One of the approaches widely used today to intensify processes is miniaturization of process equipment. Small, compact and portable devices used at the source of the raw material or at the point of application of the product have numerous applications in the chemical, pharmaceutical and food industries, biofuel production, medicine, sensor technology, environmental monitoring, etc. The wide application of microstructured devices in power generation depends on the ability to produce devices that are sustainable, durable, economically justified, and environmentally sound. Nowadays, biofuels are economically and environmentally acceptable alternatives to fossil fuels. Hydrogen is the most promising alternative energy solution because it has a high energy potential, no harmful gasses are released during its use, and the specific energy obtained is 2.75 times greater than the energy obtained from hydrocarbon-based fuels.

Compared to chemical processes of hydrogen production, biological processes are characterized by lower specific energy consumption, the process takes place under mild reaction conditions, and natural components are used as substrates.

In the proposed research, the production of biohydrogen will be carried out in a reaction system based on the oxidation of glucose by the glucose dehydrogenase, which requires the presence of the coenzyme NADP⁺ for its catalytic action (Figure 1.). Two moles of biohydrogen per mole of glucose are generated during coenzyme regeneration by hydrogenase. Hydrogenase will be produced using Ralstonia eutropha H16 and by expression of recombinant protein in E. coli. Biohydrogen production will be carried out in a batch and repetative batch reactor and in a microreactor. The medium composition and process conditions will be optimized, and natural deep eutectic solvents will be used in addition to aqueous media to stabilize enzymes and coenzymes. Finally, a kinetic and mathematical model will be developed to optimize and scale-up the process.

Figure 1. Glucose oxidation reaction catalysed by the glucose dehydrogenase (GDH) with parallel biohydrogen production catalysed by hydrogenase.

The research planned under the project aims to develop an integrated microsystem for biohydrogen production. Biohydrogen production is based on the glucose oxidation reaction catalyzed by the enzyme glucose dehydrogenase, the use of which requires the use of the coenzyme NADP^+, producing gluconic acid and NADPH as products. To justify the process economically, the coenzyme must be regenerated, which is done using the oxygen tolerant enzyme hydrogenase, with the regenerated coenzyme NADP⁺ producing two moles of hydrogen for each mole of glucose consumed. Since the oxygen tolerant hydrogenase enzyme is not commercially available, it must be produced, which is one of the main goals of the project. Therefore, the research planned under the project to develop an integrated process of biohydrogen production in a microreactor is divided into the following interconnected units:

1. production of oxygen tolerant hydrogenase 

2. measurement of enzyme activity 

3. production of natural deep eutectic solvents 

4. oxidation of glucose and production of biohydrogen in batch and repeatable batch reactors 

5. oxidation of glucose and production of biohydrogen in microreactors 

6. technological and economic analysis.