Oxygen limitation is a pitfall during screening for industrial strains

Zimmermann, Hartmut F.; Anderlei, Tibor; Büchs, Jochen; Binder, Michael

doi:10.1007/s00253-006-0414-6

Cited by 62 publications

(46 citation statements)

References 17 publications

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“…It can be assumed that in shake flasks, at least at higher cell densities (OD 600 of >10), the cells face oxygen limitation (Zimmermann et al 2006) and therefore, we tested the effect of low oxygen tension in the bioreactor from the middle to the end of the growth phase. Under these conditions, the cells showed slightly lower growth rates and biomass yields and in the production phase slightly lower production rates, significantly lower Y P/S and significantly higher glucose consumption rates (see Table 2).…”

Section: Discussionmentioning

confidence: 99%

Engineering Corynebacterium glutamicum for the production of pyruvate

Wieschalka

Blombach

Eikmanns

2012

Appl Microbiol Biotechnol

105

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A Corynebacterium glutamicum strain with inactivated pyruvate dehydrogenase complex and a deletion of the gene encoding the pyruvate:quinone oxidoreductase produces about 19 mM L: -valine, 28 mM L: -alanine and about 55 mM pyruvate from 150 mM glucose. Based on this double mutant C. glutamicum △aceE △pqo, we engineered C. glutamicum for efficient production of pyruvate from glucose by additional deletion of the ldhA gene encoding NAD(+)-dependent L: -lactate dehydrogenase (LdhA) and introduction of a attenuated variant of the acetohydroxyacid synthase (△C-T IlvN). The latter modification abolished overflow metabolism towards L: -valine and shifted the product spectrum to pyruvate production. In shake flasks, the resulting strain C. glutamicum △aceE △pqo △ldhA △C-T ilvN produced about 190 mM pyruvate with a Y (P/S) of 1.36 mol per mol of glucose; however, it still secreted significant amounts of L: -alanine. Additional deletion of genes encoding the transaminases AlaT and AvtA reduced L: -alanine formation by about 50%. In fed-batch fermentations at high cell densities with adjusted oxygen supply during growth and production (0-5% dissolved oxygen), the newly constructed strain C. glutamicum △aceE △pqo △ldhA △C-T ilvN △alaT △avtA produced more than 500 mM pyruvate with a maximum yield of 0.97 mol per mole of glucose and a productivity of 0.92 mmol g ((CDW)) (-1) h(-1) (i.e., 0.08 g g((CDW)) (-1) h(-1)) in the production phase.

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Section: Discussionmentioning

confidence: 99%

Engineering Corynebacterium glutamicum for the production of pyruvate

Wieschalka

Blombach

Eikmanns

2012

Appl Microbiol Biotechnol

105

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“…Yet, this is particularly relevant in the context of cell-based assays, since oxygen availability drastically affects the physiological state of cells. 9,10 Their genotypic distinctiveness is likely to be perturbed by phenotypic noise if oxygen cannot be provided sufficiently and homogeneously among all samples.…”

Section: Introductionmentioning

confidence: 99%

Enhanced and homogeneous oxygen availability during incubation of microfluidic droplets

et al. 2015

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Droplet microfluidic-based cell screening has the potential to surpass time-and cost efficiency of established screening platforms by several orders of magnitude, but so far lacks sufficient and homogeneous oxygen supply for large droplet numbers (>10 6 ), which is a key parameter affecting metabolism and growth of encapsulated cells. Here, we describe and validate an approach based on continuous carrier oil recirculation that ensures enhanced and homogeneous oxygen availability during mid and long-term incubation of picoliter droplets retained in a 3D-printed storage device. Using biotechnologically relevant microorganisms, we demonstrate that improved oxygen transfer results in three to eleven-fold increased biomass and highly elevated protein production with minimal interdroplet variation. In fact, obtained yields are comparable to those achieved in conventional cultivation devices, so that screening strategies commonly applied in microtiter plates or shaking flasks can now be scaled down to pL-droplets, which offer highly enhanced throughput. In contrast to mere single-cell screening, this approach allows monoclonal cell and metabolite accumulation inside droplets resulting in elevated read-out signals and reduced variability associated to stochasticity in gene expression.Additionally, the range of screening strategies is broadened, since screening for increased biomass yields or mining for microbial natural products from complex environmental samples can now be targeted with pL-droplets. This development substantially improves the robustness and versatility of droplet-based cell assays, further consolidating pL-droplets as a powerful ultrahigh-throughput experimentation platform.

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“…Otherwise wrong information about the variables under study might be obtained. 65,66 Several operational parameters affect the OTR in shaking devices, namely the shape and size of the vessel; the shaking frequency; the shaking amplitude; the shaking angle; the type of agitation (orbital or linear); and the filling volume. OTR is also influenced by the surface properties of the flask material, which may be either hydrophobic or hydrophilic; and by the physical-chemical properties of the liquid (viscosity, oxygen solubility, diffusivity and surface tension, the latter being more noticeable in MWP).…”

Section: K L a Determination In Shaken Devicesmentioning

confidence: 99%

Bioprocess scale‐up: quest for the parameters to be used as criterion to move from microreactors to lab‐scale

Marques

Cabral

Fernandes

2010

J of Chemical Tech & Biotech

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Advances in high-throughput process development and optimization involve the rational use of miniaturized stirred bioreactors, instrumented shaken flasks and microtiter plates. As expected, each one provides different levels of control and monitoring, requiring a compromise between data quantity and quality. Despite recent advances, traditional shaken flasks with nominal volumes below 250 mL and microtiter plates are still widely used to assemble wide arrays of biotransformation/bioconversion data, because of their simplicity and low cost. These tools are key assets for faster process development and optimization, provided data are representative. Nonetheless, the design, development and implementation of bioprocesses can present variations depending on intrinsic characteristics of the overall process. For each particular process, an adequate and comprehensive approach has to be established, which includes pinpointing key issues required to ensure proper scaleup. Recently, focus has been given to engineering characterization of systems in terms of mass transfer and hydrodynamics (through gaining insight into parameters such as k L a and P/V at shaken and microreactor scale), due to the widespread use of small-scale reactors in the early developmental stages of bioconversion/biotransfomation processes. Within this review, engineering parameters used as criteria for scaling-up fermentation/bioconversion processes are discussed. Particular focus is on the feasibility of the application of such parameters to small-scale devices and concomitant use for scale-up.

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Oxygen limitation is a pitfall during screening for industrial strains

Cited by 62 publications

References 17 publications

Engineering Corynebacterium glutamicum for the production of pyruvate

Engineering Corynebacterium glutamicum for the production of pyruvate

Enhanced and homogeneous oxygen availability during incubation of microfluidic droplets

Bioprocess scale‐up: quest for the parameters to be used as criterion to move from microreactors to lab‐scale

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