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Mini-bioreactors with integrated online monitoring capabilities are well established in the early stages of process development. Mini-bioreactors fulfil the demand for highthroughput-applications and a simultaneous reduction of material costs and total experimental time. One of the most essential online monitored parameters is the oxygen transfer rate (OTR). OTR-monitoring allows fast characterization of bioprocesses and process transfer to larger scales. Currently, OTR-monitoring on a smallscale is limited to shake flasks and 48-well microtiter plates (MTP). Especially, 96-deepwell MTP are used for high-throughput-experiments during early-stage bioprocess development. However, a device for OTR monitoring in 96-deepwell MTP is still not available. To determine OTR values, the measurement of the gas composition in each well of a MTP is necessary. Therefore, a new micro(µ)-scale Transfer rate Online Measurement device (µTOM) was developed. The µTOM includes 96 parallel oxygen-sensitive sensors and a single robust sealing mechanism. Different organisms (Escherichia coli, Hansenula polymorpha, and Ustilago maydis) were cultivated in the µTOM. The measurement precision for 96 parallel cultivations was 0.21 mmol•L −1 •h −1 (pooled standard deviation). In total, a more than 15-fold increase in throughput and an up to a 50-fold decrease in media consumption, compared with the shake flask RAMOS-technology, was achieved using the µTOM for OTR-monitoring.

Syngas fermentation is a potential player for future emission reduction. The first demonstration and commercial plants have been successfully established. However, due to its novelty, development of syngas fermentation processes is still in its infancy, and the need to systematically unravel and understand further phenomena, such as substrate toxicity as well as gas transfer and uptake rates, still persists. This study describes a new online monitoring device based on the respiration activity monitoring system for cultivation of syngas fermenting microorganisms with gaseous substrates. The new device is designed to online monitor the carbon dioxide transfer rate (CO2TR) and the gross gas transfer rate during cultivation. Online measured data are used for the calculation of the carbon monoxide transfer rate (COTR) and hydrogen transfer rate (H2TR). In cultivation on pure CO and CO + H2, CO was continuously limiting, whereas hydrogen, when present, was sufficiently available. The maximum COTR measured was approximately 5 mmol/L/h for pure CO cultivation, and approximately 6 mmol/L/h for cultivation with additional H2 in the gas supply. Additionally, calculation of the ratio of evolved carbon dioxide to consumed monoxide, similar to the respiratory quotient for aerobic fermentation, allows the prediction of whether acetate or ethanol is predominantly produced. Clostridium ljungdahlii, a model acetogen for syngas fermentation, was cultivated using only CO, and CO in combination with H2. Online monitoring of the mentioned parameters revealed a metabolic shift in fermentation with sole CO, depending on COTR. The device presented herein allows fast process development, because crucial parameters for scale‐up can be measured online in small‐scale gas fermentation.

Obligate aerobic organisms rely on a functional electron transport chain for energy conservation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions. We here investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the single or double knockout mutant strains displayed no, or only a weak, phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and a strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants, including quantitative physiological experiments, transcript analysis, proteomics, and enzyme activity assays revealed distinct responses to type 2 and type 1 dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes, likely by rerouting of metabolic fluxes. This metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, the possible versatile redirecting of carbon fluxes upon genetic interventions can thwart metabolic engineering efforts. IMPORTANCE While Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and, consequently, competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. This high degree of metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse toward a host with a controlled and efficient supply of redox cofactors for product synthesis.

30Obligate aerobic organisms rely on a functional electron transport chain for energy 31 generation and NADH oxidation. Because of this essential requirement, the genes of 32 this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance 33 and energy shortage under fluctuating environmental conditions. 34We here investigated the essentiality of the three NADH dehydrogenases of the 35 respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the 36 impact of the knockouts of corresponding genes on its physiology and metabolism. 37While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the 38 generated single or double knockout strains displayed none or only a marginal 39 phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear 40 phenotype with biphasic growth behavior and strongly reduced growth rate in the 41 second phase. In-depth analyses of the metabolism of the generated mutants including 42 quantitative physiological experiments, transcript analysis, proteomics and enzyme 43 activity assays revealed distinct responses to type II and type I dehydrogenase 44deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the 45 introduced genetic perturbations and maintain stable phenotypes by rerouting of 46 metabolic fluxes. 47 This metabolic adaptability has implications for biotechnological applications. While the 48 phenotypic robustness is favorable in large-scale applications with inhomogeneous 49 conditions, versatile redirecting of carbon fluxes upon genetic interventions can frustrate 50 metabolic engineering efforts. 51 52 Importance 53While Pseudomonas has the capability for high metabolic activity and the provision of 54 reduced redox cofactors important for biocatalytic applications, exploitation of this 55 3 characteristic might be hindered by high, constitutive activity of and consequently 56 competition with the NADH dehydrogenases of the respiratory chain. The in-depth 57 analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 58 presented here, provides insight into the phenotypic and metabolic response of this 59 strain to these redox metabolism perturbations. The observed great metabolic flexibility 60 needs to be taken into account for rational engineering of this promising 61 biotechnological workhorse towards a host with controlled and efficient supply of redox 62 cofactors for product synthesis. 63 64 65Many industrially relevant molecules, e. g., ethanol, butanediol or isoprene, are more 66 reduced than the industrially-used sugars glucose and sucrose or alternative, upcoming 67 carbon sources such as xylose or glycerol (1-3). The microbial production of those 68 favored compounds hence is inherently redox limited, i.e. by the supply of reduced 69 redox cofactors, generally NADH or NADPH. This bottleneck has been overcome in 70 some cases, e.g., 1,4 butanediol and 1,3-propanediol production in Escherichia coli (4, 71 5) or L-lysine synthesis in Corynebacter...