Andreas Schmid

Whole‐cell photobiocatalytic H ₂ production is constrained by hydrogenase O ₂ sensitivity, photosynthetic electron allocation, and cellular physiology. We present a systematic framework to identify and alleviate these interacting limitations using Synechocystis sp. PCC 6803 combined with Pseudomonas taiwanensis VLB120 for extracellular O ₂ microenvironment control. Rapid O ₂ diffusion over micrometer distances enabled efficient extracellular O ₂ scavenging, lowering local O ₂ exposure of the intracellular native [NiFe] hydrogenase and sustaining light‐driven H ₂ production from water. We systematically evaluated genetic, physiological, and reaction parameters affecting H ₂ production. The ndhB mutant lacking functional NDH‐1 complexes achieved the highest rates, indicating that suppressing competing electron sinks, including NDH‐1‐dependent cyclic electron flow and CO ₂ uptake, increases electron availability for H ₂ evolution. Optimization of pH, nitrogen availability, and cellular physiology enhanced performance. Under optimized conditions, H ₂ production was sustained for >72 h, reaching ~1500 µmol H ₂ g _CDW ⁻¹ , averaging ~0.35 µmol H ₂ g _CDW ⁻¹ min ⁻¹ , at least fivefold above a current Synechocystis benchmark, with estimated ~1% light‐to‐H ₂ efficiency. Inhibition of Photosystem II abolished H ₂ formation, demonstrating that water oxidation, rather than carbon metabolism, supplied the predominant electrons. Nitrogen limitation further reduced H ₂ uptake and stabilized net H ₂ accumulation. This workflow provides a transferable methodology for systematically optimizing complex whole‐cell photobiocatalytic systems.

Single‐cell technologies are emerging as a complementary approach to conventional laboratory‐scale cultivation setups. Particularly in microfluidic cultivation and screening, precisely controlled environmental conditions and defined minimal media become important for quantitative studies. Nevertheless, conventional bulk media optimized for other scales, are often employed despite differences in nutrient requirements across varying scales and cultivation modes. This study demonstrates the limitations of using bulk media for single‐cell analysis of Corynebacterium glutamicum and highlights the need for revised minimal media for microfluidic cultivations under perfusion. The commonly used CGXII medium contains protocatechuic acid (PCA), which functions both as an iron chelator and a carbon source, thereby compromising its role as a minimal medium with one limiting substrate. We systematically evaluated alternative iron chelators to replace PCA using microfluidic single‐cell cultivation. We show that not all chelators are applicable with C. glutamicum at the single‐cell level and identify citrate as a suitable replacement that ensures iron availability without supporting growth in the absence of glucose. Based on these findings, we established a revised minimal medium, termed ‘single‐cell CGXII’. This medium was validated across different scales and enables robust, reproducible investigation of cellular physiology at single‐cell resolution, improving accuracy and comparability in quantitative single‐cell research.

While photocatalysis is considered a promising sustainable technology in the field of heterogeneous catalysis as well as biocatalysis, figures of merit (FOM) for comparing catalytic performance, especially between disciplines, are not well established. Here, photocatalytic water splitting was conducted using a semiconductor (NiO/La-NaTaO₃) and a bio-photocatalyst (Synechocystis sp. PCC 6803) in the same setup under similar reaction conditions, eliminating the often ill-defined influence of the setup on the FOMs obtained. Comparing the results enables the critical evaluation of existing FOMs and a quantitative comparison of both photocatalytic systems. A single FOM is insufficient to compare the photocatalysts, instead a combination of multiple FOMs (reaction rate, photocatalytic space time yield and a redefined apparent quantum yield) is superior for assessing a variety of photocatalytic systems.

Novel cultivation technologies demand the adaptation of existing analytical concepts. Metabolic flux analysis (MFA) requires stable-isotope labeling of biomass-bound protein as the primary information source. Obtaining the required protein in cultivation set-ups where biomass is inaccessible due to low cell densities and cell immobilization is difficult to date. We developed a non-disruptive analytical concept for ¹³C-based metabolic flux analysis based on secreted protein as an information carrier for isotope mapping in the protein-bound amino acids. This “metabolic flux probe” (MFP) concept was investigated in different cultivation set-ups with a recombinant, protein-secreting yeast strain. The obtained results grant insight into intracellular protein turnover dynamics. Experiments under metabolic but isotopically nonstationary conditions in continuous glucose-limited chemostats at high dilution rates demonstrated faster incorporation of isotope information from labeled glucose into the recombinant reporter protein than in biomass-bound protein. Our results suggest that the reporter protein was polymerized from intracellular amino acid pools with higher turnover rates than biomass-bound protein. The latter aspect might be vital for ¹³C-flux analyses under isotopically nonstationary conditions for analyzing fast metabolic dynamics.