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Item type:Publication, Access status: with files , Fluxomic and metabolomic studies on the electro-fermentation of Rhodosporidium toruloides and Clostridium pasteurianum for improved bioprocesses(2022); Electrobiotechnology is a promising platform technology. The technology is expected to play a vital role in transforming the current oil-based economic system towards a sustainable and circular bioeconomy. Accordingly, this thesis deals with the influence of electro-fermentation on two bioprocesses: the microbial production of lipids by the yeast Rhodosporidium toruloides and the production of 1,3-propanediol (PDO) and n-butanol by the anaerobic bacterium Clostridium pasteurianum. In this context, metabolomics and methods from the field of systems biology are applied. This allows a reliable and quantitative description of the electricity's influence on microbial metabolism. In general, previous work has revealed that electro-fermentation has the most positive influence on a bioprocess when microorganisms can harvest electrons from a power source and make them physiologically accessible. However, other experimental studies suggest that electro-fermentation can also positively influence process performance, even if the microorganisms cannot directly take up artificially supplied electrons. In this case, the electricity is used to manipulate process parameters, in particular the oxidation-reduction redox potential (ORP). In this way, electro-fermentation can also contribute indirectly to improve the performance of bioprocesses. Hence, this work mainly focuses on evaluating the suitability of electricity for altering and controlling the fermentation broth’s ORP and the resulting effects on the microorganisms and the bioprocess. For lipid production with R. toruloides, it could be shown by elementary mode analysis that the application of electricity can theoretically increase lipid yields by a maximum of 29%, depending on the carbon source. However, this is only the case if the strain can directly harvest electrons from the electrode and simultaneously also transports protons into the cytosol. Batch cultivations showed that the ORP can, even at strictly aerobic conditions (pO2 = 50%), be reduced electrochemically by up to -600 mV. This coincided with an increase in observed lipid yields. Another interesting experimental observation concerns the degree of saturation of the lipids produced: the use of electricity with the simultaneous addition of the redox mediator neutral red led to an increase in the proportion of saturated fatty acids to more than 50%. The experimental studies with C. pasteurianum showed that the strain is not electroactive, e.g. it cannot harvest electrode-derived electrons in measurable quantities directly. Nevertheless, in fed-batch fermentations, the strain reacted to the electrochemical alteration of the ORP and produced proportionally more n-butanol than PDO. A more than four-fold increase in the intracellular NADH/NAD ratio in cathodic bioelectrical systems was observed, which resulted in the activation of reductive metabolic pathways. In addition, continuous fermentations were carried out, in which the ORP was controlled electrochemically to desired set-points at fixed dilution rate. Here, the molar product yield for PDO could be maximally improved by 57% by increasing the ORP at a dilution rate of 0.1 h-1. At the same time, however, the ORP control also led to the electrochemical production of oxygen in the bioreactor, which significantly inhibited the conversion of pyruvate to acetyl-CoA. This led to a substantial decrease in substrate uptake and biomass concentration, which resulted in a lower space-time yield for n-butanol and PDO. Furthermore, the metabolic flux analysis results indicate that C. pasteurianum possesses and uses other cellular energy generation mechanisms in addition to substrate-chain phosphorylation. The data suggest that this newly discovered mechanism for C. pasteurianum might be driven by the creation of a proton gradient with the help of intracellular hydrogenases.Publicationtype: Doctoral Thesis239 1275 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Evaluation of deep eutectic solvents as alternative reaction media in biocatalysisEnzyme catalysis is often associated with aqueous reaction media, but water is rather an inadequate solvent for hydrophobic substrates. In this study ‘deep eutectic solvents’ (DESs) were evaluated as novel reaction media for biocataylsis. For instance, a DES composed of both substrates, i.e. a DES made of (–)-Menthol:lauric acid, was used as combined solvent and substrate for the lipase catalysed synthesis of (–)-menthyl laurate, so that no additional solvent was necessary. The optimisation of the reaction resulted in a batch productivity of 443 g L−1 d−1 at 95 % conversion (24 h). Excess (–)-menthol was separated in a vacuum distillation step and was reused for a second enzymatic DES reaction.Publicationtype: Doctoral Thesis328 579