Quantifying the sensitivity of <i>G. oxydans</i> ATCC 621H and DSM 3504 to osmotic stress triggered by soluble buffers

Luchterhand, Bettina; Fischöder, Thomas; Grimm, Alexander R.; Wewetzer, Sandra J.; Wunderlich, Martin; Schlepütz, Tino; Büchs, Jochen

doi:10.1007/s10295-015-1588-7

Cited by 13 publications

(10 citation statements)

References 41 publications

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“…This observation may be caused by an altered morphology of the cells as described by Kunze et al [ 48 ]. As recently published, soluble buffers significantly increase the osmotic stress and therefore influence the oxygen consumption of G. oxydans 3504 [ 49 ]. Hence, it is highly conceivable that the osmotic pressure also influences the cell morphology.…”

Section: Resultsmentioning

confidence: 99%

Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors

et al. 2015

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BackgroundConventional experiments in small scale are often performed in a ‘Black Box’ fashion, analyzing only the product concentration in the final sample. Online monitoring of relevant process characteristics and parameters such as substrate limitation, product inhibition and oxygen supply is lacking. Therefore, fully equipped laboratory-scale stirred tank bioreactors are hitherto required for detailed studies of new microbial systems. However, they are too spacious, laborious and expensive to be operated in larger number in parallel. Thus, the aim of this study is to present a new experimental approach to obtain dense quantitative process information by parallel use of two small-scale culture systems with online monitoring capabilities: Respiration Activity MOnitoring System (RAMOS) and the BioLector device.ResultsThe same ‘mastermix’ (medium plus microorganisms) was distributed to the different small-scale culture systems: 1) RAMOS device; 2) 48-well microtiter plate for BioLector device; and 3) separate shake flasks or microtiter plates for offline sampling. By adjusting the same maximum oxygen transfer capacity (OTRmax), the results from the RAMOS and BioLector online monitoring systems supplemented each other very well for all studied microbial systems (E. coli, G. oxydans, K. lactis) and culture conditions (oxygen limitation, diauxic growth, auto-induction, buffer effects).ConclusionsThe parallel use of RAMOS and BioLector devices is a suitable and fast approach to gain comprehensive quantitative data about growth and production behavior of the evaluated microorganisms. These acquired data largely reduce the necessary number of experiments in laboratory-scale stirred tank bioreactors for basic process development. Thus, much more quantitative information is obtained in parallel in shorter time.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-015-0005-0) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors

et al. 2015

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“…Due to high product and low growth yield, G. oxydans is used for several biotechnological processes in textile, food, pharmaceutical, and fodder industries to produce valuable substances such as D-gluconate, dihydroxyacetone, L-sorbose (vitamin C production), and 6-amino-6-deoxy-Lsorbose (Miglitol production) (Hommel and Ahnert 1999;Hancock 2009;Macauley et al 2001;Claret et al 1994;De Muynck et al, 2007). G. oxydans can thrive in concentrated sugar solutions, for example, up to 30 % (w/v) glucose, but its growth and productivity declines by increased osmolarity of medium (Luchterhand et al 2015;Zahid et al 2015). Therefore, there is a need to explore the physiology of G. oxydans under osmotic stress to understand and improve its bio-catalytic efficiency.…”

Section: Discussionmentioning

confidence: 99%

Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans

Zahid¹,

Deppenmeier²

2016

Appl Microbiol Biotechnol

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Gluconobacter (G.) oxydans is able to incompletely oxidize various sugars and polyols for the production of biotechnologically important compound. Recently, we have shown that the organism produces and accumulates mannitol as compatible solute under osmotic stress conditions. The present study describes the role of two cytoplasmic mannitol dehydrogenases for osmotolerance of G. oxydans. It was shown that Gox1432 is a NADP-dependent mannitol dehydrogenase (EC 1.1.1.138), while Gox0849 uses NAD as cofactor (EC 1.1.1.67). The corresponding genes were deleted and the mutants were analyzed for growth under osmotic stress and non-stress conditions. A severe growth defect was detected for Δgox1432 when grown in high osmotic media, while the deletion of gox0849 had no effect when cells were exposed to 450 mM sucrose in the medium. Furthermore, the intracellular mannitol content was reduced in the mutant lacking the NADP-dependent enzyme Gox1432 in comparison to the parental strain and the Δgox0849 mutant under stress conditions. In addition, transcriptional analysis revealed that Gox1432 is more important for mannitol production in G. oxydans than Gox0849 as the transcript abundance of gene gox1432 was 30-fold higher than of gox0849. In accordance, the activity of the NADH-dependent enzyme Gox0849 in the cell cytoplasm was 10-fold lower in comparison to the NADPH-dependent mannitol dehydrogenase Gox1432. Overexpression of gox1432 in the corresponding deletion mutant restored growth of the cells under osmotic stress, further strengthening the importance of the NADP-dependent mannitol dehydrogenase for osmotolerance in G. oxydans. These findings provide detailed insights into the molecular mechanism of mannitol-mediated osmoprotection in G. oxydans and are helpful engineering strains with improved osmotolerance for biotechnological applications.

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“…In the work of Record et al [ 27 ] an optimum growth at approximately 0.3 OsM and the growth rate approaching to zero at values between 1.7 and 2.0 OsM was shown for E. coli cultivations. In the work by Luchterhand et al [ 28 ] calculations were presented showing that the combination of E. coli and mineral medium is a system with restrictive osmotic tolerance.…”

Section: Introductionmentioning

confidence: 99%

Parallel substrate supply and pH stabilization for optimal screening of E. coli with the membrane-based fed-batch shake flask

et al. 2018

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BackgroundScreening in the fed-batch operation mode is essential for biological cultivations facing challenges as oxygen limitation, osmotic inhibition, catabolite repression, substrate inhibition or overflow metabolism. As a screening tool on shake flask level, the membrane-based fed-batch shake flask was developed. While a controlled supply of a substrate was realized with the in-built membrane tip, the possibilities for replenishing nutrients and stabilizing pH values was not yet exploited. High buffer concentrations were initially used, shifting the medium osmolality out of the biological optimum. As the growth rate is predefined by the glucose release kinetics from the reservoir, the resulting medium acidification can be compensated with a controlled continuous supply of an alkaline compound. The focus of this research is to establish a simultaneous multi-component release of glucose and an alkaline compound from the reservoir to enable cultivations within the optimal physiological range of Escherichia coli.ResultsIn combination with the Respiratory Activity MOnitoring System, the membrane-based fed-batch shake flask enabled the detection of an ammonium limitation. The multi-component release of ammonium carbonate along with glucose from the reservoir resulted not only in the replenishment of the nitrogen source but also in the stabilization of the pH value in the culture medium. A biomass concentration up to 25 g/L was achieved, which is one of the highest values obtained so far to the best of the author’s knowledge with the utilization of a shake flask and a defined synthetic medium. Going a step further, the pH stabilization allowed the decrease of the required buffer amount to one-fourth establishing an optimal osmolality range for cultivation. As optimal physiological conditions were implemented with the multi-component release fed-batch cultivation, the supply of 0.2 g glucose in a 10 mL initial culture medium volume with 50 mM MOPS buffer resulted in a twofold higher biomass concentration than in a comparable batch cultivation.ConclusionsThe newly introduced multi-component release with the membrane-based fed-batch shake flask serves a threefold purpose of replenishing depleted substrates in the culture medium, stabilizing the pH throughout the entire cultivation time and minimizing the necessary amount of buffer to maintain an optimal osmolality range. In comparison to a batch cultivation, these settings enable to achieve higher biomass and product concentrations. Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0917-8) contains supplementary material, which is available to authorized users.

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Quantifying the sensitivity of G. oxydans ATCC 621H and DSM 3504 to osmotic stress triggered by soluble buffers

Cited by 13 publications

References 41 publications

Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors

Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors

Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans

Parallel substrate supply and pH stabilization for optimal screening of E. coli with the membrane-based fed-batch shake flask

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