John Villadsen

Oxidative phosphorylation revisited

The fundamentals of oxidative phosphorylation and photophosphorylation are revisited. New experimental data on the involvement of succinate and malate anions respectively in oxidative phosphorylation and photophosphorylation are presented. These new data offer a novel molecular mechanistic explanation for the energy coupling and ATP synthesis carried out in mitochondria and chloroplast thylakoids. The mechanism does not suffer from the flaws in Mitchells chemiosmotic theory that have been pointed out in many studies since its first appearance 50 years ago, when it was hailed as a ground‐breaking mechanistic explanation of what is perhaps the most important process in cellular energetics. The new findings fit very well with the predictions of Naths torsional mechanism of energy transduction and ATP synthesis. It is argued that this mechanism, based on at least 15 years of experimental and theoretical work by Sunil Nath, constitutes a fundamentally different theory of the energy conversion process that eliminates all the inconsistencies in Mitchells chemiosmotic theory pointed out by other authors. It is concluded that the energy‐transducing complexes in oxidative phosphorylation and photosynthesis are proton‐dicarboxylic acid anion cotransporters and not simply electrogenic proton translocators. These results necessitate revision of previous theories of biological energy transduction, coupling, and ATP synthesis. The novel molecular mechanism is extended to cover ATP synthesis in prokaryotes, in particular to alkaliphilic and haloalkaliphilic bacteria, essentially making it a complete theory addressing mechanistic, kinetic, and thermodynamic details. Finally, based on the new interpretation of oxidative phosphorylation, quantitative values for the P/O ratio, the amount of ATP generated per redox package of the reduced substrates, are calculated and compared with experimental values for fermentation on different substrates. It is our hope that the presentation of oxidative phosphorylation and photophosphorylation from a wholly new perspective will rekindle scientific discussion of a key process in bioenergetics and catalyze new avenues of research in a truly interdisciplinary field. Biotechnol. Bioeng. 2015;112: 429–437.

Mixing and mass transfer in a pilot scale U‐loop bioreactor

A system capable of handling a large volumetric gas fraction while providing a high gas to liquid mass transfer is a necessity if the metanotrophic bacterium Methylococcus capsulatus is to be used in single cell protein (SCP) production. In this study, mixing time and mass transfer coefficients were determined in a 0.15 m3 forced flow U‐loop fermenter of a novel construction. The effect on the impeller drawn power when a gas was introduced into the system was also studied. Mixing time decreased and mass transfer increased with increasing volumetric liquid flow rate and specific power input. This happened also for a large volume fraction of the gas, which was shown to have only minor effect on the power drawn from the pump impeller. Very large mass transfer coefficients, considerably higher than those obtainable in an STR and previous tubular loop reactors, could be achieved in the U‐loop fermenter equipped with static mixers at modest volumetric liquid and gas flow rates. Biotechnol. Bioeng. 2017;114: 344–354.

Use of high‐gradient magnetic fishing for reducing proteolysis during fermentation

Proteolysis during fermentation may have a severe impact on the yield and quality of a secreted product. In the current study, we demonstrate the use of high-gradient magnetic fishing (HGMF) as an efficient alternative to the more conventional methods of preventing proteolytic degradation. Bacitracin-linked magnetic affinity adsorbents were employed directly in a fermenter during Bacillus licheniformis cultivation to remove trace amounts of unwanted proteases. The constructed magnetic adsorbents had excellent, highly specific binding characteristics in the fermentation broth (K(d) = 1.94 micromolar; Q(max) = 222.8 mg/g), which obeyed the Langmuir isotherm and had rapid binding kinetics (equilibrium in <300 s). When applied directly in shake-flask cultures or in a 1-L fermenter and then removed by HGMF, the degradation of the model protein bovine serum albumin was stopped. The adsorbents could be recycled and reused during the same fermentation to remove freshly produced proteases, extending the life of the model protein in the fermenter. HGMF may provide an efficient method of stabilizing heterologous proteins produced in cultivation processes.

Reflections on the aerobic fermentation stoichiometry of crabtree positive yeasts

In this communication a stoichiometric steady state model for Crabtree positive yeasts is proposed. The model is sufficiently simple to be corroborated by experimental data on the key metabolic events around Dcrit. The key feature of the model is that the bottleneck aperture for biomass production in the model of Sonnleitner and Käppeli, 1986 shrinks abruptly at Dcrit and continues to decrease with increasing dilution rate. A black box stoichiometric analysis of experiments reported in literature indicates that production of acetaldehyde might account for the abrupt shrinkage through a severe poisoning effect on the respiratory system.

System wide cofactor turnovers can propagate metabolic stability between pathways

Metabolic homeostasis, or low-level metabolic steady state, has long been taken for granted in metabolic engineering, and research priority has always been given to understand metabolic flux control and regulation of the reaction network. In the past, this has not caused concerns because the metabolic networks studied were invariably associated with living cells. Nowadays, there are needs to reconstruct metabolic networks, and so metabolic homeostasis cannot be taken for granted. For metabolic steady state, enzyme feedback control has been known to explain why metabolites in metabolic pathways can avoid accumulation. However, we reasoned that there are further contributing mechanisms. As a new methodology developed, we separated cofactor intermediates (CIs) from non-cofactor intermediates, and identified an appropriate type of open systems for operating putative reaction topologies. Furthermore, we elaborated the criteria to tell if a multi-enzyme over-all reaction path is of in vivo nature or not at the metabolic level. As new findings, we discovered that there are interactions between the enzyme feedback inhibition and the CI turnover, and such interactions may well lead to metabolic homeostasis, an emergent property of the system. To conclude, this work offers a new perspective for understanding the role of CIs and the presence of metabolic homeostasis in the living cell. In perspective, this work might provide clues for constructing non-natural metabolic networks using multi-enzyme reactions or by degenerating metabolic reaction networks from the living cell.