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CO2 and O2 in the Sakaguchi flask headspace during culture was monitored via circulation direct monitoring and sampling system (CDMSS), a device with circulation bypass system. In static culture with Saccharomyces cerevisiae (circulation rate, 50 mL/min), a vertical CO2 concentration gradient (maximum gap ~ 2% (v/v) [height from the bottom of flask 45 mm, 7%; 155 mm, 5%]) in the Sakaguchi flask headspace was observed; no concentration O2 gradient was observed. However, shake flask culture showed vertical gradient concentrations for both CO2 and O2 (maximum gap of CO2 and O2 concentrations: 2 and 4% [heights from the bottom of flask 115 mm, 6.0 and 9.5%; 175 mm, 4.0 and 13.5%], respectively). When the CDMSS circulation rate in the Sakaguchi flask headspace was 300 or 400 mL/min, the gaseous environment was uniformly distributed so that no vertical gradient concentration was observed. In shaking culture with Escherichia coli under these conditions, CO2 was accumulated at high concentrations in the headspace and culture broth (maximum values 8%, in the headspace; 120 mg/L, in the culture broth). Most of the accumulated CO2 in the headspace could be removed by inserting a column packed with CO2 adsorbent at the bypass port of the CDMSS gaseous circulation. Thus, dissolved CO2 was maintained at a lower concentration, and the final UOD (unit optical density) value of culture was increased compared with that of the control. This study is the first to demonstrate that vertical gradients of CO2 and O2 concentrations exist in the headspace of Sakaguchi flask during culture.

About 85 years have passed since the shaking culture was devised. Since then, various monitoring devices have been developed to measure culture parameters. O2 consumed and CO2 produced by the respiration of cells in shaking cultures are of paramount importance due to their presence in both the culture broth and headspace of shake flask. Monitoring in situ conditions during shake-flask culture is useful for analysing the behaviour of O2 and CO2, which interact according to Henry’s law, and is more convenient than conventional sampling that requires interruption of shaking. In situ monitoring devices for shake-flask cultures are classified as direct or the recently developed bypass type. It is important to understand the characteristics of each type along with their unintended effect on shake-flask cultures, in order to improve the existing devices and culture conditions. Technical developments in the bypass monitoring devices are strongly desired in the future. It is also necessary to understand the mechanism underlying conventional shake-flask culture. The existing shaking culture methodology can be expanded into next-generation shake-flask cultures constituting a novel culture environment through a judicious selection of monitoring devices depending on the intended purpose of shake-flask culture. Construction and sharing the databases compatible with the various types of the monitoring devices and measurement instruments adapted for shaking culture can provide a valuable resource for broadening the application of cells with shake-flask culture.

We evaluated the ventilation ability of two types (plug-type and cap-type) of culture-stoppers having standard air permeability. The culture-stoppers were evaluated using the circulation direct monitoring and sampling system with CO2 concentration in the gas phase of a shake-flask culture as an index. The half-lives of CO2 in the headspace of the shake flask with the plug-type and cap-type stoppers were about 51.5 min and about 30.3 min, respectively. Based on these half-lives, we formulated a model equation to simulate the behaviour of CO2 with different culture-stoppers. After validating the model equation by shake-flask culture with Saccharomyces cerevisiae, we investigated the effect of different ventilation abilities of the culture-stoppers on the growth of Pelomonas saccharophila and Escherichia coli: the sensitivity of the culture-stopper to the ventilation ability was dependent on the microorganism species. In the case of P. saccharophila, when the plug-type culture-stopper was combined with controlled CO2 concentration (6%) in the flask, the maximum yield increased by twofold compared to that of the control. This study shows the importance of ventilation in headspace and conventional culture-stoppers during the shake-flask culture of microorganisms. The problems that may occur between the conventional shake-flask culture approach using a breathable culture-stopper and the next-generation shake-flask culture without a conventional culture-stopper were clarified from the evaluation of gas-permeable culture-stoppers. The importance of controlled gaseous phase in the headspace during shake-flask culture of the microorganisms was also elucidated.Key points• Ventilation capacity of culture-stoppers was evaluated using the CO2half-life concentration.• Behaviour of microorganisms varies with the type of culture-stopper.• Developed a PID system for control of CO2in flask gas phase to enhance the shake-flask culture.

Modeling and optimization were performed to enhance production of lactase through submerged fermentation by Bacillus subtilis VUVD001 using artificial neural networks (ANN) and response surface methodology (RSM). The effect of process parameters namely temperature (°C), pH, and incubation time (h) and their combinational interactions on production was studied in shake flask culture by Box–Behnken design. The model was validated by conducting an experiment at optimized process variables which gave the maximum lactase activity of 91.32 U/ml. Compared to traditional activity, 3.48-folds improved production was obtained after RSM optimization. This study clearly shows that both RSM and ANN models provided desired predictions. However, compared with RSM ( R 2  = 0.9496), the ANN model ( R 2  = 0.99456) gave a better prediction for the production of lactase.

The laboratory-scale production of plasmid DNA (pDNA) is hindered by the limitations of shake flasks, such as mass transfer capacity and lack of pH control. Consequently, better schemes for pDNA production in shake flasks are needed. pDNA production can be improved by increasing the amount of biomass, increasing the pDNA yield on biomass (Y pDNA/OD ), or both. In this study, we characterized the production of three differently sized plasmids (5.4, 6.0, and 7.8 kbp, respectively) cultured in Lysogenic Broth (LB), Terrific Broth (TB), and EnPresso B Plasmid (EBP) culture medium that releases glucose to the broth enzymatically. Higher cell densities, higher acetate accumulation, and higher pDNA titers, were obtained in cells cultured in TB than in those cultured in LB medium. The enzyme-controlled glucose release system resulted in an important increase in cell densities, while the Y pDNA/OD increased up to threefold. The most important increase in pDNA titer was observed for the 7.8-kbp plasmid, which raised from 1.6 to 4.3 mg/L in LB and TB medium, respectively, to 26.6 mg/L using the EBP medium. These results show that controlled substrate delivery is useful to increase the production of large-sized pDNA in shake flasks.

Background Limonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly. Results In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce both d-limonene and l-limonene by introducing the heterologous d-limonene synthase from Citrus limon and l-limonene synthase from Mentha spicata, respectively. However, only 0.124 mg/L d-limonene and 0.126 mg/L l-limonene were produced. To improve the limonene production by the engineered yeast Y. lipolytica strain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis in Y. lipolytica. Through the overexpression of HMGR gene, the titers of d-limonene and l-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineered Y. lipolytica strains from glucose, and the final titers of d-limonene and l-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved for d-limonene and l-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineered Y. lipolytica strains was investigated. We showed that d-limonene and l-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization. Conclusions This study represents the first report on the development of a new and efficient process to convert waste cooking oil into d-limonene and l-limonene by exploiting metabolically engineered Y. lipolytica strains for fermentation. The results obtained in this study lay the foundation for more future applications of Y. lipolytica in converting waste cooking oil into various industrially valuable products.

Lycium schweinfurthii, a wild shrub of the Solanaceae family, has received increasing attention in the last decade for its therapeutic potential in traditional medicine due to its diverse array of secondary metabolites, including phenolic substances and terpenoids. The aim of this study was to investigate the accumulation of phenolics, flavonoids, and the terpenoid lupeol in L. schweinfurthii cell suspension shake flask cultures and a single-use 2-dimensional rocking motion bioreactor. Three different media formulations were compared for in vitro cell cultures. Various parameters, such as biomass accumulation, settled cell volume, cell viability (assessed via a 2,3,5-triphenyl tetrazolium chloride assay), and sucrose consumption were determined as indicators of cell activity and growth. Total phenolic and flavonoid contents were estimated spectrophotometrically, lupeol was quantified via High-Performance Thin Layer Chromatography (HPTLC). Although a higher fresh biomass concentration of 464 g L− 1 was obtained in MS medium supplemented with a combination of each, 1 mg L− 1 of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1-Naphthaleneacetic acid (NAA), the rocking-motion bioreactor cultivation was performed with 2 mg L− 1 NAA due to its superior reproducibility in viability, productivity, and content of bioactive compounds (e.g., phenolics, flavonoids, lupeol). A final fresh biomass concentration of 185 g L− 1 was achieved in a 16 L cultivation scale with a notable increase in the concentration of phenolics (1.4-fold) and flavonoids (1.7-fold). Most importantly, the concentration of lupeol, a pentacyclic triterpenoid known for its anti-inflammatory, antibacterial, and anti-atherogenic properties, exhibited a remarkable 5.5-fold increase in the bioreactor cultivation (585 µg g− 1) compared to shake flask cultivations (106 µg g− 1). The current study demonstrated the profound impact of media composition and non-limited fed-batch conditions in a rocking-motion bioreactor on the accumulation of bioactive compounds. The findings are also relevant to other plant cell cultures.Key messageScaling-up of Lycium schweinfurthii suspension culture is possible in a rocking motion bioreactor for production of phenolics and lupeol.

•Strategies for the production of MVA virus at high-cell-densities are presented.•High-cell-density cultures can be downscaled from bioreactor to shake flasks.•Optimal MVA virus production requires a combination of fed-batch and semi-perfusion. Increasing the yield and the productivity in cell culture-based vaccine manufacturing using high-cell-density (HCD) cultivations faces a number of challenges. For example, medium consumption should be low to obtain a very high concentration of viable host cells in an economical way but must be balanced against the requirement that accumulation of toxic metabolites and limitation of nutrients have to be avoided. HCD cultivations should also be optimized to avoid unwanted induction of apoptosis or autophagy during the early phase of virus infection. To realize the full potential of HCD cultivations, a rational analysis of the cultivation conditions of the appropriate host cell line together with the optimal infection conditions for the chosen viral vaccine strain needs to be performed for each particular manufacturing process. We here illustrate our strategy for production of the modified vaccinia Ankara (MVA) virus isolate MVA-CR19 in the avian suspension cell line AGE1.CR.pIX at HCD. As a first step we demonstrate that the adjustment of the perfusion rate strictly based on the measured cell concentration and the glucose consumption rate of cells enables optimal growth in a 0.8 L bioreactor equipped with an ATF2 system. Concentrations up to 57 × 106 cells/mL (before infection) were obtained with a viability exceeding 95%, and a maximum specific cell growth rate of 0.019 h−1 (doubling time = 36.5 h). However, not only the cell-specific MVA-CR19 virus yield but also the volumetric productivity was reduced compared to infections at conventional-cell-density (CCD). To facilitate optimization of the virus propagation phase at HCD, a larger set of feeding strategies was analyzed in small-scale cultivations using shake flasks. Densities up to 63 × 106 cells/mL were obtained at the end of the cell growth phase applying a discontinuous perfusion mode (semi-perfusion) with the same cell-specific perfusion rate as in the bioreactor (0.060 nL/(cell d)). At this cell concentration, a medium exchange at time of infection was required to obtain expected virus yields during the first 24 h after infection. Applying an additional fed-batch feeding strategy during the whole virus replication phase resulted in a faster virus titer increase during the first 36 h after infection. In contrast, a semi-continuous virus harvest scheme improved virus accumulation and recovery at a rather later stage of infection. Overall, a combination of both fed-batch and medium exchange strategies resulted in similar cell-specific virus yields as those obtained for CCD processes but 10-fold higher MVA-CR19 titers, and four times higher volumetric productivity.

Vero cells are nowadays widely used in the production of human vaccines. They are considered as one of the most productive and flexible continuous cell lines available for vaccine manufacturing. However, these cells are anchorage dependent, which greatly complicates upstream processing and process scale-up. Moreover, there is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide. The use of cell lines adapted to suspension growth contributes to reach this objective. The current work describes the adaptation of Vero cells to suspension culture in different serum free media according to multiple protocols based on subsequent passages. The best one that relies on cell adaption to IPT-AFM an in-house developed animal component free medium was then chosen for further studies. Besides, as aggregates have been observed, the improvement of IPT-AFM composition and mechanical dissociation were also investigated. In addition to IPT-AFM, three chemically defined media (CD293, Hycell CHO and CD-U5) and two serum free media (293SFMII and SFM4CHO) were tested to set up a serum free culture of the suspension-adapted Vero cells (VeroS) in shake flasks. Cell density levels higher than 2 × 106 cells/mL were obtained in the assessed conditions. The results were comparable to those obtained in spinner culture of adherent Vero cells grown on Cytodex 1 microcarriers. Cell infection with LP-2061 rabies virus strain at an MOI (Multiplicity of Infection) of 0.1 and a cell density of 8 ± 0.5 × 105 cells/mL resulted in a virus titer higher than 107 FFU/mL in all media tested. Nevertheless, the highest titer equal to 5.2 ± 0.5 × 107 FFU/mL, was achieved in IPT-AFM containing a reduced amount of Ca++ and Mg++. Our results demonstrate the suitability of the obtained VeroS cells to produce rabies virus at a high titer, and pave the way to develop VeroS cells bioreactor process for rabies vaccine production.

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