Parallel online determination of ethylene release rate by Shaken Parsley cell cultures using a modified RAMOS device

Schulte, Andreas; Nolten, J.A.M.; Korona, Anna; Krömke, Hannes; Vennekötter, Jan-Bernd; Schillheim, Britta; Weßling, Matthias; Conrath, Uwe; Büchs, Jochen

doi:10.1186/s12870-018-1305-6

Cited by 16 publications

(9 citation statements)

References 35 publications

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“…The flow and stop phase durations for all cultivations conducted in the µTOM and RAMOS devices were set to 20 and 10 min, respectively (Anderlei & Büchs, 2001; Anderlei et al, 2004; Schulte et al, 2018).…”

Section: Methodsmentioning

confidence: 99%

Device for respiration activity measurement enables the determination of oxygen transfer rates of microbial cultures in shaken 96‐deepwell microtiter plates

Dinger

Lattermann²,

Flitsch³

et al. 2022

Biotech & Bioengineering

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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.

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Section: Methodsmentioning

confidence: 99%

Device for respiration activity measurement enables the determination of oxygen transfer rates of microbial cultures in shaken 96‐deepwell microtiter plates

Dinger

Lattermann²,

Flitsch³

et al. 2022

Biotech & Bioengineering

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“…The cultivations and measurements of the oxygen transfer rate (OTR) and the carbon dioxide transfer rate (CTR) were performed in a modified RAMOS System, developed by the Chair of Biochemical Engineering (RWTH Aachen University) (36, 37). The standard RAMOS for shake flasks is commercially available from the Kühner AG (Birsfelden, Switzerland) or HiTec Zang GmbH (Herzogenrath, Germany).…”

Section: Methodsmentioning

confidence: 99%

The metabolic response ofPseudomonas taiwanensisto NADH dehydrogenase deficiency

Nies

Dinger

Chen

et al. 2019

Preprint

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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...

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“…Another innovative screening devise for response-screening activity in plant cells is based on the online measurement of the defense-associated plant hormone ethylene [ 18 ]. In this approach, cultured parsley cells were grown in a RAMOS system equipped with electrochemical ethylene sensors.…”

Section: Introductionmentioning

confidence: 99%

Spotting priming-active compounds using parsley cell cultures in microtiter plates

et al. 2023

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Background Conventional crop protection has major drawbacks, such as developing pest and pathogen insensitivity to pesticides and low environmental compatibility. Therefore, alternative crop protection strategies are needed. One promising approach treats crops with chemical compounds that induce the primed state of enhanced defense. However, identifying priming compounds is often tedious as it requires offline sampling and analysis. High throughput screening methods for the analysis of priming-active compounds have great potential to simplify the search for such compounds. One established method to identify priming makes use of parsley cell cultures. This method relies on measurement of fluorescence of furanocoumarins in the final sample. This study demonstrates for the first time the online measurement of furanocoumarins in microtiter plates. As not all plants produce fluorescence molecules as immune response, a signal, which is not restricted to a specific plant is required, to extend online screening methods to other plant cell cultures. It was shown that the breathing activity of primed parsley cell cultures increases, compared to unprimed parsley cell cultures. The breathing activity can by monitored online. Therefore, online identification of priming-inducing compounds by recording breathing activity represents a promising, straight-forward and highly informative approach. However, so far breathing has been recorded in shake flasks which suffer from low throughput. For industrial application we here report a high-throughput, online identification method for identifying priming-inducing chemistry. Results This study describes the development of a high-throughput screening system that enables identifying and analyzing the impact of defense priming-inducing compounds in microtiter plates. This screening system relies on the breathing activity of parsley cell cultures. The validity of measuring the breathing activity in microtiter plates to drawing conclusions regarding priming-inducing activity was demonstrated. Furthermore, for the first time, the fluorescence of the priming-active reference compound salicylic acid and of furanocoumarins were simultaneously monitored online. Dose and time studies with salicylic acid-treated parsley cell suspensions revealed a wide range of possible addition times and concentrations that cause priming. The online fluorescence measuring method was further confirmed with three additional compounds with known priming-causing activity. Conclusions Determining the OTR, fluorescence of the priming-active chemical compound SA and of furanocoumarins in parsley suspension cultures in MTPs by online measurement is a powerful and high-throughput tool to study possible priming compounds. It allows an in-depth screening for priming compounds and a better understanding of the priming process induced by a given substance. Evaluation of priming phenomena via OTR should also be applicable to cell suspensions of other plant species and varieties and allow screening for priming-inducing chemical compounds in intact plants. These online fluorescence methods to measure the breathing activity, furanocoumarin and SA have the potential to accelerate the search for new priming compounds and promote priming as a promising, eco-friendly crop protection strategy.

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Parallel online determination of ethylene release rate by Shaken Parsley cell cultures using a modified RAMOS device

Cited by 16 publications

References 35 publications

Device for respiration activity measurement enables the determination of oxygen transfer rates of microbial cultures in shaken 96‐deepwell microtiter plates

Device for respiration activity measurement enables the determination of oxygen transfer rates of microbial cultures in shaken 96‐deepwell microtiter plates

The metabolic response ofPseudomonas taiwanensisto NADH dehydrogenase deficiency

Spotting priming-active compounds using parsley cell cultures in microtiter plates

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