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A novel approach of phenotype analysis of fermentation-based bioprocesses based on unsupervised learning (clustering) is presented. As a prior identification of phenotypes and conditional interrelations is desired to control fermentation performance, an automated learning method to output reference phenotypes (defined as vector of biomass-specific rates) was developed and the necessary computing process and parameters were assessed. For its demonstration, time series data of 90 Clostridium pasteurianum cultivations were used which feature a broad spectrum of solventogenic and acidogenic phenotypes, while 14 clusters of phenotypic manifestations were identified. The analysis of reference phenotypes showed distinct differences, where potential conditionalities were exemplary isolated. Further, cluster-based balancing of carbon and ATP or the use of reference phenotypes as indicator for bioprocess monitoring were demonstrated to highlight the perks of this approach. Overall, such analysis depends strongly on the quality of the data and experimental validations will be required before conclusions. However, the automated, streamlined and abstracted approach diminishes the need of individual evaluation of all noisy dataset and showed promising results, which could be transferred to strains with comparably wide-ranging phenotypic manifestations or as indicators for repeated bioprocesses with clearly defined target., Competing Interests: The authors have declared no conflict of interests., (© 2021 The Authors. Engineering in Life Sciences published by Wiley‐VCH GmbH.)

In this contribution, we studied the effect of electro-fermentation on the butanol production of Clostridium pasteurianum strains by a targeted metabolomics approach. Two strains were examined: an electrocompetent wild type strain (R525) and a mutant strain (dhaB mutant) lacking formation of 1,3-propanediol (PDO). The dhaB-negative strain was able to grow on glycerol without formation of PDO, but displayed a high initial intracellular NADH/NAD ratio which was lowered subsequently by upregulation of the butanol production pathway. Both strains showed a 3-5 fold increase of the intracellular NADH/NAD ratio when exposed to cathodic current in a bioelectrochemical system (BES). This drove an activation of the butanol pathway and resulted in a higher molar butanol to PDO ratio for the R525 strain. Nonetheless, macroscopic electron balances suggest that no significant amount of electrons derived from the BES was harvested by the cells. Overall, this work points out that electro-fermentation can be used to trigger metabolic pathways and improve product formation, even when the used microbe cannot be considered electroactive. Accordingly, further studies are required to unveil the underlying (regulatory) mechanisms., Competing Interests: The authors have declared no conflicts of interest., (© 2020 The Authors. Engineering in Life Sciences published by Wiley‐VCH GmbH.)

Clostridium pasteurianum produces industrially valuable chemicals such as n -butanol and 1,3-propanediol from fermentations of glycerol and glucose. Metabolic engineering for increased yields of selective compounds is not well established in this microorganism. In order to study carbon fluxes and to selectively increase butanol yields, we integrated the latest advances in genome editing to obtain an electrocompetent Clostridium pasteurianum strain for further engineering. Deletion of the glycerol dehydratase large subunit ( dhaB ) using an adapted S. pyogenes Type II CRISPR/Cas9 nickase system resulted in a 1,3-propanediol-deficient mutant producing butanol as the main product. Surprisingly, the mutant was able to grow on glycerol as the sole carbon source. In spite of reduced growth, butanol yields were highly increased. Metabolic flux analysis revealed an important role of the newly identified electron bifurcation pathway for crotonyl-CoA to butyryl-CoA conversion in the regulation of redox balance. Compared to the parental strain, the electron bifurcation pathway flux of the dhaB mutant increased from 8 to 46% of the overall flux from crotonyl-CoA to butyryl-CoA and butanol, indicating a new, 1,3-propanediol-independent pattern of glycerol fermentation in Clostridium pasteurianum ., Competing Interests: The authors have declared no conflict of interest., (© 2018 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

An autoclavable All-in-One electrolysis electrode in a rod shape assembly is developed as a new tool for bioelectrochemical systems and electricity-aided bioprocesses. It can replace the classic two-chamber bioelectrochemical system for electrolysis reactions, be inserted into conventional bioreactors and is easily adaptable as electrocatalytic surface or generator of super-fine bubbles (H 2 and O 2 ) for bioconversion processes. Whereas the bioreactor itself functions as the working electrode chamber, a well-integrated inner counter electrode chamber enables water electrolysis without the normally encountered undesired ion-transfer effect. The efficiencies of the electrode are characterized and its advantages and usefulness compared to the classic H-Cell bioelectrochemical system (BES) are demonstrated with glycerol fermentations by Clostridium pasteurianum DSM 525., (© 2018 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)