Periodic Reporting for period 1 - ABSOLUTE (Analysis of Biofilm Solid Interactions Underpinning Wastewater Treatment)
Reporting period: 2023-01-01 to 2025-06-30
Freshwater, barely enough for a world population of 7.9 billion people, is distributed unevenly, frequently wasted, polluted, and unsustainably managed. The availability of safe and sufficient water supplies is inextricably linked to how wastewater is managed. There is thus a need to develop, understand and optimise efficient and effective wastewater treatment (WWT) technologies to enhance water quality and facilitate resource recovery and water recycling. The adoption of modern biofilm technology has accelerated in recent decades as WWT in large urban centres were required to upgrade their treatment to meet more stringent standards and to expand to deal with higher inflows. Biofilm technology is the choice when process intensification is required in a WWT plant and to provide capacity to deal with stringent treatment standards for nutrients. Biofilm technology is often considered as an upgrade of the conventional activated sludge process to meet a higher effluent quality and treatment capacity without requiring expansion in the footprint of the WWT plant. However, the deployed operational efficiency of these processes is significantly below what is theoretically possible due to (a) a design process that, while using some mathematical modelling, uses rules-of-thumb, and (b) a significant knowledge gap in the understanding of several complex physio-chemical and biological mechanisms, the subject of this project.
Biofilms, the key catalysts in biofilm-based WWT processes, are structurally complex, integrated multi- cellular communities of surface-adhering microorganisms embedded within an extracellular polymeric (EPS) matrix. In the WWT context they provide a higher volumetric productivity and enhanced nutrient removal capacity compared to the Activated Sludge process. Municipal wastewater comprises soluble organic and nitrogenous matter but also a significant fraction of suspended organic particles and dissolved polymeric material/colloidal matter and some inorganic particulates. The fraction of the particulate organic matter including colloidal/polymeric matter in pre- settled municipal wastewaters is typically 40–60% of the total organic matter. Current mathematical models for biofilm processes are heavily focused on the soluble components. It is now widely accepted that one of the major limitations of biofilm models is that, while they handle soluble components reasonably well “…a general lack of basic research …has led to a severe deficiency in the ability of existing…biofilm models to describe the fate of particulate components in biofilm reactors..”. These limitations can only be addressed by developing a fundamental understanding of biofilm-particulate interactions, the focus of this project.
Objectives:
Specific Objective 1: Employing a sophisticated platform based on advanced imaging/particle tracking and fluorescent labelling techniques integrated with a continuous flow biofilm cultivation system, we will develop fundamental insights into biofilm-particulate interactions focusing on research questions that have not been previously elucidated because of the limitations of conventional approaches.
Specific Objective 2: Investigate (a) spatial profile of the reaction rates in terms of the breakdown of particulate organic matter in the biofilm (b) quantify the effect of particulate organic matter loading and spatial profile on the reaction rates of soluble components within the biofilm and translate this in quantitative terms to mathematical relationships.
Specific Objective 3: Using a combined experimental-modelling approach, develop further the IWA multispecies biofilm model framework and refine and validate the model using data from a pilot plant wastewater process.
Biofilms, the key catalysts in biofilm-based WWT processes, are structurally complex, integrated multi- cellular communities of surface-adhering microorganisms embedded within an extracellular polymeric (EPS) matrix. In the WWT context they provide a higher volumetric productivity and enhanced nutrient removal capacity compared to the Activated Sludge process. Municipal wastewater comprises soluble organic and nitrogenous matter but also a significant fraction of suspended organic particles and dissolved polymeric material/colloidal matter and some inorganic particulates. The fraction of the particulate organic matter including colloidal/polymeric matter in pre- settled municipal wastewaters is typically 40–60% of the total organic matter. Current mathematical models for biofilm processes are heavily focused on the soluble components. It is now widely accepted that one of the major limitations of biofilm models is that, while they handle soluble components reasonably well “…a general lack of basic research …has led to a severe deficiency in the ability of existing…biofilm models to describe the fate of particulate components in biofilm reactors..”. These limitations can only be addressed by developing a fundamental understanding of biofilm-particulate interactions, the focus of this project.
Objectives:
Specific Objective 1: Employing a sophisticated platform based on advanced imaging/particle tracking and fluorescent labelling techniques integrated with a continuous flow biofilm cultivation system, we will develop fundamental insights into biofilm-particulate interactions focusing on research questions that have not been previously elucidated because of the limitations of conventional approaches.
Specific Objective 2: Investigate (a) spatial profile of the reaction rates in terms of the breakdown of particulate organic matter in the biofilm (b) quantify the effect of particulate organic matter loading and spatial profile on the reaction rates of soluble components within the biofilm and translate this in quantitative terms to mathematical relationships.
Specific Objective 3: Using a combined experimental-modelling approach, develop further the IWA multispecies biofilm model framework and refine and validate the model using data from a pilot plant wastewater process.
Achievement 1 Development of advanced imaging platform
We constructed a state-of-the art platform incorporating real-time super-resolution imaging as a core tool in conjunction with a continuous-flow biofilm cultivation system to enable imaging of biofilm, aggregates/particles. The platform allows us to generate quantitative data to allow, for the first time, a fundamental understanding of particle-biofilm interactions.
Achievement 2 Super-resolution time-lapse imaging
We completed a time-lapse study on the dynamics of biofilm formation where we were able count the number of cells at each timepoint. This is in contrast to the more common use of biovolume which is not specific enough. This allows us to do a number of things including kinetic studies (to compare biofilm to planktonic growth) and to distinguish between different early stage biofilm morphologies.
Achievement 3: new modelling platform
The mechanistic understanding of particulate-biofilm interactions and the translation of that into useful mathematical models is generally lacking. We set out to re-conceptualise biofilm mathematical models using a distribution function that allows us to model morphology in a way not previously done. We have created benchmark model case studies and have undertaken comparisons between our new model and existing models.
Achievement 4: aggregate developmental dynamics
Biofilms were quantitatively compared to control studies using planktonic cell as inoculum. CLSM was employed for structure and matrix examination combined with molecular assays for biomass, viability and metabolic activity. It was found that endpoint biofilms from aggregates were quantitatively distinct from their planktonic counterpart, demonstrating greater maximum and average thickness, higher biomass and a lower roughness coefficient. We hypothesise that the resident cells within isolated aggregates are primed for biofilm development and do not need to invest the resources that are necessary for early attachment and the switch to biofilm formation by planktonic cells upon surface association.
Achievement 5: detachment dynamics
We hypothesized that different media conditions impact biofilm stability, cell viability, and reattachment efficiency, ultimately affecting the structural and functional characteristics of secondary biofilms formed from detached cells (detached matter is a type of ‘particulate organic matter’ . Detached biofilm cells exhibit a transitional phenotype between planktonic and sessile states, with cell viability and attachment experiments showing differences between primary biofilm-associated cells (PBACs) and secondary biofilm-associated cells (SBACs). The growth kinetics data indicate differences in the growth dynamics of planktonic cells and PBACs,. Morphological and structural differences were observed between primary and secondary biofilms.
We constructed a state-of-the art platform incorporating real-time super-resolution imaging as a core tool in conjunction with a continuous-flow biofilm cultivation system to enable imaging of biofilm, aggregates/particles. The platform allows us to generate quantitative data to allow, for the first time, a fundamental understanding of particle-biofilm interactions.
Achievement 2 Super-resolution time-lapse imaging
We completed a time-lapse study on the dynamics of biofilm formation where we were able count the number of cells at each timepoint. This is in contrast to the more common use of biovolume which is not specific enough. This allows us to do a number of things including kinetic studies (to compare biofilm to planktonic growth) and to distinguish between different early stage biofilm morphologies.
Achievement 3: new modelling platform
The mechanistic understanding of particulate-biofilm interactions and the translation of that into useful mathematical models is generally lacking. We set out to re-conceptualise biofilm mathematical models using a distribution function that allows us to model morphology in a way not previously done. We have created benchmark model case studies and have undertaken comparisons between our new model and existing models.
Achievement 4: aggregate developmental dynamics
Biofilms were quantitatively compared to control studies using planktonic cell as inoculum. CLSM was employed for structure and matrix examination combined with molecular assays for biomass, viability and metabolic activity. It was found that endpoint biofilms from aggregates were quantitatively distinct from their planktonic counterpart, demonstrating greater maximum and average thickness, higher biomass and a lower roughness coefficient. We hypothesise that the resident cells within isolated aggregates are primed for biofilm development and do not need to invest the resources that are necessary for early attachment and the switch to biofilm formation by planktonic cells upon surface association.
Achievement 5: detachment dynamics
We hypothesized that different media conditions impact biofilm stability, cell viability, and reattachment efficiency, ultimately affecting the structural and functional characteristics of secondary biofilms formed from detached cells (detached matter is a type of ‘particulate organic matter’ . Detached biofilm cells exhibit a transitional phenotype between planktonic and sessile states, with cell viability and attachment experiments showing differences between primary biofilm-associated cells (PBACs) and secondary biofilm-associated cells (SBACs). The growth kinetics data indicate differences in the growth dynamics of planktonic cells and PBACs,. Morphological and structural differences were observed between primary and secondary biofilms.
At this stage of the project, our advances are mostly methodological, but we are confident that over the next year we will see advances beyond state-of-the-art in the scientific understanding of biofilm-particulate interactions.