), polymer characterisation (NMR, GPC, IR). This expertise will be key for the completion of Objective 1.

Training 4-Bioreactor Design and Development: Postgraduate will receive basic training in design of packing parameters, such as support geometry, size or density. Postgraduate will also receive training in bioreactor operation and optimization, including flow rate and retention time. This expertise will be key for the completion ofObjective 3.

Contact: Dr Paco Fernandez-Trillo, University of Birmingham