Periodic Reporting for period 2 - FuturoLEAF (Leaf-inspired nanocellulose frameworks for next generation photosynthetic cell factories)
Berichtszeitraum: 2021-09-01 bis 2023-12-31
FuturoLEAF combines know-how on lignocellulosic materials and cell biology to revolutionize industrial algal biotechnology field with a new tailorable solid-state photosynthetic cell factory (SSPCF) concept. Here, photosynthetic cells are embedded within a functional architecture fabricated from lignocellulosic building blocks, designed on the principles of plant leaf anatomy and function. The project integrates knowledge on bio-based materials science and photosynthesis with achievements on synthetic biology and biomolecular engineering to conceive new technology that efficiently produces solar-driven biofuels and chemicals. The leaf-inspired platform enhances light utilization and product formation by providing controllable transport of moisture, gases, nutrients, products, and substrates to living cells in high-density thin layers. These next generation photosynthetic cell factories have significantly improved production efficiency of target chemicals compared with the current suspension systems. In addition, the solid-state systems are much less water intensive in contrast to current suspension cultures.
Targeting sustainable production of a wide spectrum of chemicals, FuturoLEAF used three industrially relevant model processes to demonstrate production of high-value products such as pharmaceutically active ingredients (APIs) and natural cosmetics, specialty chemicals such as ε-caprolactone and commodity chemicals such as hydrogen and ethylene. To this end, the project optimized multiple photosynthetic and heterotophic strains for the efficient production of volatile and soluble compounds, as well as constructed several high-performance solid-state matrix architectures tailored for different production conditions. Finally, an upscaled proof-of-concept SSPCF architecture was established for long-term ethylene production, meeting or exceeding all project key performance indicators.
FuturoLEAF contributes significantly to society by developing the biotechnology field with direct impact to existing biotechnological companies utilizing photosynthetic cells. Additionally, it is envisioned that FuturoLEAF can enable the birth of new biotechnological companies, where solid-state cell factories will be utilized with one or multiple cell types, creating new business openings in the EU. The novel technology created within FuturoLEAF will also give EU a marketing advantage over other market areas related to bioproduction. Moreover, FuturoLEAF will contribute to environmental safety and the development of green technologies. Implementation of the knowledge created in FuturoLEAF for green technology will lead to sustainable use of resources by conversion of CO2 and light into chemicals.
Targeting sustainable production of a wide spectrum of chemicals, FuturoLEAF used three industrially relevant model processes to demonstrate production of high-value products such as pharmaceutically active ingredients (APIs) and natural cosmetics, specialty chemicals such as ε-caprolactone and commodity chemicals such as hydrogen and ethylene. To this end, the project optimized multiple photosynthetic and heterotophic strains for the efficient production of volatile and soluble compounds, as well as constructed several high-performance solid-state matrix architectures tailored for different production conditions. Finally, an upscaled proof-of-concept SSPCF architecture was established for long-term ethylene production, meeting or exceeding all project key performance indicators.
FuturoLEAF contributes significantly to society by developing the biotechnology field with direct impact to existing biotechnological companies utilizing photosynthetic cells. Additionally, it is envisioned that FuturoLEAF can enable the birth of new biotechnological companies, where solid-state cell factories will be utilized with one or multiple cell types, creating new business openings in the EU. The novel technology created within FuturoLEAF will also give EU a marketing advantage over other market areas related to bioproduction. Moreover, FuturoLEAF will contribute to environmental safety and the development of green technologies. Implementation of the knowledge created in FuturoLEAF for green technology will lead to sustainable use of resources by conversion of CO2 and light into chemicals.
A palette of nanocellulose- and nanochitin -based material grades and cross-linkers were developed as wet-stable and biocompatible matrix building blocks. Thermoresponsive MIP nanogels for specific recognition and capturing of targeted photosynthetic cells were successfully synthesized and shown to bind specifically to targeted cell surface epitopes, and cell capturing onto CNF surfaces using MIPs was successfully demonstrated, showing promise for active immobilization of cells via directed self-assembly.
Natural and recombinant photoautotrophic and heterotrophic production strains were selected and optimized to efficiently produce chemicals in relevant reference systems, i.e. in suspension and immobilized in alginate. The production systems encompass a “substrate-in” approach for APIs, “substrate-in-product-out” approach for production of ε-caprolactone via whole-cell biotransformation, and “product-out” approaches for both soluble natural cosmetics and sucrose, and volatile hydrogen and ethylene. Heterotrophic microorganisms were also engineered for potential co-culturing approaches.
Tailored matrix layers were fabricated from the matrix building blocks with optimized wet strength, light distribution, and porosity for the different production systems. Wet-stable nanocellulosic hydrogels were identified as the most suitable matrices for volatile chemical production, whereas porous and wet-stable nanocellulose and nanochitin cryogels were developed for nonvolatile systems requiring higher mass transfer. Finally, the biocatalytical solid-state photosynthetic cell factory architecture was up-scaled in a photobiofilm-reactor for prolonged chemical production. In the final TRL 3 proof-of-concept, continuous production of volatile chemicals was demonstrated with nearly 300-fold increase in volumetric productivity compared to suspended cells.
The outcomes of the project were published in 14 peer reviewed articles, with additional 20 manuscripts under preparation. The results were disseminated in 54 conference talks and 21 poster presentations. The project also resulted in 1 PhD thesis, with 6 PhD works under way, 4 Master’s theses, and 1 Bachelor’s thesis. Additionally, FuturoLEAF teamed up with scientific journalist and professional screenwriter Nina Pulkkis form Photino Science Communication to produce promotional video material for the project. Market analysis suggested that production of high-value compounds with reproducible conditions and low batch variation is the primary market for the SSPCF production platform. Moreover, few relevant patents were found in the IPR analysis, ensuring freedom-to-operate for the continued development of the FuturoLEAF technologies.
Natural and recombinant photoautotrophic and heterotrophic production strains were selected and optimized to efficiently produce chemicals in relevant reference systems, i.e. in suspension and immobilized in alginate. The production systems encompass a “substrate-in” approach for APIs, “substrate-in-product-out” approach for production of ε-caprolactone via whole-cell biotransformation, and “product-out” approaches for both soluble natural cosmetics and sucrose, and volatile hydrogen and ethylene. Heterotrophic microorganisms were also engineered for potential co-culturing approaches.
Tailored matrix layers were fabricated from the matrix building blocks with optimized wet strength, light distribution, and porosity for the different production systems. Wet-stable nanocellulosic hydrogels were identified as the most suitable matrices for volatile chemical production, whereas porous and wet-stable nanocellulose and nanochitin cryogels were developed for nonvolatile systems requiring higher mass transfer. Finally, the biocatalytical solid-state photosynthetic cell factory architecture was up-scaled in a photobiofilm-reactor for prolonged chemical production. In the final TRL 3 proof-of-concept, continuous production of volatile chemicals was demonstrated with nearly 300-fold increase in volumetric productivity compared to suspended cells.
The outcomes of the project were published in 14 peer reviewed articles, with additional 20 manuscripts under preparation. The results were disseminated in 54 conference talks and 21 poster presentations. The project also resulted in 1 PhD thesis, with 6 PhD works under way, 4 Master’s theses, and 1 Bachelor’s thesis. Additionally, FuturoLEAF teamed up with scientific journalist and professional screenwriter Nina Pulkkis form Photino Science Communication to produce promotional video material for the project. Market analysis suggested that production of high-value compounds with reproducible conditions and low batch variation is the primary market for the SSPCF production platform. Moreover, few relevant patents were found in the IPR analysis, ensuring freedom-to-operate for the continued development of the FuturoLEAF technologies.
We have shown that novel bio-based nanomaterials assemblies based on nanocellulose and other bio-sourced carbohydrates are viable as cell immobilization matrixes ta ilored for different photoproduciton conditions. Moreover, the developed solid-state immobilisation systems display prolonged cell viability and chemicals production, especially with volatile compounds. In the beginning of the FuturoLEAF project, we expected that the engineered multi-layer architecture will convert light energy to the targeted product with maximum efficiency of around 4-6 % under low light conditions. We achieved 4% values of light to H2 conversion already in the first year of the project by developing the films with double-layer structure with truncated photosynthetic antennae strain placed a top of layer with wild-type cells. Moreover, we envisoned a 50-fold increase in volumetric cell density and 200-300-fold increase in volumetric productivity compared to suspended cells, together with an increase in long-term cell functionality as biocatalysts from weeks to months. At the end of the project, we demonstrated a 100-130-fold increase in cell density and 280-fold increase in productivity, as well as cell functionality of over 4 months. The findings in FuturoLEAF can be used to improve the efficiency and reproducibility of biocatalytic chemicals production, which enables taking steps away from dependency of fossil sources and towards sustainable energy and chemicals production, while contributing to the green production and climate change mitigation.