Periodic Reporting for period 1 - MicroBioMem (Untangling the biophysical interactions governing biofilm hydraulic resistance using cyrogel membrane microfluidics)
Periodo di rendicontazione: 2021-10-01 al 2023-09-30
Access to potable water is a basic human right; however, for one third of the world population this right is not met. Membrane ultrafiltration is increasingly used to remove particulate matter, bacteria and large viruses from greywater, rainwater, and wastewater. A simple, low cost, portable variant of membrane ultrafiltration is gravity-driven membrane (GDM) filtration, which uses elevation head to impose hydrostatic pressure and induce flow, eliminating the need for expensive external pumping systems. GDM filtration inherently operates within a low hydrostatic pressure range (10–200 mbar), meaning the permeate flux is determined by the membrane hydraulic resistance, a parameter that increases over time due to membrane biofouling. GDM systems exploit the long-term stabilisation of biofouling layers to yield a stable permeate flux, a phenomenon known as flux stabilisation, enabling low-maintenance long-term system operation (5–8 years). However, GDM flux stabilisation values are typically an order of magnitude lower than conventional ultrafiltration systems, a major obstacle inhibiting community-scale adoption of GDM systems. Therefore, developing approaches to increase flux stabilisation values are critical to the furtherance of this technology.
The biofouling layer in GDM filters is a mixture of passive particulate and living biological matter, the major constituent being microbial biofilms. Ubiquitous in nature, biofilms are microbial communities bound together by a matrix of self-secreted extracellular polymeric substances (EPS). The EPS matrix is primarily composed of proteins, polysaccharides, and extracellular DNA, which together form a hydrated viscoelastic gel. This polymer network provides mechanical stability and acts as a small molecule diffusion barrier, contributing over 80% of the total GDM system hydraulic resistance. Biofilm hydraulic resistance is primarily a function of EPS pore size; however, EPS composition and spatial distribution is heterogeneous, with regulation of EPS secretion determined by local microbial responses to biological and chemical cues. Significantly, the application of external stresses invokes structural rearrangement through stress relaxation, and there is emerging evidence suggesting that mechanical cues can trigger EPS secretion. Biofilm hydraulic resistance has been shown to correlate with changes in mesoscale morphology, EPS volume and viscoelastic behaviour, factors seemingly shaped by hydrostatic pressure. However, despite these mesoscale observations, the microscale biophysical mechanisms and microstructural rearrangements that shape biofilm hydraulic resistance remain underexplored.
Currently, membrane-bound biofilm studies are performed at the meso- and macro-scale, and so require extended growth periods (days to months) and must rely upon ex-situ sacrificial analysis techniques with limited temporal and spatial resolution. Despite the important contribution of biofilm microstructure and EPS composition to the determination of hydraulic resistance, a systematic understanding of how hydrostatic pressure shapes biofilm microstructural development, EPS composition and mechanical properties is still lacking. This knowledge gap stems primarily from the difficulty of directly probing microscale rearrangements of biofilm structure and EPS composition under controlled hydrodynamic conditions. This project will fill this gap.
The aim of this project was to understand how hydrostatic pressure shapes the biophysical development of biofilm microstructure and EPS composition to affect biofilm hydraulic resistance. Insights gained at the microscale enabled the synthesis and rapid testing of pressure control strategies to engineer biofilm hydraulic resistance. To achieve these aims the project was split into two main objectives (O).
O1. To determine how hydrostatic pressure shapes microscale EPS distribution and composition of membrane-bound biofilms and how these properties interact to determine hydraulic resistance.
O2. To establish a scalable relation between hydrostatic pressure and biofilm hydraulic resistance based on the microscale investigations of O1 and demonstrate its validity at the macroscale using membrane biofouling simulators.
The biofouling layer in GDM filters is a mixture of passive particulate and living biological matter, the major constituent being microbial biofilms. Ubiquitous in nature, biofilms are microbial communities bound together by a matrix of self-secreted extracellular polymeric substances (EPS). The EPS matrix is primarily composed of proteins, polysaccharides, and extracellular DNA, which together form a hydrated viscoelastic gel. This polymer network provides mechanical stability and acts as a small molecule diffusion barrier, contributing over 80% of the total GDM system hydraulic resistance. Biofilm hydraulic resistance is primarily a function of EPS pore size; however, EPS composition and spatial distribution is heterogeneous, with regulation of EPS secretion determined by local microbial responses to biological and chemical cues. Significantly, the application of external stresses invokes structural rearrangement through stress relaxation, and there is emerging evidence suggesting that mechanical cues can trigger EPS secretion. Biofilm hydraulic resistance has been shown to correlate with changes in mesoscale morphology, EPS volume and viscoelastic behaviour, factors seemingly shaped by hydrostatic pressure. However, despite these mesoscale observations, the microscale biophysical mechanisms and microstructural rearrangements that shape biofilm hydraulic resistance remain underexplored.
Currently, membrane-bound biofilm studies are performed at the meso- and macro-scale, and so require extended growth periods (days to months) and must rely upon ex-situ sacrificial analysis techniques with limited temporal and spatial resolution. Despite the important contribution of biofilm microstructure and EPS composition to the determination of hydraulic resistance, a systematic understanding of how hydrostatic pressure shapes biofilm microstructural development, EPS composition and mechanical properties is still lacking. This knowledge gap stems primarily from the difficulty of directly probing microscale rearrangements of biofilm structure and EPS composition under controlled hydrodynamic conditions. This project will fill this gap.
The aim of this project was to understand how hydrostatic pressure shapes the biophysical development of biofilm microstructure and EPS composition to affect biofilm hydraulic resistance. Insights gained at the microscale enabled the synthesis and rapid testing of pressure control strategies to engineer biofilm hydraulic resistance. To achieve these aims the project was split into two main objectives (O).
O1. To determine how hydrostatic pressure shapes microscale EPS distribution and composition of membrane-bound biofilms and how these properties interact to determine hydraulic resistance.
O2. To establish a scalable relation between hydrostatic pressure and biofilm hydraulic resistance based on the microscale investigations of O1 and demonstrate its validity at the macroscale using membrane biofouling simulators.
The project was split into two main work packages: WP1 - Design and implementation of a microfluidic platform towards the quantification of bacterial biofilm hydraulic resistance. Application of the aforementioned platform for the microscale characterisation of bacterial biofilm hydraulic resistance as a function of; extracellular matrix composition, hydrostatic pressure and environmental conditions such as nutrient concentration and pH. WP2 - Quantification of bacterial biofilm hydraulic resistance on the mesoscale.
WP1 - A microfluidic platform was successfully established. The technology that allowed for the successful implementation of in-situ microfluidic membranes is now the focus of a patent application, and was the focus of an SNSF Bridge fellowship which was successfully awarded. The platform allowed for the characterisation of the aformented physicochemical conditions on bacterial biofilm hydraulic resistance. Quantified the per um3 hydraulic resistance of Bacillus subtilis, Pseudomonas aeruginosa, Staphyloccocus. epidermidis and a library of mutants. Results revealed the additive contribution of EPS components on hydraulic resistance. Rich nutrient environments produce biofilms with lower hydraulic resistance and a lower density biofilm. pH influences the permeability of bacterial biofilms, impacting the eDNA secretion. These results will be the focus of a peer reviewed article to be submitted following the action.
WP2 - Mesoscale hydraulic resistance experiments were performed using a membrane fouling simulator setup. These experiments focused upon Bacillus subtilis biofilms and a library of extracellular matrix mutants. The biofilms were grown at three different pressures.
WP1 - A microfluidic platform was successfully established. The technology that allowed for the successful implementation of in-situ microfluidic membranes is now the focus of a patent application, and was the focus of an SNSF Bridge fellowship which was successfully awarded. The platform allowed for the characterisation of the aformented physicochemical conditions on bacterial biofilm hydraulic resistance. Quantified the per um3 hydraulic resistance of Bacillus subtilis, Pseudomonas aeruginosa, Staphyloccocus. epidermidis and a library of mutants. Results revealed the additive contribution of EPS components on hydraulic resistance. Rich nutrient environments produce biofilms with lower hydraulic resistance and a lower density biofilm. pH influences the permeability of bacterial biofilms, impacting the eDNA secretion. These results will be the focus of a peer reviewed article to be submitted following the action.
WP2 - Mesoscale hydraulic resistance experiments were performed using a membrane fouling simulator setup. These experiments focused upon Bacillus subtilis biofilms and a library of extracellular matrix mutants. The biofilms were grown at three different pressures.
The project successfully designed a microscale platform for high-fidelity measurements of biofilm hydraulic resistance. The technology developed within this platform enables the integration of extra sensing modalities. This platform allows researchers to measure the density, thickness, and activity of bacterial biofilms within a microfluidic channel, opening up the possibility of testing the efficacy of antimicrobial and anti-extracellular matrix compounds.
The data acquired towards the characterization of biofilm hydraulic resistance is a step beyond the state of the art as it is of higher fidelity and removes the influence of heterogeneity from biofilm hydraulic resistance measurements, furthermore, the system provides direct optical access to membrane-grown biofilms, allowing for the application of advanced microscopy and spectroscopy methodologies.
Our results investigated the influence of hydrostatic pressure regimes on biofilm growth and hydraulic resistance, as well as testing matrix-degrading enzymes, such as DNase. The results can inform operation regimes for plant operators and engineers designing decentralised water treatment systems.
The data acquired towards the characterization of biofilm hydraulic resistance is a step beyond the state of the art as it is of higher fidelity and removes the influence of heterogeneity from biofilm hydraulic resistance measurements, furthermore, the system provides direct optical access to membrane-grown biofilms, allowing for the application of advanced microscopy and spectroscopy methodologies.
Our results investigated the influence of hydrostatic pressure regimes on biofilm growth and hydraulic resistance, as well as testing matrix-degrading enzymes, such as DNase. The results can inform operation regimes for plant operators and engineers designing decentralised water treatment systems.