Bioelectrochemical TiN|Tsfdh Catalyst for CO2 Reduction to Hcooh
Authors: Arena Federica , Giuffredi Giorgio , Perego Andrea , Donini Stefano , Parisini Emilio , DiFonzo Fabio 
Published: February 2020

The ever-increasing worldwide energy demand and anthropogenic emissions produced by the combustion of fossil fuels, along with the consequent increment in the global average temperature, have pushed the scientific community towards the development of energy vectors with minimal carbon footprint.
Electrochemical -reduction is an approach that holds potential for exploiting the cyclic reduction and oxidation of carbon-based fuels. As of today, high efficiency in these systems can only be achieved by employing precious metals, which suffer from both low natural availability and high costs as well as unsatisfactory energy efficiencies and lack of conversion product specificity.
Recently, the development of a novel hybrid technology based on the combined use of biological organisms or molecules and nanomaterials has created great opportunities to produce renewable fuels and chemicals from the reduction of with a minimal overpotential. According to this, the enzymatic electrosynthesis (EES) exploits pure enzymes to catalyze reactions with higher transformation efficiency, higher activity under controlled experimental conditions and higher selectivity towards both specific substrates and products.
The first step of CO2 reduction enzymatic process consists of the production of formic acid using the catalytic properties of the enzyme formate dehydrogenase (FDH) (EC 1.2.1.2.). Unlike most metallic catalysts, this enzyme reversibly catalyzes the transformation of CO2 to formate as the only product of the reaction. In this contest, we used the NAD-dependent FDH from the aerobic bacterium Thiobacillus sp. KNK65MA (TsFDH) for its superior -reducing activity (Kcat= 0.318 s-1)[1].
Surface chemistry and surface morphology play a key role in the interaction between the enzyme and the electrode, ultimately affecting the successful development of a bioelectrochemical system (BES). In fact, the atomic composition of the material and its structure affect enzyme immobilization, which in turn influences the electrochemical performance of the EES. In this context, titanium nitride (TiN) can be efficiently used as a scaffold for enzyme immobilization by exploiting its surface-exposed Ti4+ atoms for the binding. As such, TiN has been described as a promising general catalyst support material. Indeed, TiN features high electrical conductivity, good biocompatibility, as well as outstanding oxidation and acid corrosion resistance, together with a hybrid metallic/ceramic behaviour[2].
The aim of this work is to realize a nanostructured TiN scaffold featuring an increased available surface area to enhance the interface between the enzyme and the electrode. To this end, we developed a novel hybrid device where the FDH enzyme from Thiobacillus sp. KNK65MA (EC 1.2.1.2) is deposited on a nanostructured mesoporous support of TiN fabricated by Pulsed Laser Deposition (Fig.1a). This deposition method allows the production of a biocompatible nanostructured support with high surface area and a tree-like morphology. Its high porosity and high specific area maximize the contact with the enzyme, thus improving the efficiency of the EES for reduction.
In order to identify the most suitable film morphology, the immobilization process on TiN support with different porosity was assessed by enzymatic assay after drop-casting (active area of 1 cm2). The results show that there is an increase in the amount of TsFDH that is bound to the TiN nanostructure when the porosity, and therefore the surface area, of the nanostructure increases. For the more porous morphology the percentage of adsorbed TsFDH is 48%, corresponding to 1 µg of specifically-adsorbed TsFDH.
After, a calibration curve was performed to determine the maximum binding capacity of the TiN support. The result shows that, by increasing the concentration of the drop-casted TsFDH, the enzyme unit calculated on the surface increases until a TsFDH loading quantity of 1,14 mg is used, corresponding to 59 µg of immobilized protein.
To assess the CO2 reduction electrochemical performance of the TiN|TsFDH electrode, chronoamperometric measurements were done at a constant potential of -0.45VRHE. The TiN support alone shows a stable reduction current (-130 µA ), due to hydrogen evolution (HER) occurring on the surface of the electrode (blue line, Figure 1b). Then, we tested the CO2 reduction activity of the enzyme-conjugated electrode (green trace, Figure 1b). A reductive current density with higher magnitude of -190 µA was registered, showing the actual operation of the BES system.
After test, the solution was examined by 1H NMR spectroscopy. The peak signal at 8.455 ppm in NMR spectra demonstrates that the formic acid is the only product of the catalytic reaction performed by TsFDH. This result was used to quantify the amount of formic acid synthetized in the BES. During a 3 h reaction period, the electrosynthesis of formic acid was about 5.3±0.4 μmol under potential applied of -0.45VRHE.
[1]H.Choe;PLOS ONE(2014).
[2]P.H.;Electrochimica Acta(2010)

Figure 1