Analysis of the influence of flame sterilization included in sampling operations on shake-flask cultures of microorganisms.
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ABSTRACT:
SUBMITTER: Takahashi M
PROVIDER: S-EPMC7326993 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
| S-EPMC8614854 | biostudies-literature
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.
| S-EPMC3072307 | biostudies-literature
Project description:Silk-elastin-like polymers (SELPs) are protein-based polymers composed of repetitive amino acid sequence motifs found in silk fibroin (GAGAGS) and mammalian elastin (VPGVG). These polymers are of much interest, both from a fundamental and applied point of view, finding potential application in biomedicine, nanotechnology and as materials. The successful employment of such polymers in such diverse fields, however, requires the ready availability of a variety of different forms with novel enhanced properties and which can be simply prepared in large quantities on an industrial scale. In an attempt to create new polymer designs with improved properties and applicability, we have developed four novel SELPs wherein the elastomer forming sequence poly(VPGVG) is replaced with a plastic-like forming sequence, poly(VPAVG), and combined in varying proportions with the silk motif. Furthermore, we optimised a simplified production procedure for these, making use of an autoinduction medium to reduce process intervention and with the production level obtained being 6-fold higher than previously reported for other SELPs, with volumetric productivities above 150?mg/L. Finally, we took advantage of the known enhanced stability of these polymers in developing an abridged, non-chromatographic downstream processing and purification protocol. A simple acid treatment allowed for cell disruption and the obtention of relative pure SELP in one-step, with ammonium sulphate precipitation being subsequently used to enable improved purity. These simplified production and purification procedures improve process efficiency and reduce costs in the preparation of these novel polymers and enhances their potential for application.
| S-EPMC3599559 | biostudies-literature
Project description:BackgroundProduction of 2,3-butanediol from renewable resources is a promising measure to decrease the consumption of fossil resources in the chemical industry. One of the most influential parameters on biotechnological 2,3-butanediol production is the oxygen availability during the cultivation. As 2,3-butanediol is produced under microaerobic process conditions, a well-controlled oxygen supply is the key parameter to control biomass formation and 2,3-butanediol production. As biomass is on the one hand not the final product, but on the other hand the essential biocatalyst, the optimal compromise between biomass formation and 2,3-butanediol production has to be defined.ResultsA shake flask methodology is presented to evaluate the effects of oxygen availability on 2,3-butanediol production with Bacillus licheniformis DSM 8785 by variation of the filling volume. A defined two-stage cultivation strategy was developed to investigate the metabolic response to different defined maximum oxygen transfer capacities at equal initial growth conditions. The respiratory quotient was measured online to determine the point of glucose depletion, as 2,3-butanediol is consumed afterwards. Based on this strategy, comparable results to stirred tank reactors were achieved. The highest space-time yield (1.3 g/L/h) and a 2,3-butanediol concentration of 68 g/L combined with low acetoin concentrations and avoided glycerol formation were achieved at a maximum oxygen transfer capacity of 13 mmol/L/h. The highest overall 2,3-butanediol concentration of 78 g/L was observed at a maximum oxygen transfer capacity of 4 mmol/L/h.ConclusionsThe presented shake flask approach reduces the experimental effort and costs providing a fast and reliable methodology to investigate the effects of oxygen availability. This can be applied especially on product and by-product formation under microaerobic conditions. Utilization of the maximum oxygen transfer capacity as measure for the oxygen availability allows for an easy adaption to other bioreactor setups and scales.
| S-EPMC6498610 | biostudies-literature
Project description:Since natural resources for the bioproduction of commodity chemicals are scarce, waste animal fats (WAF) are an interesting alternative biogenic residual feedstock. They appear as by-product from meat production, but several challenges are related to their application: first, the high melting points (up to 60 °C); and second, the insolubility in the polar water phase of cultivations. This leads to film and clump formation in shake flasks and microwell plates, which inhibits microbial consumption. In this study, different flask and well designs were investigated to identify the most suitable experimental set-up and further to create an appropriate workflow to achieve the required reproducibility of growth and product synthesis. The dissolved oxygen concentration was measured in-line throughout experiments. It became obvious that the gas mass transfer differed strongly among the shake flask design variants in cultivations with the polyhydroxyalkanoate (PHA) accumulating organism Ralstonia eutropha. A high reproducibility was achieved for certain flask or well plate design variants together with tailored cultivation conditions. Best results were achieved with bottom baffled glass and bottom baffled single-use shake flasks with flat membranes, namely, >6 g L-1 of cell dry weight (CDW) with >80 wt% polyhydroxybutyrate (PHB) from 1 wt% WAF. Improved pre-emulsification conditions for round microwell plates resulted in a production of 14 g L-1 CDW with a PHA content of 70 wt% PHB from 3 wt% WAF. The proposed workflow allows the rapid examination of fat material as feedstock, in the microwell plate and shake flask scale, also beyond PHA production. KEY POINTS: • Evaluation of shake flask designs for cultivating with hydrophobic raw materials • Development of a workflow for microwell plate cultivations with hydrophobic raw materials • Production of polyhydroxyalkanoate in small scale experiments from waste animal fat.
| S-EPMC10313549 | biostudies-literature
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.
| S-EPMC5514527 | biostudies-literature
Project description:BackgroundBiotechnological development in shake flask necessitates vital engineering parameters e.g. volumetric power input, mixing time, gas liquid mass transfer coefficient, hydromechanical stress and effective shear rate. Determination and optimization of these parameters through experiments are labor-intensive and time-consuming. Computational Fluid Dynamics (CFD) provides the ability to predict and validate these parameters in bioprocess engineering. This work provides ample experimental data which are easily accessible for future validations to represent the hydrodynamics of the fluid flow in the shake flask.ResultsA non-invasive measuring technique using an optical fluorescence method was developed for shake flasks containing a fluorescent solution with a waterlike viscosity at varying filling volume (VL = 15 to 40 mL) and shaking frequency (n = 150 to 450 rpm) at a constant shaking diameter (do = 25 mm). The method detected the leading edge (LB) and tail of the rotating bulk liquid (TB) relative to the direction of the centrifugal acceleration at varying circumferential heights from the base of the shake flask. The determined LB and TB points were translated into three-dimensional (3D) circumferential liquid distribution plots. The maximum liquid height (Hmax) of the bulk liquid increased with increasing filling volume and shaking frequency of the shaking flask, as expected. The toroidal shapes of LB and TB are clearly asymmetrical and the measured TB differed by the elongation of the liquid particularly towards the torus part of the shake flask.ConclusionThe 3D liquid distribution data collected at varying filling volume and shaking frequency, comprising of LB and TB values relative to the direction of the centrifugal acceleration are essential for validating future numerical solutions using CFD to predict vital engineering parameters in shake flask.
| S-EPMC5541408 | biostudies-literature
Project description:Computational fluid dynamics (CFD) has recently become a pivotal tool in the design and scale-up of bioprocesses. While CFD has been extensively utilized for stirred tank reactors (STRs), there exists a relatively limited body of literature focusing on CFD applications for shake flasks, almost exclusively concentrated on fluids at waterlike viscosity. The importance of CFD model validation cannot be overstated. While techniques to elucidate the internal flow field are necessary for model validation in STRs, the liquid distribution, caused by the orbital shaking motion of shake flasks, can be exploited for model validation. An OpenFOAM CFD model for shake flasks has been established. Calculated liquid distributions were compared to suitable, previously published experimental data. Across a broad range of shaking conditions, at waterlike and moderate viscosity (16.7 mPa∙s), the CFD model's liquid distributions align excellently with the experimental data, in terms of overall shape and position of the liquid relative to the direction of the centrifugal force. Additionally, the CFD model was used to calculate the volumetric power input, based on the energy dissipation. Depending on the shaking conditions, the computed volumetric power inputs range from 0.1 to 7 kW/m3 and differed on average by 0.01 kW/m3 from measured literature data.
| S-EPMC10864319 | biostudies-literature
Project description: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.
| S-EPMC5941677 | biostudies-literature
Project description:Saccharopolyspora erythraea NRRL2338 (wildtype) and K41-135 (overproducer strain from Kosan Biosciences, KOVP) were grown in shake flask cultures in rich medium R5. RNA samples were harvested for each strain over 5 days. 12 h samples taken for each strain were used as their respective reference ("time zero") samples. The genomic DNA control compares NRRL2338 gDNA to NRRL2338 12 h RNA to show relative gene expression at the start of the timecourse. The 12 h RNA control compares initial gene expression between the wildtype and overproducer strains.
2005-07-21 | E-SMDB-2243 | biostudies-arrayexpress