Microbial biofilms can be both cause and cure to a range of emerging societal problems including antimicrobial tolerance, water sanitation, water scarcity and pollution. The identities of extracellular polymeric substances (EPS) responsible for the establishment and function of biofilms are poorly understood. The lack of information on the chemical and physical identities of EPS limits the potential to rationally engineer biofilm processes, and impedes progress within the water and wastewater sector towards a circular economy and resource recovery. Here, a multidisciplinary roadmap for addressing this EPS identity crisis is proposed. This involves improved EPS extraction and characterization methodologies, cross-referencing between model biofilms and full-scale biofilm systems, and functional description of isolated EPS with in situ techniques (e.g. microscopy) coupled with genomics, proteomics and glycomics. The current extraction and spectrophotometric characterization methods, often based on the principle not to compromise the integrity of the microbial cells, should be critically assessed, and more comprehensive methods for recovery and characterization of EPS need to be developed.

The advantage of using non-invasive imaging such as optical coherence tomography (OCT) to asses material properties from deformed biofilm geometries can be compromised by the assumptions made on fluid forces acting on the biofilm. This study developed a method for the determination of elastic properties of biofilms by modelling the biofilm deformation recorded by OCT imaging with poroelastic fluid-structure interaction computations. Two-dimensional biofilm geometries were extracted from OCT scans of non-deformed and deformed structures as a result of hydrodynamic loading. The biofilm geometries were implemented in a model coupling fluid dynamics with elastic solid mechanics and Darcy flow in the biofilm. The simulation results were compared with real deformed geometries and a fitting procedure allowed estimation of the Young's modulus in given flow conditions. The present method considerably improves the estimation of elastic moduli of biofilms grown in mini-fluidic rectangular channels. This superior prediction is based on the relaxation of several simplifying assumptions made in past studies: shear stress is not anymore taken constant over the biofilm surface, total stress including also pressure is accounted for, any biofilm shape can be used in the determinations, and non-linear behavior of mechanical properties can be estimated. Biofilm elastic moduli between 70 and 700 Pa were obtained and biofilm hardening at large applied stress due to increasing flow velocity was quantified. The work performed here opens the way for in-situ determination of other mechanical properties (e.g., viscoelastic properties, relaxation times, plastic yields) and provides data for modelling biofilm deformation and detachment with eventual applications in biofilm control and removal strategies.

This study focused on characterizing the structure of biofilms developed on carriers used in lab-scale moving bed biofilm reactors. Both light microscopy (2D) and optical coherence tomography (OCT) were employed to track the biofilm development on carriers of different geometry and under different aeration rates. Biofilm structure was further characterized with respect to average biofilm thickness, biofilm growth velocity, biomass volume, compartment filling degree, surface area, etc. The results showed that carriers with a smaller compartment size stimulated a quick establishment of biofilms. Low aeration rates favored fast development of biofilms. Comparison between the results derived from 2D and 3D images revealed comparable results with respect to average biofilm thickness and compartment filling degree before the carrier compartments were fully willed with biomass. However, 3D imaging with OCT was capable of visualizing and quantifying the heterogeneous structure of biofilms, which cannot be achieved using 2D imaging.