Shake flask culture

Once methanol-using organisms had been isolated they were screened in small-volume shake-flask cultures to determine their ability to grow in methanol-minimal-medium (such as Medium B described in the previous section) to produce high yields at high growth rates. Optimum growth temperatures and pHs were also determined. 

The temperature optima of isolated organisms would be measured early on in development, usually in shake-flask culture. 

Fig. 2.1 Root growth (O) and accumulation of tobacco mosaic virus (TMV) ( ) in hairy roots of N. benthamiana. TMV concentrations were measured by ELISA. The error bars indicate standard errors from four replicate shake-flask cultures.

Biocatalytic Synthesis of 6-Hydroxy Fluvastatin using Mortierella rammaniana DSM 62752 in Shake Flask Culture and on Multi-gram Scale using a Wave Bioreactor... 

The synthesis of 6-hydroxy fluvastatin with M. rammaniana DSM 62752 gave high conversion (>95 %) in shake flask culture on 400 mL scale with 0.1 g L of fluvastatin as well as on 22 L scale in a Wave bioreactor-fed batch process at a final substrate concentration of 0.4 g L Instead of the partial purification by a second solid-phase extraction described above, 6-hydroxy fluvastatin can be obtained in high purity ( 95 %) by, for example, preparative medium-pressure liquid chromatography (MPLC) on RP18 silica gel. ... 

Hydroxy fluvastatin could be prepared analogously via biotransformation in shake flask culture with Streptomyces violascens ATCC 31560. Different media and minor variations of the process schedule had to be applied. Before supplementation of... 

Broth extracts were prepared by ethyl-acetate (EtOAc) extraction of 7-day-old shake-flask cultures using three extractions of EtOAc with volumes equivalent to the original culture volume. The samples were dried using reduced pressure and the aliquots (400 mg) of the dried materials were reconstituted in 10 ml of the HSCCC solvent system (5 ml of the lower phase and 5 ml of upper phase). 

In shake-flask cultures, pH control is usually limited to either addition of buffering salts such as phosphates, or periodic manual pH adjustment, which is obviously tedious and less effective. The use of ammonium sulfate as the major nitrogen source in Mandels medium (10) requires a more compelling buffering system. Without buffering, the pH drops quickly... 

In the present study, pH-controlling strategies were executed in both shake-flask cultures and a fermentor. Furthermore, various organic acid buffer systems were studied and their efficiency was evaluated in shake-flask cultures. 

Application of Organic Acid Buffer Systems in Shake-Flask Cultures... 

Figure 5 shows the effect of different lengths of root tips in shake-flask cultures. By inoculating shake flasks with root tips of different lengths (5-25 mm), the highest growth rate was obtained for 5-mm-long root tips. 

We investigated the effect of inoculum conditions such as the part, number, and length of root tips age of the hairy roots and size of inoculant on the growth and metabolite biosynthesis of P. ginseng C. A. Meyer hairy root culture. The growth of P. ginseng hairy roots in shake-flask cultures was found to vary depending on the inoculum conditions. The end part, whose apical meristem of root tip had been excised prior to inoculation,... 

A shake flask culture may have the following composition in g/L meat extract 3.0 yeast extract 10.0 calcium carbonate 4.0 Starch 25.0 tap water q.s. to 1000 ml. The flasks are shaken for about 24 h at about 28°-30°C an then the pre-cultures 1 L are used to inoculate jar fermentors each containing 10 L of the following nutrient medium, g meat extract 40.0 peptone 40.0 yeast extract 10.0 sodium chloride 25.0 soybean meal 100.0 glucose 500.0 calcium carbonate 50.0 tap water q.s. to 10 L. 

In plant cell cultures, shake flask culture is an indispensable stage of cultivation. Investigations in a shake flask are very essential and critical to bioprocess scale-up and optimization. We have developed a simple and convenient technique based on the principle of the Warburg manometric method to measure 02 uptake rate (OUR) and C02 evolution rate (CER) of suspended cells in a shake flask culture. This technique has been successfully applied to suspension cultures of Panax notoginseng cells, and some important bioprocess parameters, such as OUR, CER, respiratory quotient (RQ), SOUR and specific CER (SCER), were quantitatively obtained [99]. As long as the environment temperature is strictly controlled to within an error of 0.1 °C, the measuring system is accurate and reproducible, is easy to operate, is economical, and is also able to treat many samples simultaneously. 

Organism. Candida utilis strain ATCC 26387 was used in this work. The organism was stored on agar slants containing the medium in Table I and 1% dextrose. The culture was initially propagated in 50 ml of media in 250 ml shake flasks on a gyrator shaker at 28°C and 100 rev/min. Four hundred ml of shake flask culture were used to inoculate 7 liters of medium in a 14 liter New Brunswick fermenter. Four liters of this culture were asep-tically transferred after 7.5 hours to 40 liters of medium in a 70 liter computer (PDP-11/34 with 3 discs and 96K words of core) -controlled fermentation system. This system has been described in detail elsewhere (12). The details are shown in Figure 1. 

In shake flask cultures, there is only one reasonable possibility to keep pH within a narrow range, namely the use of a very strong buffer, usually phosphate buffer. This is the major reason why culture media often contain a tremendous excess of phosphate. Insertion of multiple pH probes and titrant-addition tubes into shakers has, however, been proposed and marketed [66]. 

Besides addition of heme, the influence of culture temperature on heterologous production of peroxidases has also been reported. For example, lowering the culture temperature from 28 to 19°C enhanced the level of active versatile peroxidase of P. eryngii 5.8-fold and reduced the effective proteolytic activity of the A. nidulans host strain by 2-fold. In this way, a maximum peroxidase activity of 466 U/L was reached [42]. Efficient heterologous production of peroxidases is not always dependent on the availability of heme. The heterologous production of Arthromyces ramosus peroxidase (ARP) has been analyzed in A. awamori under the control of the inducible endoxylanase promoter. Secretion of active ARP was achieved at up to 800 mg/L in shake flask cultures without addition of hemin [43]. This represents a 1,600-fold increase in production compared to ARP production in S. cerevisiae and 38-fold increase compared to ARP production in P. pastoris (see Sect. 12.2). These observations support that several filamentous fungi are more effective secretors of proteins than yeast strains like S. cerevisiae and P. pastoris. Also for... 

A fermentor or a shaker unit for shake flask cultures in a 37°C incubator. 

The solubilized inclusion bodies obtained from small scale purifications yielding approx 20-30 g E. coli cell pastes (12 x 800 mL shake flask cultures)... 

With shikonin, shear effects bein to limit productivity at a kLa of only 10 hr 1 in a shake flask culture. Therefore, if shear limitations can be overcome, at least a 30-fold improvement in productivity should be possible. This is the challenge b This represents 25% utilization of O2 in sparged air. c This represents 20% of saturation dissolved oxygen level. 

Seed preparation The primary seed fermentation is performed using shaking-flask culture techniques. Once grown, the suspension is then transferred to further seed stages. The purpose of the seed preparation is to generate enough inoculums for the production of a fermenter. 

Commercial Production The commercial production of ARA-rich SCO starts with the thawing of a cryovial of a certified stock culture of the production strain of Mart, alpina. The thawed cryovial is used to inoculate a series of shake-flask cultures and seed fermenters of increasing volume to maintain a 5-10% inoculum for each successive stage of the seed train. Final production scale is typically 50 to 200 m (see Figure 2) (50). 

The model-based dynamic optimisation results that were obtained from a fixed feed composition, same initial condition as the batch culture, and a feeding interval of 6-12 h, suggested an optimal cell cycle-arrest time at 126 h and supplementation with feed from 48 h onwards. The results of three different fed-batch cultures with identical supplementation strategies but various cell cycle-arrest times are shown in Fig.3. The viable cell concentration, Xy, was closely predicted up to about 80 h. However, after lOOh, Xv decreased significantly in all three cultures. The predicted MAb concentration was in accordance with the experimental results with only a slight under-prediction around 80-100 h. Both model predictions and experimental results indicated a small difference in MAb yield when the cultures were arrested at different times. The optimised fed-batch experiments involved a total of 9 shake flask cultures so the... 

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