Dataset Information

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

ABSTRACT: Screening 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.In 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.The 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.

SUBMITTER: Philip P

PROVIDER: S-EPMC5941677 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Philip P P Kern D D Goldmanns J J Seiler F F Schulte A A Habicher T T Büchs J J

Microbial cell factories 20180509 1

<h4>Background</h4>Screening 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 exp ...[more]

PMID: 29743073

Similar Datasets

Project description:BackgroundThe initial part of process development involves extensive screening programs to identify optimal biological systems and cultivation conditions. For a successful scale-up, the operation mode on screening and production scale must be as close as possible. To enable screening under fed-batch conditions, the membrane-based fed-batch shake flask was developed. It is a shake flask mounted with a central feed reservoir with an integrated rotating membrane tip for a controlled substrate release. Building on the previously provided proof of principle for this tool, this work extends its application by constructive modifications and improved methodology to ensure reproducible performance.ResultsThe previously limited operation window was expanded by a systematic analysis of reservoir set-up variations for cultivations with the fast-growing organism Escherichia coli. Modifying the initial glucose concentration in the reservoir as well as interchanging the built-in membrane, resulted in glucose release rates and oxygen transfer rate levels during the fed-batch phase varying up to a factor of five. The range of utilizable membranes was extended from dialysis membranes to porous microfiltration membranes with the design of an appropriate membrane tip. The alteration of the membrane area, molecular weight cut-off and liquid volume in the reservoir offered additional parameters to fine-tune the duration of the initial batch phase, the oxygen transfer rate level of the fed-batch phase and the duration of feeding. It was shown that a homogeneous composition of the reservoir without a concentration gradient is ensured up to an initial glucose concentration of 750 g/L. Finally, the experimental validity of fed-batch shake flask cultivations was verified with comparable results obtained in a parallel fed-batch cultivation in a laboratory-scale stirred tank reactor.ConclusionsThe membrane-based fed-batch shake flask is a reliable tool for small-scale screening under fed-batch conditions filling the gap between microtiter plates and scaled-down stirred tank reactors. The implemented reservoir system offers various set-up possibilities, which provide a wide range of process settings for diverse biological systems. As a screening tool, it accurately reflects the cultivation conditions in a fed-batch stirred tank reactor and enables a more efficient bioprocess development.

Project description:Simplicity renders shake flasks ideal for strain selection and substrate optimization in biotechnology. Uncertainty during initial experiments may, however, cause adverse growth conditions and mislead conclusions. Using growth models for online predictions of future biomass (BM) and the arrival of critical events like low dissolved oxygen (DO) levels or when to harvest is hence important to optimize protocols. Established knowledge that unfavorable metabolites of growing microorganisms interfere with the substrate suggests that growth dynamics and, as a consequence, the growth model parameters may vary in the course of an experiment. Predictive monitoring of shake flask cultures will therefore benefit from estimating growth model parameters in an online and adaptive manner. This paper evaluates a newly developed particle filter (PF) which is specifically tailored to the requirements of biotechnological shake flask experiments. By combining stationary accuracy with fast adaptation to change the proposed PF estimates time-varying growth model parameters from iteratively measured BM and DO sensor signals in an optimal manner. Such proposition of inferring time varying parameters of Gompertz and Logistic growth models is to our best knowledge novel and here for the first time assessed for predictive monitoring of Escherichia coli (E. coli) shake flask experiments. Assessments that mimic real-time predictions of BM and DO levels under previously untested growth conditions demonstrate the efficacy of the approach. After allowing for an initialization phase where the PF learns appropriate model parameters, we obtain accurate predictions of future BM and DO levels and important temporal characteristics like when to harvest. Statically parameterized growth models that represent the dynamics of a specific setting will in general provide poor characterizations of the dynamics when we change strain or substrate. The proposed approach is thus an important innovation for scientists working on strain characterization and substrate optimization as providing accurate forecasts will improve reproducibility and efficiency in early-stage bioprocess development.

Project description:BackgroundPichia pastoris is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast.ResultsAddition of defined concentrations of Antifoam A (Sigma), Antifoam C (Sigma), J673A (Struktol), P2000 (Fluka) or SB2121 (Struktol) to shake-flask cultures of P. pastoris increased the total amount of recombinant GFP in the culture medium (the total yield) and in the case of P2000, SB2121 and J673A almost doubled it. When normalized to the culture density, the GFP specific yield (μg OD₅₉₅⁻¹) was only increased for Antifoam A, Antifoam C and J673A. Whilst none of the antifoams affected the growth rate of the cells, addition of P2000 or SB2121 was found to increase culture density. There was no correlation between total yield, specific yield or specific growth rate and the volumetric oxygen mass transfer coefficient (k(L)a) in the presence of antifoam. Moreover, the antifoams did not affect the dissolved oxygen concentration of the cultures. A comparison of the amount of GFP retained in the cell by flow cytometry with that in the culture medium by fluorimetry suggested that addition of Antifoam A, Antifoam C or J673A increased the specific yield of GFP by increasing the proportion secreted into the medium.ConclusionsWe show that addition of a range of antifoaming agents to shake flask cultures of P. pastoris increases the total yield of the recombinant protein being produced. This is not only a simple method to increase the amount of protein in the culture, but our study also provides insight into how antifoams interact with microbial cell factories. Two mechanisms are apparent: one group of antifoams (Antifoam A, Antifoam C and J673A) increases the specific yield of GFP by increasing the total amount of protein produced and secreted per cell, whilst the second (P2000 or SB2121) increases the total yield by increasing the density of the culture.

Project description:BackgroundLack of accounting for proton uptake and secretion has confounded interpretation of the stoichiometry of photosynthetic growth of algae. This is also problematic for achieving growth of microalgae to high cell concentrations which is necessary to improve productivity and the economic feasibility of commercial-scale chemical production systems. Since microalgae are capable of consuming both nitrate and ammonium, this represents an opportunity to balance culture pH based on a nitrogen feeding strategy that does not utilize gas-phase CO₂ buffering. Stoichiometry suggests that approximately 36 weight%NH₄⁺ (balance nitrogen as NO₃⁻) would minimize the proton imbalance and permit high-density photoautotrophic growth as it does in higher plant tissue culture. However, algal media almost exclusively utilize nitrate, and ammonium is often viewed as 'toxic' to algae.ResultsThe microalgae Chlorella vulgaris and Chlamydomonas reinhardtii exclusively utilize ammonium when both ammonium and nitrate are provided during growth on excess CO₂. The resulting proton imbalance from preferential ammonium utilization causes the pH to drop too low to sustain further growth when ammonium was only 9% of the total nitrogen (0.027 gN-NH₄⁺/L). However, providing smaller amounts of ammonium sequentially in the presence of nitrate maintained the pH of a Chlorella vulgaris culture for improved growth on 0.3 gN/L to 5 gDW/L under 5% CO₂ gas-phase supplementation. Bioreactor pH dynamics are shown to be predictable based on simple nitrogen assimilation as long as there is sufficient CO₂ availability.ConclusionsThis work provides both a media formulation and a feeding strategy with a focus on nitrogen metabolism and regulation to support high-density algal culture without buffering. The instability in culture pH that is observed in microalgal cultures in the absence of buffers can be overcome through alternating utilization of ammonium and nitrate. Despite the highly regulated array of nitrogen transporters, providing a nitrogen source with a balanced degree of reduction minimizes pH fluctuations. Understanding and accommodating the behavior of nitrogen utilization in microalgae is key to avoiding 'culture crash' and reliance on gas phase CO₂ buffering, which becomes both ineffective and cost-prohibitive for commercial-scale algal culture.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets