Elucidating Electron Transfer Kinetics and Optimizing System Performance for Escherichia coli-Based Semi-Artificial H₂ Production

Abstract

Both photo-and biocatalysis are well-established and intensively studied. The combination of these two approaches is also an emerging research field, commonly referred to as semi-artificial photosynthesis. Semi-artificial photosynthesis aims at combining highly efficient synthetic light harvesters with the self-healing and potent catalytic properties of biocatalysis. In this study, a semi-artificial photocatalytic system featuring Escherichia coli bacteria, which heterologously express the [FeFe] hydrogenase enzyme HydA1 from green algae, is employed as a hydrogen gas production catalyst. To probe the influence of photochemistry on overall system performance, the E. coli whole-cell catalyst is combined with two different photosensitizers and redox mediators. The addition of a redox mediator greatly improves the rates and longevity of the photocatalytic system, as reflected in increases of both the turn-over number (0.777 vs 10.9 μmol H 2 mL −1 OD 600 −1) and the turn-over frequency (175 vs 334 μmol H 2 mL −1 h −1 OD 600 −1). The redox mediator is found to both protect from photobleaching and enable electron transport to the hydrogenase from an extracellular photosensitizer. However, E. coli cells are strongly affected by the photocatalytic system, leading to a decrease in cell integrity and cell viability, possibly due to toxic decomposition products formed during the photocatalytic process. We furthermore employed steady-state and transient absorption spectroscopy to investigate solution potentials and the kinetics of electron transfer processes between the sacrificial electron donor, photosensitizer, redox mediator, and the [FeFe] hydrogenase as the final electron acceptor. These results allowed us to rationalize the different activities observed in photocatalytic assays and offer a better understanding of the factors that influence the photocatalytic performance of E. coli-based whole-cell systems.

E. coli-based semi-artificial photosynthesis: biocompatibility of redox mediators and electron donors in [FeFe] hydrogenase driven hydrogen evolution

“…In an in vivo context, a wide range of combinations of hydrogenase-containing microorganisms and photosensitizers (PSs) have been reported for light-driven hydrogen gas production. 13–20…”

“…Considering molecular photosensitizers, we and others have shown that Eosin Y (EY) can enter Escherichia coli and serve as an intracellular PS to drive heterologously expressed [FeFe] hydrogenase in the presence of triethanolamine (TEOA) as a sacrificial electron donor (SED). 19–22 The addition of a redox mediator (RM) such as methyl viologen (MV) improves the overall efficiency of the photochemistry, and also facilitates transmembrane electron transfer and thus enables the use of extracellular PSs such as ruthenium tris-bipyridine ([Ru(bpy) 3 ] 2+ ). 19 However, the active photosystem, or components thereof, exhibits cytotoxicity, hence decreasing E. coli's viability or ability to reproduce.…”

“…19–22 The addition of a redox mediator (RM) such as methyl viologen (MV) improves the overall efficiency of the photochemistry, and also facilitates transmembrane electron transfer and thus enables the use of extracellular PSs such as ruthenium tris-bipyridine ([Ru(bpy) 3 ] 2+ ). 19 However, the active photosystem, or components thereof, exhibits cytotoxicity, hence decreasing E. coli's viability or ability to reproduce. Our previous results suggested that both MV and by-products of the hydrogen evolution could lead to this observed cytotoxicity.…”

“…Our previous results suggested that both MV and by-products of the hydrogen evolution could lead to this observed cytotoxicity. 19 As a case in point, oxidation of the common SED TEOA can lead to the formation of aldehydes such as glycoaldehyde. 23–26 Due to the high reactivity of such aldehydes, TEOA is potentially toxic for E. coli .…”

“…Indeed, our previous studies showed that cell growth was entirely inhibited already within hours for samples that were photocatalytically producing hydrogen with TEOA as the SED. 19 The issue of toxicity is not restricted to molecular PSs or specific SEDs, and cell-toxicity has been observed also with e.g. CdS nanoparticles which are commonly employed in semi-artificial photosynthesis assemblies.…”