Use of Ionic Liquids and Support Materials for High Performance Enzymatic Conversion of CO₂ into Formic Acid and Formaldehyde

Enzymatic conversion of CO₂ to fuels and valuable chemicals is a promising way to reduce increasing CO₂ emissions. The research in this thesis was mainly divided into three sequential parts: First, the single enzymatic reactions, CO₂ → formic acid, was studied in detail. Later on, two additional multi-enzymatic reactions were added to the latter (CO₂ → formic acid → formaldehyde → methanol). Lastly, a cofactor (NADH) regeneration system was added to the main sequential reaction, so external cofactor addition would not be required.
There are several limitations that render the inefficiently enzymatic conversion of CO₂ nowadays. One of them is the low solubility of CO₂ in water or buffer. To address this limitation, ionic liquids (ILs), with a higher solubility than water, were explored as alternative co-solvents for increasing CO₂ capture. In studying on this reaction, we found that degradation of NADH, which occurs during the enzymatic reaction, causes overestimation of the CO₂ conversion results when using the conventional analytical method (quantification of NADH reduction – so-called method N). Therefore we proposed and established a new detection method (so-called method C), further investigated the degradation mechanism of NADH in order to select different kinds of ILs that are suitable for our enzymatic system. As a result, by stabilizing NADH with ILs and optimizing the variables, the yield of formic acid in BmimBF4 was increased two-fold compared to the yield of formic acid in phosphate buffer.
To further increase conversion of CO₂, two more sequential reactions were added to the main one, so the product from one reaction was consumed in the subsequent one and the equilibrium could be switched towards higher conversion. To that purpose, formaldehyde hydrogenase (FaldDH), and alcohol dehydrogenase (ADH), which catalyzed the conversion of formic acid to formaldehyde and methanol, respectively, were added. It was indeed observed that particularly the addition of ADH (with very high activity) contributed significantly to the overall conversion of CO₂. To conduct such a sequential reaction though, several ionic liquids compatible with enzymes were evaluated, a high concentration of CO₂ could be kept in the system. After evaluating the activity of the three enzymes in different ILs, IL [CH][Glu] presented by far the best performance. By using such IL system, CO₂ concentration was detected to be 15 times higher compared with the amount in water. Subsequently, in order to make the platform for the multi-enzymatic reaction even more efficient, a membrane reactor design enabling enzyme immobilization, which should increase enzyme stability, was developed. For that purpose, the so-called “fouling induced immobilization method” was used, which enable to maintain the activity of the immobilization enzymes at approximately that of the free enzymes, due to mild and fast immobilization procedure.
Additionally, a high enzyme loading could also be attained, and the contact time for the substrate-enzyme complex could be controlled by changing pressure. Finally, the yield of product (methanol) in [CH][Glu] was increased three-fold compared to the yield in conventional buffer.
When talking about the economic viability of enzymatically converting CO₂ at large scale, the cost of the cofactor (NADH) is the biggest bottlenecks because three molar equivalents of NADH are consumed to transform one molar equivalent of CO₂ to methanol. NADH regeneration through a photocatalytic method was envisaged as a promising way to decrease the cost because limitless solar energy is available for utilizaiton. Inspired by the natural light-harvesting pigments, the porphyrins, porphyrin-based ionic liquid photosensitizers were synthesized and evaluated for NADH regeneration. After evaluating several photosensitizers, ZnTPyPBr showed the best performance in NADH regenerations. After the regeneration system was developed, a photocatalytic membrane was developed, so the multi-enzymatic reaction could be coupled with in-situ NADH regeneration. The selected photosensitizer, enzymes, and electron mediator were immobilized in the surface of the membrane and the multi-enzymatic reaction was performed in the photocatalytic membrane under visible light. The artificial photocatalytic system was successfully developed and investigated. Cost of NADH could be therefore significantly decreased and the overall system showed promise as a preliminary platform for efficient and inexpensive CO₂ conversion.