Periodic Reporting for period 1 - NIBIOX (Heterogeneous biocatalysts for oxygen-independent oxidations using inorganic salts)
Berichtszeitraum: 2023-12-01 bis 2025-05-31
NIBIOX aims at surpassing the productivity limits derived from the reliance of current industrial oxidative processes on molecular oxygen to produce aldehydes and acids, massively used as intermediates in the manufacture of cosmetics, agrochemicals and pharmaceuticals. Current industrial oxidative reactions are dependent on molecular oxygen and thus limited by oxygen solubility. To overcome this limitation, existing technologies increase the oxygen solubility in reaction media by decreasing the polarity of the media, bubbling air into the reaction milieu or increasing agitation rate, which damage the catalysts, thus reducing their operational stability. NIBIOX proposes a disruptive approach to surpass productivity limits: we will use our proprietary self-sufficient and facultative anaerobic heterogeneous biocatalysts to oxidize alcohols into high-value fine chemicals without requiring any oxygen supply
The research activities have been mostly focused on the development of the three main objectives of the NIBIOX project:
Objective 1. Technological development and fabrication of self-sufficient and facultative anaerobic heterogeneous biocatalysts. This family of biocatalysts is considered what we call the minimum value product (MVP) NIBIOX will deliver. Different Nitrate reductases were selected, and their activities for nitrate oxidation in the presence of either NADPH or NADH were tested. As a result, we selected the most versatile one to oxidize nitrate with both NADH and NADPH. This enzyme was then co-immobilized with an alcohol dehydrogenase and NADH cofactor on different carriers, and its functional properties (activity and stability) were characterized. The carrier that retained the highest activity and stability of both enzymes upon immobilization was selected for process intensification in objective 2.
Objective 2. Validation of optimal self-sufficient heterogeneous biocatalyst (MVP) as an enabling technology to oxidize alcohols in the absence of oxygen. The MVP was tested for the oxidation of benzyl alcohol as a model oxidation, both in batch and in flow under oxygen-limited conditions. Using nitrate as oxidant and the MVP herein developed, we achieved similar volumetric productivity and product yield as using the conventional biooxidation approach using NADH oxidase and molecular oxygen as ultimate electron donor, but higher
Objective 3. Market potential and sustainability assessment of MVP through estimation of fabrication costs and sustainable metrics of a model alcohol oxidation performed with the MVP. Our assessment has provided guidelines to improve the sustainability of PoC technology based on the MVP developed. Higher substrate loadings and conversions at lower reaction times must be achieved to enable proper resource use, with better economic and environmental outcomes. Moreover, solvent optimization at the extractive downstream should focus on the solvent-to-reactor-volume ratio, on enabling solvent recycling, and on promoting biogenic solvents, to ensure higher carbon neutrality in the inevitable incineration of the (largely recycled) spent organic solvents. Regarding the IP and market analysis, we concluded that the NIBIOX concept is highly innovative and holds significant promise for various applications. Given its current proof-of-concept stage, besides IP protection, further development through follow-up projects is recommended – both from academic funding to industrial-driven cooperations –, to explore the full potential and practical implementation.
Objective 1. Technological development and fabrication of self-sufficient and facultative anaerobic heterogeneous biocatalysts. This family of biocatalysts is considered what we call the minimum value product (MVP) NIBIOX will deliver. Different Nitrate reductases were selected, and their activities for nitrate oxidation in the presence of either NADPH or NADH were tested. As a result, we selected the most versatile one to oxidize nitrate with both NADH and NADPH. This enzyme was then co-immobilized with an alcohol dehydrogenase and NADH cofactor on different carriers, and its functional properties (activity and stability) were characterized. The carrier that retained the highest activity and stability of both enzymes upon immobilization was selected for process intensification in objective 2.
Objective 2. Validation of optimal self-sufficient heterogeneous biocatalyst (MVP) as an enabling technology to oxidize alcohols in the absence of oxygen. The MVP was tested for the oxidation of benzyl alcohol as a model oxidation, both in batch and in flow under oxygen-limited conditions. Using nitrate as oxidant and the MVP herein developed, we achieved similar volumetric productivity and product yield as using the conventional biooxidation approach using NADH oxidase and molecular oxygen as ultimate electron donor, but higher
Objective 3. Market potential and sustainability assessment of MVP through estimation of fabrication costs and sustainable metrics of a model alcohol oxidation performed with the MVP. Our assessment has provided guidelines to improve the sustainability of PoC technology based on the MVP developed. Higher substrate loadings and conversions at lower reaction times must be achieved to enable proper resource use, with better economic and environmental outcomes. Moreover, solvent optimization at the extractive downstream should focus on the solvent-to-reactor-volume ratio, on enabling solvent recycling, and on promoting biogenic solvents, to ensure higher carbon neutrality in the inevitable incineration of the (largely recycled) spent organic solvents. Regarding the IP and market analysis, we concluded that the NIBIOX concept is highly innovative and holds significant promise for various applications. Given its current proof-of-concept stage, besides IP protection, further development through follow-up projects is recommended – both from academic funding to industrial-driven cooperations –, to explore the full potential and practical implementation.
The NIBIOX project has achieved significant advancements in the field of oxidative biocatalysts because the outcome technology has allowed the operation of enzymatic oxidations in the absence of oxygen using an inorganic electron acceptor. To the best of our knowledge, this is the first example of an oxygen-independent biotransformation catalyzed by enzyme through a continuous process. This technology eliminates productivity limitations derived from oxygen diffusion, avoids the accumulation of hydrogen peroxide and competes in terms of productivity and product yields with oxygen-independent biotransformations. The further uptake and success of this technology relies on further research to scale-up the manufacturing process of MVP through a cost-effective analysis of the starting materials. One the major bottlenecks for the manufacturing scaleup is the supply of the nitrate reductase. This supply is very limited and discontinuous from time to time. So to further advance with the technology, we need to either set a pipeline to produce and purify nitrate reductase in-house or find a partner to overexpress this enzyme at a large scale and cooperate with us to manufacture 0.1 Kg of heterogeneous biocatalyst. This amount will be needed to test a 5 L scale biotransformation. With this data in hand, we will be in place for the demonstration of our technology. Without this demonstration, we will not be able to access markets and finance the further development of the MVP.