Periodic Reporting for period 2 - Fungateria (Enlisting synthetic fungal-bacterial consortia to produce multi-cellular mycelium-based ELMs with computational capability)
Periodo di rendicontazione: 2023-11-01 al 2025-04-30
Alongside rapid developments in the ELM field, there is a rapidly increasing interest in the research and development of mycelium materials, which are composed of the vegetative part of filamentous fungi. It can be envisaged that mycelium-based ELMs have a great potential given the rapid growth to various scales, the robustness of fungi in harsh conditions as opposed to mammalian cells and their advanced biological properties, including environmental sensing and transmission of electronic signals. Nevertheless, research of living mycelium-based materials has been limited.
In line with the production and implementation of ELMs in a resource and environmentally conscious manner, Fungateria addresses this development gap and targets three primary objectives:
(1) to develop a portfolio of mycelium-based ELMs composed of a synthetic co-cultivation consortium of a filamentous fungus and a bacterial strain;
(2) to develop an autonomous bottom-up manufacturing technology that enables an engineered morphogenesis of mycelium materials;
(3) to probe the emerging ethical, social and environmental issues for ELM technologies.
We identify four key areas that make up the project's pathway to impact:
(1) Technological impacts - the development of the unique bottom-up autonomous fabrication platform will not only generate a foundation for the production of mycelium-based ELMs, but will also generate insights and principles for similar production platforms for other ELM organism types;
(2) Economic impacts - because of the modular nature of the fabrication platform, upscaling becomes feasible thereby enabling the valorisation of high-volume agricultural or industrial side streams. A biobased value chain can be envisaged, involving various sectors such as waste management and logistics on the side of feedstock providers, industrial biotechnology companies on the side of technology providers and various consumer good sectors (packaging, textiles/fashion/design, automotive, construction, healthcare) on the side of end users;
(3) Sustainability impacts - by making better use of biological raw material, by-products and waste streams (e.g. forestry and agricultural residues, food waste) as feedstocks for fungal based ELM production, sustainable material production based on circular economy principles is viable;
(4) Societal impacts - given that it has been demonstrated that standard mycelium materials are an easily accessible technology for the general public, Fungateria’s focus on mycelium-based ELMs will lead to opportunities of creating public awareness regarding ELMs, thereby paving the way for social acceptance other ELM types.
In line with the production and implementation of ELMs in a resource and environmentally conscious manner, Fungateria addresses this development gap and targets three primary objectives:
(1) to develop a portfolio of mycelium-based ELMs composed of a synthetic co-cultivation consortium of a filamentous fungus and a bacterial strain;
(2) to develop an autonomous bottom-up manufacturing technology that enables an engineered morphogenesis of mycelium materials;
(3) to probe the emerging ethical, social and environmental issues for ELM technologies.
We identify four key areas that make up the project's pathway to impact:
(1) Technological impacts - the development of the unique bottom-up autonomous fabrication platform will not only generate a foundation for the production of mycelium-based ELMs, but will also generate insights and principles for similar production platforms for other ELM organism types;
(2) Economic impacts - because of the modular nature of the fabrication platform, upscaling becomes feasible thereby enabling the valorisation of high-volume agricultural or industrial side streams. A biobased value chain can be envisaged, involving various sectors such as waste management and logistics on the side of feedstock providers, industrial biotechnology companies on the side of technology providers and various consumer good sectors (packaging, textiles/fashion/design, automotive, construction, healthcare) on the side of end users;
(3) Sustainability impacts - by making better use of biological raw material, by-products and waste streams (e.g. forestry and agricultural residues, food waste) as feedstocks for fungal based ELM production, sustainable material production based on circular economy principles is viable;
(4) Societal impacts - given that it has been demonstrated that standard mycelium materials are an easily accessible technology for the general public, Fungateria’s focus on mycelium-based ELMs will lead to opportunities of creating public awareness regarding ELMs, thereby paving the way for social acceptance other ELM types.
Co-cultivation
Strong progress has been made toward establishing a co-cultivation toolbox for engineered living materials (ELMs). Fungal-bacterial co-cultivation systems were developed and supported by standardised protocols that enhance throughput and reproducibility. A key achievement is the implementation of an inducible lacZ reporter system, enabling real-time, visual tracking of bacterial function and localisation within the fungal matrix. Initial plans for melanin-based photoprotection and antimicrobial enzymes were revised in favour of more feasible, high-impact functionalities, such as PAH degradation.
Growth Composing Platform
Significant progress has made toward developing a modular GC platform for controlled fungal-bacterial morphogenesis. A GC setup has been developed demonstrating clear effects of different light wavelengths on mycelial yield and morphology. Photomasking techniques further enabled spatial control of growth. For 3D morphogenesis, molding strategies using 3D-printed forms, polypropylene fabric and hydrogel biotubes have bben tested, enabling structured and volumetric mycelium growth.
Sensing and computing
Extracellular electrophysiology has been previously identified as of relevance in functionalisation of the ELM in electrogenetic and similar applications including electogenetic logic switches. A wide spectral bandwidth (0.001- 6000 Hz) of fungal and ELM extracellular electrophysiology has been recorded and analysed using customisable microelectrode arrays (MEAs) and signal processing pipelines. Stimuli experiments, with a focus on electrical stimulus and evoked transmembrane potentials, are underway together with the implementation of logic gates.
Modelling
The modelling framework has been extended with the capability to represent environmental light, and the utility and accessibility of the model has been increased by migration to a web-based application with a flexible interface capable of representing a wide range of experimental setups. The basic infrastructure for integration between digital representation and live experiments has been established. Model validation and refinement processes are set up to compare the model against the diverse experiments found in the literature as well as incorporate newly generated data.
Digital Twin
A hardware Digital Twin implementation of the ELM is under development with the aim of accelerating exploration and experimentation as well as providing methods of direct interfacing in potential use-case scenarios. A suite of modelling methods have been developed to replicate the growth and electrical characteristics of mycelium-based ELMs. Circuit-level simulations have been conducted to confirm that operational behavior closely mirrors actual ELM activity. A hardware compatible Cellular Automata framework for modelling ELMs behavior has been developed.
Strong progress has been made toward establishing a co-cultivation toolbox for engineered living materials (ELMs). Fungal-bacterial co-cultivation systems were developed and supported by standardised protocols that enhance throughput and reproducibility. A key achievement is the implementation of an inducible lacZ reporter system, enabling real-time, visual tracking of bacterial function and localisation within the fungal matrix. Initial plans for melanin-based photoprotection and antimicrobial enzymes were revised in favour of more feasible, high-impact functionalities, such as PAH degradation.
Growth Composing Platform
Significant progress has made toward developing a modular GC platform for controlled fungal-bacterial morphogenesis. A GC setup has been developed demonstrating clear effects of different light wavelengths on mycelial yield and morphology. Photomasking techniques further enabled spatial control of growth. For 3D morphogenesis, molding strategies using 3D-printed forms, polypropylene fabric and hydrogel biotubes have bben tested, enabling structured and volumetric mycelium growth.
Sensing and computing
Extracellular electrophysiology has been previously identified as of relevance in functionalisation of the ELM in electrogenetic and similar applications including electogenetic logic switches. A wide spectral bandwidth (0.001- 6000 Hz) of fungal and ELM extracellular electrophysiology has been recorded and analysed using customisable microelectrode arrays (MEAs) and signal processing pipelines. Stimuli experiments, with a focus on electrical stimulus and evoked transmembrane potentials, are underway together with the implementation of logic gates.
Modelling
The modelling framework has been extended with the capability to represent environmental light, and the utility and accessibility of the model has been increased by migration to a web-based application with a flexible interface capable of representing a wide range of experimental setups. The basic infrastructure for integration between digital representation and live experiments has been established. Model validation and refinement processes are set up to compare the model against the diverse experiments found in the literature as well as incorporate newly generated data.
Digital Twin
A hardware Digital Twin implementation of the ELM is under development with the aim of accelerating exploration and experimentation as well as providing methods of direct interfacing in potential use-case scenarios. A suite of modelling methods have been developed to replicate the growth and electrical characteristics of mycelium-based ELMs. Circuit-level simulations have been conducted to confirm that operational behavior closely mirrors actual ELM activity. A hardware compatible Cellular Automata framework for modelling ELMs behavior has been developed.
Co-culitvation
Fungal-bacterial co-cultivation was limited by low reproducibility, throughput challenges, and rudimentary control over microbial function. Integration of visual biosensing in a mixed-species ELM and the focus on reproducibility mark a substantial leap over prior ad hoc, low-resolution approaches.
Growth Composing Platform
Growth control in ELMs was limited to basic containment or substrate patterning; little spatial control over morphogenesis existed. The GCP introduces a modular and multimodal system for precise spatial-temporal control of fungal growth, enabling architectural and structural programming not previously demonstrated.
Sensing and Computing
Fungal electrophysiology was underexplored, with limited bandwidth analysis and little application in computing or logic. FUNGATERIA is laying the ground for observation of fungal electrophysiology to programmable, logic-based signal processing, bridging biology and unconventional computing.
Modelling
ELM modelling was typically static, lab-bound, or limited to isolated biological parameters. The modelling framework evolves into a real-time, interactive and environmental-context-aware system, enabling predictive and adaptive experimental design as well as providing the basis for closed-loop production of designs thorugh the GCP.
DIgital Twin
Digital Twins for biological systems were conceptual, with limited physical modeling and interfacing capacity. This positions ELMs in the realm of cyber-biological systems, where software, hardware, and living systems co-evolve—a vision largely unexplored in current literature.
Fungal-bacterial co-cultivation was limited by low reproducibility, throughput challenges, and rudimentary control over microbial function. Integration of visual biosensing in a mixed-species ELM and the focus on reproducibility mark a substantial leap over prior ad hoc, low-resolution approaches.
Growth Composing Platform
Growth control in ELMs was limited to basic containment or substrate patterning; little spatial control over morphogenesis existed. The GCP introduces a modular and multimodal system for precise spatial-temporal control of fungal growth, enabling architectural and structural programming not previously demonstrated.
Sensing and Computing
Fungal electrophysiology was underexplored, with limited bandwidth analysis and little application in computing or logic. FUNGATERIA is laying the ground for observation of fungal electrophysiology to programmable, logic-based signal processing, bridging biology and unconventional computing.
Modelling
ELM modelling was typically static, lab-bound, or limited to isolated biological parameters. The modelling framework evolves into a real-time, interactive and environmental-context-aware system, enabling predictive and adaptive experimental design as well as providing the basis for closed-loop production of designs thorugh the GCP.
DIgital Twin
Digital Twins for biological systems were conceptual, with limited physical modeling and interfacing capacity. This positions ELMs in the realm of cyber-biological systems, where software, hardware, and living systems co-evolve—a vision largely unexplored in current literature.